YB-1: A NEW PLAYER IN FSH REGULATION OF
GENE EXPRESSION IN GRANULOSA CELLS
By
ELYSE MARIE DONAUBAUER
A dissertation submitted in partial fulfillment of
the requirements for the degree of
DOCTOR OF PHILOSOPHY
WASHINGTON STATE UNIVERSITY
School of Molecular Biosciences
MAY 2016
© Copyright by ELYSE MARIE DONAUBAUER, 2016
All Rights Reserved
© Copyright by ELYSE MARIE DONAUBAUER, 2016
All Rights Reserved
To the Faculty of Washington State University:
The members of the Committee appointed to examine the dissertation of ELYSE MARIE
DONAUBAUER find it satisfactory and recommend that it be accepted.
_________________________________________ Mary Hunzicker-Dunn, Ph.D., Chair
_________________________________________ John Nilson, Ph.D.
_________________________________________ Joseph Harding, Ph.D.
_________________________________________ Michael Konkel, Ph.D.
_________________________________________ Eric Shelden, Ph.D.
ii
ACKNOWLEDGEMENT
I would like to acknowledge my mentor, Mary Hunzicker-Dunn, for her constant
encouragement and support in the completion of this dissertation.
iii
YB-1: A NEW PLAYER IN FSH REGULATION OF
GENE EXPRESSION IN GRANULOSA CELLS
Abstract
by Elyse Marie Donaubauer, Ph.D.
Washington State University
May 2016
Chair: Mary Hunzicker-Dunn, Ph.D.
Within the ovarian follicle, immature oocytes are surrounded and supported by granulosa cells
(GCs). Stimulation of GCs by follicle stimulating hormone (FSH) promotes their proliferation and
differentiation, events that are necessary for fertility. FSH activates multiple signaling pathways to
regulate genes necessary for follicular maturation. This research focuses on the regulation by FSH of
extracellular signal-regulated kinase (ERK) and identifies Y-box binding protein-1 (YB-1) as a
downstream target of ERK signaling. FSH-dependent ERK(Thr202/Tyr204) phosphorylation is protein
kinase A (PKA) dependent, yet requires the constitutively active upstream ERK signaling pathway
proteins. Treatment with EGFR inhibitor AG1478, a dominant-negative RAS, and the MEK inhibitor
PD98059 all blocked FSH-dependent ERK(Thr202/Tyr204) phosphorylation. We hypothesized that FSH
via PKA regulates ERK phosphorylation by inhibiting the activity of a protein phosphatase that
dephosphorylates ERK in the absence of FSH. Results show that treatment with MAP kinase phosphatase
3
(dual
specificity
phosphatase
6)
(MKP3(DUSP6))
inhibitors
increase
ERK(Thr202/Tyr204)
phosphorylation in the absence of FSH to levels comparable to ERK phosphorylated in the presence of
FSH. Further, ERK coimmunoprecipitated with MKP3(DUSP6), and treatment with MKP3(DUSP6)
iv
inhibitors blocked dephosphorylation of recombinant phospho-ERK2-GST. Together these results
indicate that MKP3(DUSP6) is the phosphatase that dephosphorylates ERK in the absence of FSH. We
further identified YB-1 as a downstream target of ERK. YB-1 is a nucleic acid binding protein that
regulates transcription and translation. Our results show that FSH promotes an increase in the
phosphorylation of YB-1 on Ser102 within 15 min that is maintained until ~8 h post treatment. FSHstimulated phosphorylation of YB-1(Ser102) is prevented by pretreatment of GCs with the PKA-selective
inhibitor PKI, the MEK inhibitor PD98059, and the ribosomal S6 kinase-2 (RSK-2) inhibitor BI-D1870.
Transduction of GCs with the dephospho-adenoviral-YB-1(S102A) mutant prevented the FSH-dependent
induction of Egfr, Cyp19a1, and Inha mRNAs. Collectively, these results demonstrate novel regulation of
ERK(Thr202/Tyr204) phosphorylation by FSH and identify ERK-dependent gene targets that are required
for follicular maturation. These results reveal that the ERK signaling pathways contributes to GC
maturation by promoting the phosphorylation of the transcriptional activator YB-1 that is required for
expression of at least three crucial FSH target genes.
v
TABLE OF CONTENTS
Page
ACKNOWLEDMENT ........................................................................................ iii
ABSTRACT ........................................................................................................ iv
LIST OF FIGURES ............................................................................................ vii
LIST OF COMMONLY ABBREVIATED WORDS ......................................... ix
CHAPTER ONE: Introduction ............................................................................................. 1
CHAPTER TWO: FSH-dependent regulation of ERK phosphorylation by MKP3 ........... 38
CHAPTER THREE: ERK–dependent phosphorylation of Y-Box binding protein 1
(YB-1) enhances gene expression in granulosa cells in response to FSH ................... 71
CHAPTER FOUR: YB-1 and Translation ....................................................................... 115
CHAPTER FIVE: Conclusions and Future Directions..................................................... 136
vi
LIST OF FIGURES
Page
CHAPTER 1
1. FIGURE 1. Overview of Signaling in Rat Granulosa Cells ................................... 17
2. FIGURE 2. Overview of Initiation of Translation .................................................. 19
CHAPTER 2
3. FIGURE 1. Canonical MAPK signaling pathway is active in GCs independent
of FSH stimulation and is necessary for phosphorylation of ERK1/2 .................... 60
4. FIGURE 2. Phosphorylation of ERK1/2 on Thr202/Tyr204 is dependent on
PKA activity............................................................................................................ 62
5. FIGURE 3. Effect of Ser/Thr and Tyr phosphatase inhibitors on
ERK1/2(Thr202/Tyr204) phosphorylation ................................................................. 64
6. FIGURE 4. Effect of DUSP inhibitors on ERK1/2phosphorylation ...................... 66
7. FIGURE 5. MKP3(DUSP6) interacts with ERK1/2 in GCs................................... 68
8. FIGURE 6. Active ERK2-GST dephosphorylation is blocked by
MKP3(DUSP6) inhibitors....................................................................................... 70
CHAPTER 3
9. FIGURE 1. Treatment of GCs with FSH leads to prolonged phosphorylation
of YB-1 on Ser102 .................................................................................................... 98
10. FIGURE 2. Phosphorylation of YB-1 on Ser102 is mediated by cAMP and
PKA and independent of exchange proteins activated by cAMP ......................... 100
vii
LIST OF FIGURES, CONT.
Page
11. FIGURE 3. FSH- stimulated phosphorylation of YB-1 on Ser102 is via ERK
signaling, not PI3K/AKT ...................................................................................... 102
12. FIGURE 4. FSH- stimulated phosphorylation of YB-1 on Ser102 is
RSK-2 dependent ................................................................................................. 104
13. FIGURE 5. Prolonged FSH- stimulated phosphorylation of YB-1 on Ser102
is dependent on inhibition of the Ser/Thr phosphatase PP1 ................................. 106
14. FIGURE 6. YB-1 and phospho-YB-1(Ser102) are detected in the both the
nuclear and cytosolic fractions; phosphorylation of YB-1 on Ser102
promotes nuclear localization ............................................................................... 108
15. FIGURE 7. ERK signaling is necessary for FSH-stimulated target gene
mRNA expression ................................................................................................. 110
16. FIGURE 8. Phospho - YB-1 regulates FSH and ERK dependent-target gene
mRNA expression ................................................................................................. 112
17. FIGURE 9. Proposed signaling pathway by which FSH promotes the
PKA- dependent phosphorylation of YB-1 on Ser102 to enhance expression
of select FSH target genes..................................................................................... 114
CHAPTER FOUR
18. FIGURE 1. YB-1 interacts with 5’cap and initiation factor complex .................. 131
19. FIGURE 2. YB-1 binds to PABP1, but not eIF4E ............................................... 133
20. FIGURE 3. YB-1 binding to mRNAs increases in response to
FSH-stimulation .................................................................................................... 135
viii
LIST OF COMMONLY ABBREVIATED WORDS
Common abbreviations used are:
GCs, granulosa cells
FOXO1, forkhead box- containing protein O1
FSH, follicle stimulating hormone
cAMP, cyclic adenosine monophosphate
PKA, protein kinase A
CREB, cAMP-response binding element protein
PKI, PKA inhibitor
EGFR, epidermal growth factor receptor
RSK-2, ribosomal S6 kinase-2
MEK, mitogen-activated protein kinase kinase PP1, protein phosphatase 1
Egr1, early growth response factor – 1
GPCR, G protein-coupled receptor
RTK, receptor tyrosine kinase
AKT, protein kinase B
JNK, c-Jun N-terminal kinase
PI3K, phosphatidylinositol-3- kinase
PTP, protein tyrosine phosphatase
YB-1, Y-box binding protein-1
eIF4E, eukaryotic initiation factor 4E
IGF1, insulin-like growth factor 1
CDC25, cell division cycle 25
Myr, myristoylated
MAPK, mitogen activated protein kinase
LH, luteinizing hormone
RTK, receptor tyrosine kinases
E2, estradiol-17β
MKP, MAP kinase phosphatase
P, penicillin
PTP, protein tyrosine phosphatase
S, streptomycin
ERK, extracellular signal-regulated kinase
PP2, protein phosphatase 2
DUSP, dual specificity phosphatase
mRNP, mRNA-protein complex
SHP2, SRC homology-2 (SH2) domain-containing tyrosine phosphatase
ix
DEDICATION
This dissertation is dedicated to my parents who provided unlimited and much needed emotional
support. They will have no idea what this dissertation is about, but will still attempt to read it.
That is unconditional love and support.
x
CHAPTER ONE
INTRODUCTION
FOLLICULAR MATURATION
In females, the ovarian follicle has a distinct role in fertility. The ovarian follicle serves to
produce a mature oocyte that can be fertilized, to prepare the uterus to accept the fertilized oocyte, and to
sustain the development of the embryo (reviewed in (1)). These processes are accomplished through
response to and regulation by multiple endocrine and paracrine hormones. Endocrine hormones follicle
stimulating hormone (FSH) and luteinizing hormone (LH) are required for follicular maturation and
ovulation of the oocyte (2,3). Granulosa cells (GCs) within the follicle produce and secrete estradiol (4).
The resulting estradiol circulating in the blood will act on the pituitary to regulate LH production, on GCs
to stimulate growth, and on the uterus to prepare for and sustain the embryo after implantation (reviewed
in (5) and (6)). Following ovulation, terminally differentiated GCs produce and secrete progesterone (4),
which is also essential for the uterus to support the embryo and maintain pregnancy. Thus, the ovarian
follicle has a central role in regulation of all these events.
Within the ovary, the follicle surrounds and supports the female gamete and is comprised of an
oocyte surrounded by support GCs, basal lamina, and an external layer of theca cells that contains the
vascular supply for the follicle. Follicular maturation occurs within the follicle in two distinct stages, the
gonadotropin – independent stage and the gonadotropin – dependent stage (reviewed in (1)). During each
reproductive cycle, a cohort of follicles undergoes maturation from immature primordial follicles to
preantral follicles in the gonadotropin – independent stage.
Primordial follicles are formed prenatally following germ cell cyst breakdown. Germ cell cysts
consist of clusters of dividing oogonia, and these oogonia become oocytes when they enter mitosis. As
the germ cell cysts breakdown, there is programed cell death of some of the oocytes, and the bordering
GCs will surround single oocytes, forming primordial follicles (reviewed in (7)). Primordial follicles
1 develop into primary, then secondary follicles, which are marked by the growth of the oocyte, addition of
multiple layers of GCs, and formation of the theca cell layer. Although the regulation of germ cell cyst
breakdown and primordial follicle formation to primary and secondary follicle formation is still being
elucidated, mouse knockout models have identified many essential regulators in the pathway (reviewed in
(7)). For example, growth differentiation factor 9 (Gdf9)- knockout mice have ovaries that only contain
primary follicles, indicating that GDF-9 is necessary for primary follicles to develop into secondary
follicles (8). Anti-Müllerian hormone (Amh)-knockout mice have fewer primordial follicles, leading to
premature follicle depletion (9). As AMH treatment caused a decrease in primordial follicle assembly, it
is thought that the Amh-knockout mice have fewer primordial follicles to start with (10). These results
indicate that both GDF-9 and AMH are a few of the important regulators of the maturation of primordial
follicles to primary and secondary follicles.
During the gonadotropin-dependent stage the follicle responds to the gonadotropin hormones
FSH and LH. Maturation of preantral follicles occurs in response to FSH and only a few follicles mature
completely while most of the cohort undergoes apoptosis and atresia (11-13). Response to FSH promotes
production and secretion of estrogen, proliferation and differentiation of GCs, and expansion of the
follicle in size. Response to LH promotes ovulation of the oocyte and initiation of meiosis and formation
of the corpus luteum that serves to support the oocyte upon fertilization. Only GCs express the FSH
receptor, and in response to FSH, GCs undergo proliferation and differentiation, causing the follicle to
increase in size. A fluid filled antrum forms within the center of the follicle, and two distinct populations
of GCs develop. The cumulus cells surround the oocyte and provide it with nutrients, metabolites, and
essential regulatory signals (14). The mural GCs line the interior adjacent to the basal lamina, and
produce essential steroids (15,16) and express LH receptors (17,18). Interestingly, the cumulus GCs are
distinguished from mural GCs by the low expression of Lhcgr mRNA (19). FSH initiates this transition
through regulation of hundreds of genes within GCs (20,21), leading to changes in many cellular
processes such as intracellular signaling, steroid production, and cell cycle regulation.
2 FSH facilitates regulation of the transcription of target genes through a variety of mechanisms,
such as an increase in the expression of the transcription factors early growth response factor – 1 (Egr1)
(22) and c-Fos (23). FSH promotes posttranslational modification of common transcription factors like
cAMP response element binding protein (CREB) (24), FOXO1 (25), and Smad2/3 (26), as well as
promotes an increase in phosphorylated and acetylated histone H3 bound to FSH target genes (27). These
modifications, and others similar, lead to an FSH-dependent increase in transcription of genes that
encode, for example, epiregulin (28), LH receptor (29), regulatory (R) IIβ subunit of protein kinase A
(PKA) (30), and serum glucocorticoid kinase (SGK) (31).
FSH also promotes an increase in the paracrine and autocrine hormones produced by the GCs
through activating gene targets (32,33). Examples include: increases in production of inhibin - α, the
hormone necessary to negatively feedback and repress production of FSH at the pituitary and
hypothalamus (27,34); increases in progesterone due to increased production of P-450 cholesterol side
chain cleavage (SCC) necessary for progesterone production (35,36); and importantly increases in
estrogen due to increased production of P-450 aromatase (aromatase) necessary in estrogen biosynthesis
(37,38).
FSH triggers proliferation of the GCs. One way this is achieved is through regulation of the
expression of the cell cycle protein cyclin D2. Cyclin D2 is necessary for follicular maturation, as
demonstrated by the infertility of the cyclin D2-null mouse model (39). Interestingly, FSH alone is not
sufficient to promote cyclin D2 expression in rat GCs in culture; a TGFβ agonist, such as activin, is
required in addition to FSH (40,41). FSH and activin regulates the transcription factors FOXO1 and
Smad2/3, respectively, to activate transcription of cyclin D2 (26), and to promote granulosa cell
proliferation.
Collectively, these data indicate that the action of FSH on GCs promotes coordinated regulation
of hundreds of genes. This regulation requires released repression of negative regulators, as well as
3 activation of multiple signaling pathways, and is essential to maturation of the follicle resulting in
ovulation of the oocyte.
SIGNALING IN GCs
In order to regulate expression of essential target genes, FSH activates a variety of signaling
cascades such as the phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT) pathway and mitogenactivated protein kinases (MAPK)/ extracellular signal-regulated kinase (ERK) pathway (see Fig 1). FSH
accomplishes these activations mainly through an increase in cAMP levels and activation of PKA. FSH
binds to its G-protein coupled receptor, a seven transmembrane receptor protein that undergoes a
conformational change after binding to FSH. The FSH receptor (FSHR) is only expressed in GCs, is
coupled to the stimulatory G protein Gs that in turn activates adenylyl cyclase. Activation of adenylyl
cyclase promotes an increase in intracellular cAMP levels. The role of cAMP in GCs is demonstrated by
treatment with forskolin, an adenylyl cyclase activator. Forskolin treatment increases cellular levels of
cAMP, mimicking the response in GCs to FSH, including activation of PKA and phosphorylation of
CREB (42,43). PKA is a tetrameric holoenzyme that has two regulatory and two catalytic subunits. The
regulatory subunits contain a pseudosubstrate motif that binds the substrate-binding site of the PKA
catalytic domain, blocking its activity. cAMP binds to the regulatory subunit of PKA, inducing a
conformational change and releasing the active catalytic subunits (44). PKA activates multiple signaling
cascades, as well as stimulates phosphorylation of CREB, histone H3, and β – catenin (25,27) (Fig 1).
Treatment with either the adenoviral – PKA inhibitor (PKI), a pseudosubstrate for the catalytic subunit of
PKA, or the myristoylated (Myr) – PKI peptide blocks FSH mediated phosphorylation of direct PKA
targets CREB(Ser133) (25,43) and β – catenin(Ser552) (25), blocks activation of both the PI3K/AKT and
MAPK/ERK pathways (43,45), and blocks induction of FSH-dependent gene targets Map2D and Inha
(27,43) as well as Lhcgr and Cyp11a1 (46). Additionally, expression of a constitutively active PKA
(PKA-CQR) in GCs effectively mimics FSH treatment, including regulation of FSH target genes and
4 activation of estrogen and progesterone biosynthetic pathways necessary for follicular maturation (20),
indicating that PKA is both necessary and sufficient to regulate target gene expression. Taken together,
these results demonstrate that FSH acts via PKA to regulate targets and promote maturation of the
follicle.
FSH promotes activation of PI3K/AKT (27,47,48) primarily through regulation of insulin
receptor substrate 1 (IRS1) (45). PI3K is canonically activated with ligand binding to either the insulin
receptor or insulin – like growth factor 1 receptor (IGF1R) receptor tyrosine kinases (RTKs) (reviewed in
(49)). After ligand binding, the receptor dimerizes and autophosphorylates tyrosine residues within NPXY
motif. These pTyr binding domains on the RTKs allow for binding of adaptor proteins (IRS1/2 or and
GRB2 (growth factor receptor binding protein 2) associated binding protein (GAB)GAB1/2) and, in turn,
the adaptor proteins are phosphorylated by RTKs on specific tyrosine resides in the carboxy terminus
(50). The pTyr residues on either IRS1/2 or GAB1/2 create SRC homology-2 (SH2) binding domains
(dual phosphorylated TyrMetXMet motifs) for PI3K (51). Dual SH2 domains on the 85 kDa regulatory
subunit of PI3K (p85) bind dual phospho-YMXM motifs on IRS1/2 or GAB1/2, resulting in activation of
the 100 kD catalytic subunit (p100) of PI3K (52). In GCs, FSH promotes phosphorylation of IRS1 on
Tyr989 (a phospho-YMXM motif) (45) that activates PI3K.
Active PI3K generates phosphatidylinositol-3,4,5-trisphosphate (PIP3) through addition of a
phosphate to phosphatidylinositol-4,5-bisphosphate (PIP2). Increased levels of PIP3 in the plasma
membrane facilitate the binding of both phosphoinositide-dependent kinase 1 (PDK1) and AKT via their
pleckstrin homology domains (53,54). With PDK1 in proximity to AKT, PDK1 will phosphorylate AKT
on Thr308 (55,56). Full activation of AKT occurs after mammalian target of rapamycin (mTOR)/Rictor
complex (mTORC2) phosphorylates AKT on Ser473 (57), and in GCs, is PKA dependent (45). Active
AKT will translocate to other cellular locations, including the nucleus, to promote cellular proliferation,
enhance translation and initiate differentiation (58).
5 Phosphorylated/active AKT facilitates GC proliferation and differentiation through regulation of
a variety of substrates. In response to FSH, AKT inhibits tuberin, also called tuberous sclerosis complex 2
(TSC2), a negative regulator of cell growth. In the absence of stimuli, tuberin acts as a GTPase activating
protein (GAP) for ras-homologue enriched in brain (RHEB), keeping inactive RHEBGDP levels high (59).
In response to FSH, AKT inhibits tuberin, allowing active RhebGTP levels to increase and to activate
mTOR (60). mTOR will then phosphorylate p70 S6 kinase (p70S6K) and 4E-binding protein (BP)1
(4EBP1) (60), promoting an increase in translation. AKT also phosphorylates the transcription factor
FOXO1 in response to FSH (25,26,61,62). FOXO1 is a member of the forkhead box-containing O
subfamily and binds DNA in an unphosphorylated state. FOXO transcription factors have been shown to
both activate and repress transcription in a gene dependent manner and FOXO1 is no exception. FOXO1
has been shown to activate the cell cycle inhibitor p27Kip1 (63), glucose-6-phosphatase (64), as well as to
repress cyclin D1 and D2 (65). In GCs, in the absence of FSH, FOXO1 has been shown to be bound to
the cyclin D2 promoter, and treatment with FSH promoted its dissociation (26). Additionally, treatment of
GCs with a constitutively active adenoviral-FOXO1 resulted in the inhibition of the induction of the
necessary follicular maturation gene targets Cyp19a1 and Cyp11a1, Inha, and Ereg (26). Additional genes
were identified as FOXO1 targets including the LH receptor (Lhcgr) and genes required for cholesterol
biosynthesis and steroidogenesis (66).
FSH also regulates mitogen-activated protein kinase kinase (MEK)/ERK signaling in GCs
through regulation of ERK phosphorylation (43,67,68). As presented in Chapter 3, my recent results show
that ERK activation is necessary for the induction of genes that are required for follicular maturation,
including Inha (one subunit of the hormone inhibin that functions to inhibit FSH expression by the
pituitary as follicles mature), Egfr (the epidermal growth factor receptor, that is required for LH-induced
oocyte maturation), and Cyp19a1(that encodes aromatase, the rate limiting enzyme in estrogen
biosynthesis) (Fig 1) (69). Canonical MEK/ERK signaling originates with activation of RTKs at the
plasma membrane (reviewed in (70)). After ligand binding, autophosphorylation of the RTKs creates
6 docking sites for Src homology 2 (SH2) containing proteins such as GRB2 (71). GRB2 is in complex
with the guanine nucleotide exchange factor SOS (72), and after binding to the RTK, SOS activates RAS
through release of GDP (73), triggering the MAPK signaling cascade. The tyrosine phosphatase SRC
homology-2 (SH2) domain-containing tyrosine phosphatase (SHP2) is also recognized to contribute to the
activation of RAS, although the mechanism by which SHP2 enhances RAS is poorly understood (74-76).
Active RAS activates the Ser/Thr kinase RAF-1 (77), that subsequently phosphorylates the dual
specificity kinase MEK (78). MEK will phosphorylate ERK on Thr202 and Tyr204, and ERK will
phosphorylate the downstream kinase ribosomal S6 kinase (RSK-2) (79). Additionally, ERK activity has
been shown necessary for many cellular processes, such as cell cycle regulation, transcription, and
translation.
ERK activity is generally necessary for progression of cells through G1 into the S phase of the
cell cycle. This is accomplished in part through regulation of the expression of cyclin D, part of the cyclin
D-cyclin-dependent kinase (CDK) 4/6 complex, which is necessary for cell entry into S phase (80,81).
Additionally, ERK activity promotes downregulation of p27kip1, a CDK inhibitor (82), required for the
late G1 phase activation of cyclin E-CDK2 (83). Activation of cyclin E promotes induction of cyclin A
and formation of active cyclin A-CDK2 complex. Both cyclin E and cyclin A are required for S phase
entry and cyclin A is required for progression through S phase (reviewed in (81)). Thus, cyclin D is an
integral player in this sequence of events, and the regulation of its expression by ERK demonstrates one
way in which ERK contributes to cell cycle regulation.
ERK regulates transcription through altering the activities of transcription factors in many ways,
including altering their cellular location, their expression or stability, and/or their ability to bind to other
transcription factors or DNA (reviewed in (84,85)). For example, ERK phosphorylates the transcription
factors JunD (86), c-Fos (87), GATA4 (88), Elk-1 (89), and c-Myc (90), enhancing their activity as
transcriptional activators. Additionally phosphorylation of SF-1 (Nr5a1 gene product) by ERK promotes
recruitment of coactivators and ultimately results in an active conformation (91). Alternatively,
7 phosphorylation by ERK
of the progesterone receptor (92) and GATA-1 (93) promotes their
ubiquitination and subsequent degradation. Through these mechanisms, and others, ERK is able to
regulate transcription of target genes.
Regulation of translation in response to ERK activity is accomplished through regulation of a few
essential translational regulator proteins by either ERK itself or, more commonly, by its downstream
kinase RSK. First, both ERK and RSK have been shown to phosphorylate and inactivate the translational
inhibitor protein TSC1/2 (94). Additionally, both kinases phosphorylate mTORC1 protein Raptor, leading
to increased mTORC1 activity (95,96). Phosphorylation of the ribosomal protein S6 by RSK has been
well established, and this phosphorylation correlates with eIF4F complex assembly and cap-dependent
translation (97-99). RSK phosphorylates the additional components of the translational machinery, such
as the initiation factor protein eIF4B (100) and the elongation factor 2 kinase (eEF2) (101). Thus, through
regulation of multiple components necessary for translation, ERK is able to effect translational activity.
In response to FSH, there is a rapid, transient increase in the phosphorylation of ERK on Thr202
and Tyr204 and its downstream kinase RSK2 on Ser380 (27,43,69). ERK activation is required for the
induction of MAP2D (43), Egr-1 (22) and cyclin D2 (102) in GCs, based on the use of MEK inhibitors.
Additionally, the importance of ERK signaling in GCs is demonstrated by the expression of a
constitutively active RAS (KRAS-Gly12Asp) in preantral GCs that resulted in subfertility and the
formation of abnormal follicles and blocked induction of the Lhcgr (103).
Previous results from our lab have shown that FSH-stimulated ERK phosphorylation is both
PKA and MEK dependent, as demonstrated using the inhibitors Myr-PKI and PD98059, respectively
(43). However, MEK is phosphorylated in untreated GCs, and the phosphorylation level remains constant
in response to FSH. RAF-1 and RAS were also found to be active, and unresponsive to FSH stimulation
(43). Additionally, both RAS and EGFR activity are necessary for FSH-dependent phosphorylation of
ERK on Thr202 and Tyr204, based on experiments using a dominant negative adenoviral-RAS and the
EGFR inhibitor AG1487, respectively (see Chapter 2, (43). These results demonstrate that while the
8 upstream components of the MEK/ERK signaling cascade are necessary for FSH-dependent ERK
phosphorylation, the activity of the upstream pathway is not regulated by FSH directly, and is most likely
activated in an FSH independent manner.
Because MEK is still necessary for ERK(Thr202 and Tyr204) phosphorylation, the FSH-dependent
regulation of ERK(Thr202 and Tyr204) phosphorylation is most likely mediated through inhibition of a
phosphatase. In the absence of FSH, the phosphatase would be active and dephosphorylating ERK, but
following FSH treatment the phosphatase would be inactivated, allowing for MEK dependent ERK(Thr202
and Tyr204) phosphorylation to procced and accumulate. Although a 100 kD protein tyrosine phosphatase
(PTP), detected using a PTP-SL antibody (104), was identified to interact with ERK in GCs (43), further
experiments utilizing a pan-PTP inhibitor Na2VO3 failed to raise ERK(Thr202 and Tyr204) phosphorylation
in untreated cells (see Chapter 2). Additionally, different antibodies against PTP-SL failed to detect the
100 kD protein (see Chapter 2), indicating that the signal detected at 100 kD might not be the phosphatase
that dephosphorylates ERK in the absence of FSH. My preliminary studies indicated that FSH-dependent
ERK phosphorylation could be regulated by a member of the dual specificity phosphatases (DUSP)
family (see Chapter 2).
DUSPs
DUSPs are members of the PTP family, and are grouped together because they are able to
dephosphorylate both tyrosine and serine/threonine residues on the same substrate. There are different
subgroups of DUSPs; those DUSPs that act on MAPK proteins (also called mitogen-activated protein
kinase phosphatase [MKPs]) comprise one subfamily. Individual MKPs show substrate specificity
preference among ERK, JNK (c-Jun N-terminal kinase), and p38 MAPK in response to stimuli, as well as
differential cellular location (reviewed in (105)). Additionally, individual DUSPs are regulated
differently, and can be induced or phosphorylated or degraded to alter their phosphatase activity
(reviewed in (106)). While DUSPs in GCs are rather understudied, previous results can be used to focus
9 on the best candidates for examination. Out of the 61 DUSPs identified (reviewed in (105)), 10 are
MKPs, and of those 10, GCs only express MKP1(DUSP1), MKP3(DUPS6), MKPX/PYST2(DUSP7),
MKP5(DUSP10), MKP7(DUSP16), in addition to cell cycle dual specificity phosphatases cell division
cycle 25A (CDC25A), CDC25B, and CDC25C (20) (Herndon et al., manuscript in preparation). MKP5
and MKP7 are selective for p38 MAPK and JNK over ERK, and were eliminated from the investigation
list. Unfortunately, at the time of publishing, MKPX did not have readily available inhibitors, and could
not be easily investigated.
MKP1(DUSP1) was first isolated from skin cells (107) and was first shown to preferentially
dephosphorylates p38 and JNK over ERK (108,109). However, MKP1(DUSP1) has since been shown to
dephosphorylate ERK as well (110,111). MKP1(DUSP1) is generally localized to the nucleus in cells,
and is regulated through induction in response to a variety of different stimuli (reviewed in (112)).
MKP3(DUSP6) shows substrate preference for ERK over either p38 or JNK (113,114) and is localzed to
the cytoplasm. MKP3(DUSP6) is most commonly regulated through phosphorylations on Ser159 and
Ser197 downstream of ERK that cause MKP3(DUSP6) degradation through the proteasome (115).
CDC25A-C are cell cycle regulators that mainly dephosphorylated and activate cyclin-CDK complexes.
CDK complexes are held inactive through inhibitory Thr and Tyr phosphorylations on the ATP binding
loop, and will become active after dephosphorylation by CDC25. Thus, expression and activity of CDC25
is highly regulated and dependent on multiple mechanisms. Interestingly, CDC25 inhibitors have been
shown to promote increased ERK(Thr202 and Tyr204) phosphorylation (116,117), and CDC25 has been
shown to interact and dephosphorylate ERK directly (118). For these reasons, investigations focused on
MKP1(DUPS1), MKP3(DUSP6), and CDC25A-C.
YB-1
Y-box-binding protein 1 (YB-1) is a multifunctional vertebrate protein that is a member of the
cold shock domain (CDS) family. YB-1 is a nucleic acid chaperone protein, binding both DNA and RNA
10 (119,120). Because of this trait, YB-1 is involved with most DNA- and mRNA- dependent processes,
such as DNA replication, transcription, pre-mRNA processing, and translation (reviewed in (121,122)).
Found in both the cytosolic and nuclear compartments of the cell, YB-1 interacts with an assortment of
diverse proteins and is involved with many cellular processes including differentiation and proliferation
(123-125).
YB-1 was described as a major component of the mRNA- protein complex (mRNP) first in birds
(119,126), followed by mammals (rabbits) (127). When YB-1 binds to RNA, it melts the secondary
structure (128), and acts as an mRNA chaperone protein. At a high YB-1:mRNA ratio, YB-1 binds along
the whole mRNA strand and stabilizes the mRNA, protects mRNA from degradation by exonucleases,
and blocks association with the translation initiation factors (129-131). However, at low YB-1:mRNA
ratios, YB-1 stimulates the initiation of translation (132), possibly through promoting the movement of
the small ribosomal subunit along the mRNA to the initiation codon (133).
YB-1 was first identified binding to DNA via a ‘Y-box-binding site‘ (5’-CTGATTGGC/TC/TAA–
3’) (134), but it was later shown that YB-1 could interact with a variety of sequences (135,136). YB-1
activates Egfr (137), Mdr1 (multidrug resistance gene 1) (138), Ptpn1 (protein tyrosine phosphatase 1B)
(139), and Smad7 (140) genes and inactivates p53 (141), Vegf (142), and other genes (143,144) in
response to various stimuli in multiple cancer cell lines. YB-1 has been shown to be misregulated in many
oncogenic studies (145-150), and depending on the cancer cell line, is identified as either an oncoprotein
or a tumor suppressor. Overexpression of YB-1 in cancer cells increases induction of MMP2 (matrix
metalloproteinase 2) and is thought to contribute to the increase in capability for cancer cell migration and
invasion (151,152). Overexpression of YB-1 in breast epithelial cells results in epithelial-mesenchymal
transition (EMT) (153), possibly through an increase in cap-dependent translation of EMT regulators
(154). The EMT is a significant stage in tumor metastasis, characterized by a loss of cellular cuboidal
shape, apical basal polarity, and a near complete loss of E-cadherin and other epithelial cell markers
(155).
11 YB-1 can regulate the transcription of genes through binding with other transcription factors. For
example, in the promoter region for Mmp2, YB-1 is found in complex with AP-2 and p53, and this
complex activates induction of the gene (156). YB-1 is often found in complex with purine-rich element
binding protein A (PurA) and PurB, with the complex binding many targets genes, acting as both
activator and repressor complexes (157-159).
YB-1 has been shown to be posttranscriptionally modified in a variety of different ways, most
commonly through phosphorylation. YB-1 can be phosphorylated both in vitro and in vivo on Ser102 by
the kinase Akt (160,161) and by the kinase RSK (162). Also, both ERK2 and GSK3β are able to
phosphorylate YB-1 on Thr38 and/or Ser41 and/or Ser45 (142). These phosphorylations have been shown to
alter the binding of YB-1 to both DNA and RNA (142,160), providing one possible mechanism for YB-1
participation in both transcription and translation. Additionally, mass – spectrometric studies have
identified that YB-1 has multiple phosphorylation sites in addition to those previously mentioned,
including Tyr162, Ser165 and/or Ser167, Ser174 and/or Ser176, and Ser313 and/or Ser314 (163).
As described in Chapter 3, my results show that YB-1 is phosphorylated on Ser102 in response to
FSH stimulation beginning approximately 15 min and lasting until approximately 8 hours following
treatment. Phosphorylation of YB-1 on Ser102 is PKA-, ERK-, and RSK-dependent. Additionally, use of a
dephospho-adenoviral-YB-1(S102A) mutant blocked induction by FSH of Egfr, Inha, and Cyp19a1
mRNAs (69). These results expand on the current knowledge of the role of YB-1 as a transcriptional
regulator, reveal that phosph-YB-1(Ser102) is crucial regulator of genes in GCs that define the
preovulatory phenotype, and verify the regulation of YB-1 phosphorylated on Ser102 by RSK.
TRANSLATION
Regulation of translation occurs through regulation of the different protein components that make
up the translational machinery. Translation occurs in three stages: initiation, elongation, and termination.
12 Although all three stages are regulated, most of the regulation occurs during initiation and the assembly of
the different protein components (164).
Before initiation, pre-mRNA is processed before leaving the nucleus. The poly-(A) tail and the
5’-7-methyl guanine (m7G)-cap are added to the pre-mRNA to protect the free 5’ and 3’ ends (reviewed
in (165)). Introns are removed from the pre-mRNA by the spliceosome (166), and following removal,
mRNA is shuttled to mRNA export proteins for movement out of the nucleus (167). As mRNA leaves the
nucleus, mRNA binding proteins, such as poly(A)-binding protein (PABP), will bind mRNA as it moves
to different compartments of the cell (168).
Initiation begins with eIF4E binding to the 5’-m7G cap of mRNA, and recruitment of eIF4G and
eIF4A, forming eIF4F (Fig. 2). Simultaneously, the 40S ribosome binds eIF1, eIF3 and eIF2-GTP-MettRNAMet, forming the 43S pre-initiation complex (reviewed in (169)). eIF4F promotes binding of the
pre-initiation complex to the 5’-end of mRNA. eIF4F will also interact with PABP proteins binding to the
poly-(A) tail, forming a circular ring structure that promotes continuous and efficient translation (170).
The 43S pre-initiation complex will scan along the mRNA in the 5’ to 3’ direction until the start AUG
codon is located (171). Upon recognition of the start AUG sequence, the 43S pre-initiation complex stalls,
and eIF2-GTP is hydrolyzed to eIF2-GDP, promoting recruitment to the 60S ribosomal subunit (169).
eIF5B facilitates the joining of the 60S ribosomal subunit, and eIF1, eIF2-GDP and eIF5B are released
(172). The translational machinery is ready to continue down the mRNA strand, adding amino acids to the
growing polypeptide chain.
Elongation occurs as tRNA-aminoacyl (AA) move through three sites on the ribosome, resulting
in transfer of the AA from the tRNA to the growing polypeptide chain. tRNA-AA is bound to elongation
factor 1A (eEF1A)-GTP and will bind the A site on the ribosome (173). Hydrolysis of GTP to GDP
facilitates the release of eEF1A-GDP from the ribosome, leaving the tRNA-AA in the A site. Elongation
factor eEF1B recycles eEF1A, through hydrolysis of GTP to GDP. Elongation factor 2 facilitates
movement of the tRNAs into the next site on the ribosome (174). eIF5A is required to correctly position
13 the initiator tRNA-Met in the P-active site of the ribosomal complex (175). Addition of AA to the
polypeptide chain will continue until the ribosomal complex encounters a stop codon.
Termination occurs upon recognition by the ribosome of one of three termination codons and
binding of two eukaryotic release factors (eRFs) (176). eRF1 binds to the ribosome, and this binding
promotes the ribosome to release the polypeptide chain. eRF3-GTP binds to the ribosome and hydrolysis
of GTP to GDP releases eRF1 (177). A second hydrolysis of GTP to GDP releases eRF3 from the
ribosome. The remaining components are disassembled in a virtually unknown process, freeing the small
40S ribosomal subunit to reassemble and start translation again.
The different ways in which FSH promotes translation were covered briefly in previous
sections. FSH regulates translation through regulation of specific components of the translational
complex, most notability through regulation of the phosphorylation of 4EPB1 and p70S6K via mTORC1
(Fig.1). FSH stimulates inhibition of TSC2, allowing for an increase in mTORC1 activity (178).
Following FSH stimulation, mTORC1 stimulates phosphorylation of p70S6K and the 4EBP1 (60). FSH
regulation of translation could thus promote a rapid increase in protein expression, allowing for rapid
cellular responses.
SUMMARY
Herein, we show in Chapter 2 that while ERK signaling upstream of MEK is constitutively
active, it is necessary for FSH-dependent ERK(Thr
ERK(Thr
202
202
and Tyr204) phosphorylation. Results show that
and Tyr204) phosphorylation is selectively inhibited by DUSP inhibitors. Inhibitors of
MKP3(DUSP6) increase ERK(Thr
202
and Tyr204) phosphorylation in the absence of FSH to levels
comparable to FSH-stimulated ERK(Thr
202
and Tyr204) phosphorylation. ERK co-immunoprecipitates
with MKP3(DUSP6), and MKP3(DUSP6) inhibitors block dephosphorylation of ERK2-GST. These
results indicated that in the absence of FSH in GCs, ERK is dephosphorylated by MKP3(DUSP6).
14 Results presented in Chapter 3 show that YB-1 is phosphorylated on Ser102 in response to FSHstimulation. YB-1 phosphorylation on Ser102 is PKA-, ERK-, and RSK-2-dependent. Additionally, YB1(Ser102) phosphorylation is dependent on protein phosphatase 1 (PP1), and YB-1 interacts with both
PP1βc and RSK-2. Expression of a dephospho-adenoviral-YB-1(S102A) mutant prevented the FSHstimulated induction of Egfr, Cyp19a1, and Inha mRNA. Together these results reveal an unexpected
role for the MEK/ERK/RSK pathway and for YB-1 in target gene induction by FSH in GCs.
In Chapter 4, we demonstrate that YB-1 interacts with select mRNA binding proteins and
initiation factor proteins. As a result of the isolation of proteins binding the 5’ cap using an m7GTPSepharose bead, we detect total YB-1 protein, and YB-1 increases binding following FSH stimulation.
Using a Flag-tagged YB-1 in immunoprecipitation studies, we detected PABP1 binding to YB-1, and this
binding is FSH-independent. In an investigation of mRNAs bound to YB-1, we observe an increase in
Hif1α and Egr1 mRNAs bound to YB-1 over IgG control. We also observe an increase Egr1 mRNA
bound to YB-1 in response to FSH stimulation. Together these results suggest that in response to FSH,
YB-1 increases binding to mRNAs and the initiation factor complex.
Overall, the research presented in this document focuses on FSH-stimulated ERK signaling and
the role of its downstream target YB-1. We present results that expand the current knowledge of ERKdependent gene targets in GCs and demonstrate that their expression is also YB-1-dependent. These gene
targets, Egfr, Cyp19a1, and Inha, are all necessary for FSH-dependent follicular maturation. These results
demonstrate the critical role that both ERK and YB-1 play in GC maturation in response to FSH
stimulation.
15 FIGURE LEGENDS AND FIGURES
FIGURE 1. Overview of FSH-regulated signaling in GCs. This schematic represents our understanding
of FSH-dependent signaling via PKA to direct gene targets and to the PI3K/AKT and ERK signaling
cascades.
16 17 FIGURE 2. Overview of initiation of translation. Reused with permission from Sonenberg and
Hinnebusch (2009) (169).
18 19 REFERENCES
1.
Hunzicker-Dunn, M., and Mayo, K. E. (2015) Gonadotropin Signaling in the Ovary. in KNOBIL
AND NEILL’S PHYSIOLOGY OF REPRODUCTION (Plant, T. M., and Zeleznik, A. J. eds.),
Fourth Ed., Elsevier. pp 895 - 946
2.
Kumar, T. R., Wang, Y., Lu, N., and Matzuk, M. M. (1997) Follicle stimulating hormone is
required for ovarian follicle maturation but not male fertility. Nat. Genet. 15, 201-204
3.
Zhang, F. P., Poutanen, M., Wilbertz, J., and Huhtaniemi, I. (2001) Normal prenatal but arrested
postnatal sexual development of luteinizing hormone receptor knockout (LuRKO) mice. Mol.
Endocrinol. 15, 172-183
4.
Sluss, P. M., Lee, K., Mattox, J. H., Smith, P. C., Graham, M. C., and Partridge, A. B. (1994)
Estradiol and progesterone production by cultured granulosa cells cryopreserved from in vitro
fertilization patients. Eur. J. Endocrinol. 130, 259-264
5.
Christensen, A., Bentley, G. E., Cabrera, R., Ortega, H. H., Perfito, N., Wu, T. J., and Micevych,
P. (2012) Hormonal regulation of female reproduction. Hormone and metabolic research =
Hormon- und Stoffwechselforschung = Hormones et metabolisme 44, 587-591
6.
Carson, D. D., Bagchi, I., Dey, S. K., Enders, A. C., Fazleabas, A. T., Lessey, B. A., and
Yoshinaga, K. (2000) Embryo implantation. Dev. Biol. 223, 217-237
7.
Pepling, M. E. (2012) Follicular assembly: mechanisms of action. Reproduction 143, 139-149
8.
Dong, J., Albertini, D. F., Nishimori, K., Kumar, T. R., Lu, N., and Matzuk, M. M. (1996)
Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature 383, 531535
9.
Durlinger, A. L., Kramer, P., Karels, B., de Jong, F. H., Uilenbroek, J. T., Grootegoed, J. A., and
Themmen, A. P. (1999) Control of primordial follicle recruitment by anti-Mullerian hormone in
the mouse ovary. Endocrinology 140, 5789-5796
10.
Nilsson, E. E., Schindler, R., Savenkova, M. I., and Skinner, M. K. (2011) Inhibitory actions of
Anti-Mullerian Hormone (AMH) on ovarian primordial follicle assembly. PloS one 6, e20087
11.
Amsterdam, A., Keren-Tal, I., Aharoni, D., Dantes, A., Land-Bracha, A., Rimon, E., Sasson, R.,
and Hirsh, L. (2003) Steroidogenesis and apoptosis in the mammalian ovary. Steroids 68, 861867
20 12.
Pru, J. K., and Tilly, J. L. (2001) Programmed cell death in the ovary: insights and future
prospects using genetic technologies. Mol. Endocrinol. 15, 845-853
13.
McGee, E. A. (2000) The regulation of apoptosis in preantral ovarian follicles. Biol Signals
Recept 9, 81-86
14.
Sutton-McDowall, M. L., Gilchrist, R. B., and Thompson, J. G. (2010) The pivotal role of
glucose metabolism in determining oocyte developmental competence. Reproduction 139, 685695
15.
Whitelaw, P. F., Smyth, C. D., Howles, C. M., and Hillier, S. G. (1992) Cell-specific expression
of aromatase and LH receptor mRNAs in rat ovary. Journal of molecular endocrinology 9, 309312
16.
Zlotkin, T., Farkash, Y., and Orly, J. (1986) Cell-specific expression of immunoreactive
cholesterol side-chain cleavage cytochrome P-450 during follicular development in the rat ovary.
Endocrinology 119, 2809-2820
17.
Eppig, J. J., Wigglesworth, K., Pendola, F., and Hirao, Y. (1997) Murine oocytes suppress
expression of luteinizing hormone receptor messenger ribonucleic acid by granulosa cells. Biol.
Reprod. 56, 976-984
18.
Mehlmann, L. M. (2005) Stops and starts in mammalian oocytes: recent advances in
understanding the regulation of meiotic arrest and oocyte maturation. Reproduction 130, 791-799
19.
Jeppesen, J. V., Kristensen, S. G., Nielsen, M. E., Humaidan, P., Dal Canto, M., Fadini, R.,
Schmidt, K. T., Ernst, E., and Yding Andersen, C. (2012) LH-receptor gene expression in human
granulosa and cumulus cells from antral and preovulatory follicles. J. Clin. Endocrinol. Metab.
97, E1524-1531
20.
Escamilla-Hernandez, R., Little-Ihrig, L., Orwig, K. E., Yue, J., Chandran, U., and Zeleznik, A. J.
(2008) Constitutively active protein kinase A qualitatively mimics the effects of folliclestimulating hormone on granulosa cell differentiation. Mol. Endocrinol. 22, 1842-1852
21.
Sasson, R., Dantes, A., Tajima, K., and Amsterdam, A. (2003) Novel genes modulated by FSH in
normal and immortalized FSH-responsive cells: new insights into the mechanism of FSH action.
FASEB J. 17, 1256-1266
22.
Russell, D. L., Doyle, K. M., Gonzales-Robayna, I., Pipaon, C., and Richards, J. S. (2003) Egr-1
induction in rat granulosa cells by follicle-stimulating hormone and luteinizing hormone:
21 combinatorial regulation by transcription factors cyclic adenosine 3',5'-monophosphate regulatory
element binding protein, serum response factor, sp1, and early growth response factor-1. Mol.
Endocrinol. 17, 520-533
23.
Sharma, S. C., and Richards, J. S. (2000) Regulation of AP1 (Jun/Fos) factor expression and
activation in ovarian granulosa cells. Relation of JunD and Fra2 to terminal differentiation. J.
Biol. Chem. 275, 33718-33728
24.
Mukherjee, A., Park-Sarge, O. K., and Mayo, K. E. (1996) Gonadotropins induce rapid
phosphorylation of the 3',5'-cyclic adenosine monophosphate response element binding protein in
ovarian granulosa cells. Endocrinology 137, 3234-3245
25.
Law, N. C., Weck, J., Kyriss, B., Nilson, J. H., and Hunzicker-Dunn, M. (2013) Lhcgr Expression
in Granulosa Cells: Roles for PKA-Phosphorylated beta-Catenin, TCF3, and FOXO1. Mol.
Endocrinol. 27, 1295-1310
26.
Park, Y., Maizels, E. T., Feiger, Z. J., Alam, H., Peters, C. A., Woodruff, T. K., Unterman, T. G.,
Lee, E. J., Jameson, J. L., and Hunzicker-Dunn, M. (2005) Induction of cyclin D2 in rat granulosa
cells requires FSH-dependent relief from FOXO1 repression coupled with positive signals from
Smad. J. Biol. Chem. 280, 9135-9148
27.
Salvador, L. M., Park, Y., Cottom, J., Maizels, E. T., Jones, J. C., Schillace, R. V., Carr, D. W.,
Cheung, P., Allis, C. D., Jameson, J. L., and Hunzicker-Dunn, M. (2001) Follicle-stimulating
hormone stimulates protein kinase A-mediated histone H3 phosphorylation and acetylation
leading to select gene activation in ovarian granulosa cells. J. Biol. Chem. 276, 40146-40155
28.
Sekiguchi, T., Mizutani, T., Yamada, K., Yazawa, T., Kawata, H., Yoshino, M., Kajitani, T.,
Kameda, T., Minegishi, T., and Miyamoto, K. (2002) Transcriptional regulation of the epiregulin
gene in the rat ovary. Endocrinology 143, 4718-4729
29.
Shi, H., and Segaloff, D. L. (1995) A role for increased lutropin/choriogonadotropin receptor
(LHR) gene transcription in the follitropin-stimulated induction of the LHR in granulosa cells.
Mol. Endocrinol. 9, 734-744
30.
Ratoosh, S. L., Lifka, J., Hedin, L., Jahsen, T., and Richards, J. S. (1987) Hormonal regulation of
the synthesis and mRNA content of the regulatory subunit of cyclic AMP-dependent protein
kinase type II in cultured rat ovarian granulosa cells. J. Biol. Chem. 262, 7306-7313
31.
Gonzalez-Robayna, I. J., Alliston, T. N., Buse, P., Firestone, G. L., and Richards, J. S. (1999)
Functional and subcellular changes in the A-kinase-signaling pathway: relation to aromatase and
22 Sgk expression during the transition of granulosa cells to luteal cells. Mol. Endocrinol. 13, 13181337
32.
Richards, J. S. (1994) Hormonal control of gene expression in the ovary. Endocr. Rev. 15, 725751
33.
McGee, E. A., and Hsueh, A. J. (2000) Initial and cyclic recruitment of ovarian follicles. Endocr.
Rev. 21, 200-214
34.
Woodruff, T. K., Meunier, H., Jones, P. B., Hsueh, A. J., and Mayo, K. E. (1987) Rat inhibin:
molecular cloning of alpha- and beta-subunit complementary deoxyribonucleic acids and
expression in the ovary. Mol. Endocrinol. 1, 561-568
35.
Goldring, N. B., Durica, J. M., Lifka, J., Hedin, L., Ratoosh, S. L., Miller, W. L., Orly, J., and
Richards, J. S. (1987) Cholesterol side-chain cleavage P450 messenger ribonucleic acid: evidence
for hormonal regulation in rat ovarian follicles and constitutive expression in corpora lutea.
Endocrinology 120, 1942-1950
36.
Hedin, L., Gaddy-Kurten, D., Kurten, R., DeWitt, D. L., Smith, W. L., and Richards, J. S. (1987)
Prostaglandin endoperoxide synthase in rat ovarian follicles: content, cellular distribution, and
evidence for hormonal induction preceding ovulation. Endocrinology 121, 722-731
37.
Carlone, D. L., and Richards, J. S. (1997) Functional interactions, phosphorylation, and levels of
3',5'-cyclic adenosine monophosphate-regulatory element binding protein and steroidogenic
factor-1 mediate hormone-regulated and constitutive expression of aromatase in gonadal cells.
Mol. Endocrinol. 11, 292-304
38.
Parakh, T. N., Hernandez, J. A., Grammer, J. C., Weck, J., Hunzicker-Dunn, M., Zeleznik, A. J.,
and Nilson, J. H. (2006) Follicle-stimulating hormone/cAMP regulation of aromatase gene
expression requires beta-catenin. Proceedings of the National Academy of Sciences of the United
States of America 103, 12435-12440
39.
Sicinski, P., Donaher, J. L., Geng, Y., Parker, S. B., Gardner, H., Park, M. Y., Robker, R. L.,
Richards, J. S., McGinnis, L. K., Biggers, J. D., Eppig, J. J., Bronson, R. T., Elledge, S. J., and
Weinberg, R. A. (1996) Cyclin D2 is an FSH-responsive gene involved in gonadal cell
proliferation and oncogenesis. Nature 384, 470-474
40.
El-Hefnawy, T., and Zeleznik, A. J. (2001) Synergism between FSH and activin in the regulation
of proliferating cell nuclear antigen (PCNA) and cyclin D2 expression in rat granulosa cells.
Endocrinology 142, 4357-4362
23 41.
Miro, F., and Hillier, S. G. (1996) Modulation of granulosa cell deoxyribonucleic acid synthesis
and differentiation by activin. Endocrinology 137, 464-468
42.
Hsueh, A. J., Adashi, E. Y., Jones, P. B., and Welsh, T. H., Jr. (1984) Hormonal regulation of the
differentiation of cultured ovarian granulosa cells. Endocr. Rev. 5, 76-127
43.
Cottom, J., Salvador, L. M., Maizels, E. T., Reierstad, S., Park, Y., Carr, D. W., Davare, M. A.,
Hell, J. W., Palmer, S. S., Dent, P., Kawakatsu, H., Ogata, M., and Hunzicker-Dunn, M. (2003)
Follicle-stimulating hormone activates extracellular signal-regulated kinase but not extracellular
signal-regulated kinase kinase through a 100-kDa phosphotyrosine phosphatase. J. Biol. Chem.
278, 7167-7179
44.
Taylor, S. S., Buechler, J. A., and Yonemoto, W. (1990) cAMP-dependent protein kinase:
framework for a diverse family of regulatory enzymes. Annu. Rev. Biochem. 59, 971-1005
45.
Hunzicker-Dunn, M. E., Lopez-Biladeau, B., Law, N. C., Fiedler, S. E., Carr, D. W., and Maizels,
E. T. (2012) PKA and GAB2 play central roles in the FSH signaling pathway to PI3K and AKT
in ovarian granulosa cells. Proceedings of the National Academy of Sciences of the United States
of America 109, E2979-2988
46.
Law, N. C., and Hunzicker-Dunn, M. E. (2015) Insulin Receptor Substrate 1: The Hub Linking
Follicle-stimulating Hormone to Phosphatidylinositol-3 Kinase Activation. J. Biol. Chem.
47.
Zeleznik, A. J., Saxena, D., and Little-Ihrig, L. (2003) Protein kinase B is obligatory for folliclestimulating hormone-induced granulosa cell differentiation. Endocrinology 144, 3985-3994
48.
Gonzalez-Robayna, I. J., Falender, A. E., Ochsner, S., Firestone, G. L., and Richards, J. S. (2000)
Follicle-Stimulating hormone (FSH) stimulates phosphorylation and activation of protein kinase
B (PKB/Akt) and serum and glucocorticoid-lnduced kinase (Sgk): evidence for A kinaseindependent signaling by FSH in granulosa cells. Mol. Endocrinol. 14, 1283-1300
49.
Vanhaesebroeck, B., and Alessi, D. R. (2000) The PI3K-PDK1 connection: more than just a road
to PKB. Biochem. J. 346 Pt 3, 561-576
50.
McGettrick, A. J., Feener, E. P., and Kahn, C. R. (2005) Human insulin receptor substrate-1 (IRS1) polymorphism G972R causes IRS-1 to associate with the insulin receptor and inhibit receptor
autophosphorylation. J. Biol. Chem. 280, 6441-6446
24 51.
Rordorf-Nikolic, T., Van Horn, D. J., Chen, D., White, M. F., and Backer, J. M. (1995)
Regulation of phosphatidylinositol 3'-kinase by tyrosyl phosphoproteins. Full activation requires
occupancy of both SH2 domains in the 85-kDa regulatory subunit. J. Biol. Chem. 270, 3662-3666
52.
Vanhaesebroeck, B., Guillermet-Guibert, J., Graupera, M., and Bilanges, B. (2010) The emerging
mechanisms of isoform-specific PI3K signalling. Nat. Rev. Mol. Cell Biol. 11, 329-341
53.
Welch, H., Eguinoa, A., Stephens, L. R., and Hawkins, P. T. (1998) Protein kinase B and rac are
activated in parallel within a phosphatidylinositide 3OH-kinase-controlled signaling pathway. J.
Biol. Chem. 273, 11248-11256
54.
Anderson, K. E., Coadwell, J., Stephens, L. R., and Hawkins, P. T. (1998) Translocation of PDK1 to the plasma membrane is important in allowing PDK-1 to activate protein kinase B. Curr.
Biol. 8, 684-691
55.
Meier, R., Alessi, D. R., Cron, P., Andjelkovic, M., and Hemmings, B. A. (1997) Mitogenic
activation, phosphorylation, and nuclear translocation of protein kinase Bbeta. J. Biol. Chem. 272,
30491-30497
56.
Stephens, L., Anderson, K., Stokoe, D., Erdjument-Bromage, H., Painter, G. F., Holmes, A. B.,
Gaffney, P. R., Reese, C. B., McCormick, F., Tempst, P., Coadwell, J., and Hawkins, P. T. (1998)
Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent
activation of protein kinase B. Science 279, 710-714
57.
Alessi, D. R., Andjelkovic, M., Caudwell, B., Cron, P., Morrice, N., Cohen, P., and Hemmings,
B. A. (1996) Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J. 15,
6541-6551
58.
Manning, B. D., and Cantley, L. C. (2007) AKT/PKB signaling: navigating downstream. Cell
129, 1261-1274
59.
Fingar, D. C., and Blenis, J. (2004) Target of rapamycin (TOR): an integrator of nutrient and
growth factor signals and coordinator of cell growth and cell cycle progression. Oncogene 23,
3151-3171
60.
Alam, H., Maizels, E. T., Park, Y., Ghaey, S., Feiger, Z. J., Chandel, N. S., and Hunzicker-Dunn,
M. (2004) Follicle-stimulating hormone activation of hypoxia-inducible factor-1 by the
phosphatidylinositol 3-kinase/AKT/Ras homolog enriched in brain (Rheb)/mammalian target of
rapamycin (mTOR) pathway is necessary for induction of select protein markers of follicular
differentiation. J. Biol. Chem. 279, 19431-19440
25 61.
Richards, J. S., Sharma, S. C., Falender, A. E., and Lo, Y. H. (2002) Expression of FKHR,
FKHRL1, and AFX genes in the rodent ovary: evidence for regulation by IGF-I, estrogen, and the
gonadotropins. Mol. Endocrinol. 16, 580-599
62.
Cunningham, M. A., Zhu, Q., Unterman, T. G., and Hammond, J. M. (2003) Follicle-stimulating
hormone promotes nuclear exclusion of the forkhead transcription factor FoxO1a via
phosphatidylinositol 3-kinase in porcine granulosa cells. Endocrinology 144, 5585-5594
63.
Burgering, B. M., and Kops, G. J. (2002) Cell cycle and death control: long live Forkheads.
Trends Biochem. Sci. 27, 352-360
64.
Schmoll, D., Walker, K. S., Alessi, D. R., Grempler, R., Burchell, A., Guo, S., Walther, R., and
Unterman, T. G. (2000) Regulation of glucose-6-phosphatase gene expression by protein kinase
Balpha and the forkhead transcription factor FKHR. Evidence for insulin response unit-dependent
and -independent effects of insulin on promoter activity. J. Biol. Chem. 275, 36324-36333
65.
Van Der Heide, L. P., Hoekman, M. F., and Smidt, M. P. (2004) The ins and outs of FoxO
shuttling: mechanisms of FoxO translocation and transcriptional regulation. Biochem. J. 380,
297-309
66.
Liu, Z., Rudd, M. D., Hernandez-Gonzalez, I., Gonzalez-Robayna, I., Fan, H. Y., Zeleznik, A. J.,
and Richards, J. S. (2009) FSH and FOXO1 regulate genes in the sterol/steroid and lipid
biosynthetic pathways in granulosa cells. Mol. Endocrinol. 23, 649-661
67.
Andric, N., and Ascoli, M. (2006) A delayed gonadotropin-dependent and growth factormediated activation of the extracellular signal-regulated kinase 1/2 cascade negatively regulates
aromatase expression in granulosa cells. Mol. Endocrinol. 20, 3308-3320
68.
Das, S., Maizels, E. T., DeManno, D., St Clair, E., Adam, S. A., and Hunzicker-Dunn, M. (1996)
A stimulatory role of cyclic adenosine 3',5'-monophosphate in follicle-stimulating hormoneactivated mitogen-activated protein kinase signaling pathway in rat ovarian granulosa cells.
Endocrinology 137, 967-974
69.
Donaubauer, E. M., and Hunzicker-Dunn, M. Extracellular signal-regulated kinase (ERK)–
dependent phosphorylation of Y-Box binding protein 1 (YB1) enhances gene expression in
granulosa cells in response to follicle stimulating hormone (FSH). J. Biol. Chem. in revision
70.
Roskoski, R., Jr. (2012) ERK1/2 MAP kinases: structure, function, and regulation. Pharmacol.
Res. 66, 105-143
26 71.
Cobb, M. H., and Goldsmith, E. J. (1995) How MAP kinases are regulated. J. Biol. Chem. 270,
14843-14846
72.
Okada, S., and Pessin, J. E. (1996) Interactions between Src homology (SH) 2/SH3 adapter
proteins and the guanylnucleotide exchange factor SOS are differentially regulated by insulin and
epidermal growth factor. J. Biol. Chem. 271, 25533-25538
73.
Mor, A., and Philips, M. R. (2006) Compartmentalized Ras/MAPK signaling. Annu. Rev.
Immunol. 24, 771-800
74.
Dance, M., Montagner, A., Salles, J. P., Yart, A., and Raynal, P. (2008) The molecular functions
of Shp2 in the Ras/Mitogen-activated protein kinase (ERK1/2) pathway. Cell. Signal. 20, 453-459
75.
De Rocca Serra-Nedelec, A., Edouard, T., Treguer, K., Tajan, M., Araki, T., Dance, M., Mus, M.,
Montagner, A., Tauber, M., Salles, J. P., Valet, P., Neel, B. G., Raynal, P., and Yart, A. (2012)
Noonan syndrome-causing SHP2 mutants inhibit insulin-like growth factor 1 release via growth
hormone-induced ERK hyperactivation, which contributes to short stature. Proceedings of the
National Academy of Sciences of the United States of America 109, 4257-4262
76.
Mashima, R., Hishida, Y., Tezuka, T., and Yamanashi, Y. (2009) The roles of Dok family
adapters in immunoreceptor signaling. Immunol. Rev. 232, 273-285
77.
Jelinek, T., Dent, P., Sturgill, T. W., and Weber, M. J. (1996) Ras-induced activation of Raf-1 is
dependent on tyrosine phosphorylation. Mol. Cell. Biol. 16, 1027-1034
78.
Gardner, A. M., Vaillancourt, R. R., Lange-Carter, C. A., and Johnson, G. L. (1994) MEK-1
phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: analysis of
phosphopeptides and regulation of activity. Molecular biology of the cell 5, 193-201
79.
Dalby, K. N., Morrice, N., Caudwell, F. B., Avruch, J., and Cohen, P. (1998) Identification of
regulatory phosphorylation sites in mitogen-activated protein kinase (MAPK)-activated protein
kinase-1a/p90rsk that are inducible by MAPK. J. Biol. Chem. 273, 1496-1505
80.
Weber, J. D., Raben, D. M., Phillips, P. J., and Baldassare, J. J. (1997) Sustained activation of
extracellular-signal-regulated kinase 1 (ERK1) is required for the continued expression of cyclin
D1 in G1 phase. Biochem. J. 326 ( Pt 1), 61-68
81.
Sherr, C. J., and Roberts, J. M. (1999) CDK inhibitors: positive and negative regulators of G1phase progression. Genes Dev. 13, 1501-1512
27 82.
Rivard, N., Boucher, M. J., Asselin, C., and L'Allemain, G. (1999) MAP kinase cascade is
required for p27 downregulation and S phase entry in fibroblasts and epithelial cells. The
American journal of physiology 277, C652-664
83.
Sherr, C. J. (1994) Growth factor-regulated G1 cyclins. Stem Cells 12 Suppl 1, 47-55; discussion
55-47
84.
Whitmarsh, A. J. (2007) Regulation of gene transcription by mitogen-activated protein kinase
signaling pathways. Biochim. Biophys. Acta 1773, 1285-1298
85.
Whitmarsh, A. J., and Davis, R. J. (1996) Transcription factor AP-1 regulation by mitogenactivated protein kinase signal transduction pathways. Journal of molecular medicine 74, 589-607
86.
Vinciguerra, M., Vivacqua, A., Fasanella, G., Gallo, A., Cuozzo, C., Morano, A., Maggiolini, M.,
and Musti, A. M. (2004) Differential phosphorylation of c-Jun and JunD in response to the
epidermal growth factor is determined by the structure of MAPK targeting sequences. J. Biol.
Chem. 279, 9634-9641
87.
Murphy, L. O., Smith, S., Chen, R. H., Fingar, D. C., and Blenis, J. (2002) Molecular
interpretation of ERK signal duration by immediate early gene products. Nat. Cell Biol. 4, 556564
88.
Liang, Q., Wiese, R. J., Bueno, O. F., Dai, Y. S., Markham, B. E., and Molkentin, J. D. (2001)
The transcription factor GATA4 is activated by extracellular signal-regulated kinase 1- and 2mediated phosphorylation of serine 105 in cardiomyocytes. Mol. Cell. Biol. 21, 7460-7469
89.
Buchwalter, G., Gross, C., and Wasylyk, B. (2004) Ets ternary complex transcription factors.
Gene 324, 1-14
90.
Murphy, L. O., MacKeigan, J. P., and Blenis, J. (2004) A network of immediate early gene
products propagates subtle differences in mitogen-activated protein kinase signal amplitude and
duration. Mol. Cell. Biol. 24, 144-153
91.
Hammer, G. D., Krylova, I., Zhang, Y., Darimont, B. D., Simpson, K., Weigel, N. L., and
Ingraham, H. A. (1999) Phosphorylation of the nuclear receptor SF-1 modulates cofactor
recruitment: integration of hormone signaling in reproduction and stress. Mol. Cell 3, 521-526
92.
Shen, T., Horwitz, K. B., and Lange, C. A. (2001) Transcriptional hyperactivity of human
progesterone receptors is coupled to their ligand-dependent down-regulation by mitogenactivated protein kinase-dependent phosphorylation of serine 294. Mol. Cell. Biol. 21, 6122-6131
28 93.
Hernandez-Hernandez, A., Ray, P., Litos, G., Ciro, M., Ottolenghi, S., Beug, H., and Boyes, J.
(2006) Acetylation and MAPK phosphorylation cooperate to regulate the degradation of active
GATA-1. EMBO J. 25, 3264-3274
94.
Roux, P. P., Ballif, B. A., Anjum, R., Gygi, S. P., and Blenis, J. (2004) Tumor-promoting phorbol
esters and activated Ras inactivate the tuberous sclerosis tumor suppressor complex via p90
ribosomal S6 kinase. Proceedings of the National Academy of Sciences of the United States of
America 101, 13489-13494
95.
Carriere, A., Romeo, Y., Acosta-Jaquez, H. A., Moreau, J., Bonneil, E., Thibault, P., Fingar, D.
C., and Roux, P. P. (2011) ERK1/2 phosphorylate Raptor to promote Ras-dependent activation of
mTOR complex 1 (mTORC1). J. Biol. Chem. 286, 567-577
96.
Carriere, A., Cargnello, M., Julien, L. A., Gao, H., Bonneil, E., Thibault, P., and Roux, P. P.
(2008) Oncogenic MAPK signaling stimulates mTORC1 activity by promoting RSK-mediated
raptor phosphorylation. Curr. Biol. 18, 1269-1277
97.
Roux, P. P., Shahbazian, D., Vu, H., Holz, M. K., Cohen, M. S., Taunton, J., Sonenberg, N., and
Blenis, J. (2007) RAS/ERK signaling promotes site-specific ribosomal protein S6
phosphorylation via RSK and stimulates cap-dependent translation. J. Biol. Chem. 282, 1405614064
98.
Chung, J., Kuo, C. J., Crabtree, G. R., and Blenis, J. (1992) Rapamycin-FKBP specifically blocks
growth-dependent activation of and signaling by the 70 kd S6 protein kinases. Cell 69, 1227-1236
99.
Angenstein, F., Greenough, W. T., and Weiler, I. J. (1998) Metabotropic glutamate receptorinitiated translocation of protein kinase p90rsk to polyribosomes: a possible factor regulating
synaptic protein synthesis. Proceedings of the National Academy of Sciences of the United States
of America 95, 15078-15083
100.
Shahbazian, D., Roux, P. P., Mieulet, V., Cohen, M. S., Raught, B., Taunton, J., Hershey, J. W.,
Blenis, J., Pende, M., and Sonenberg, N. (2006) The mTOR/PI3K and MAPK pathways converge
on eIF4B to control its phosphorylation and activity. EMBO J. 25, 2781-2791
101.
Wang, X., Li, W., Williams, M., Terada, N., Alessi, D. R., and Proud, C. G. (2001) Regulation of
elongation factor 2 kinase by p90(RSK1) and p70 S6 kinase. EMBO J. 20, 4370-4379
102.
Kayampilly, P. P., and Menon, K. M. (2004) Inhibition of extracellular signal-regulated protein
kinase-2 phosphorylation by dihydrotestosterone reduces follicle-stimulating hormone-mediated
cyclin D2 messenger ribonucleic acid expression in rat granulosa cells. Endocrinology 145, 17861793
29 103.
Fan, H. Y., Shimada, M., Liu, Z., Cahill, N., Noma, N., Wu, Y., Gossen, J., and Richards, J. S.
(2008) Selective expression of KrasG12D in granulosa cells of the mouse ovary causes defects in
follicle development and ovulation. Development 135, 2127-2137
104.
Pulido, R., Zuniga, A., and Ullrich, A. (1998) PTP-SL and STEP protein tyrosine phosphatases
regulate the activation of the extracellular signal-regulated kinases ERK1 and ERK2 by
association through a kinase interaction motif. EMBO J. 17, 7337-7350
105.
Patterson, K. I., Brummer, T., O'Brien, P. M., and Daly, R. J. (2009) Dual-specificity
phosphatases: critical regulators with diverse cellular targets. Biochem. J. 418, 475-489
106.
Theodosiou, A., and Ashworth, A. (2002) MAP kinase phosphatases. Genome biology 3,
REVIEWS3009
107.
Keyse, S. M., and Emslie, E. A. (1992) Oxidative stress and heat shock induce a human gene
encoding a protein-tyrosine phosphatase. Nature 359, 644-647
108.
Hirsch, D. D., and Stork, P. J. (1997) Mitogen-activated protein kinase phosphatases inactivate
stress-activated protein kinase pathways in vivo. J. Biol. Chem. 272, 4568-4575
109.
Franklin, C. C., and Kraft, A. S. (1997) Conditional expression of the mitogen-activated protein
kinase (MAPK) phosphatase MKP-1 preferentially inhibits p38 MAPK and stress-activated
protein kinase in U937 cells. J. Biol. Chem. 272, 16917-16923
110.
Niisato, N., Ohta, M., Eaton, D. C., and Marunaka, Y. (2012) Hypotonic stress upregulates betaand gamma-ENaC expression through suppression of ERK by inducing MKP-1. American
journal of physiology. Renal physiology 303, F240-252
111.
Medhora, M., Dhanasekaran, A., Pratt, P. F., Jr., Cook, C. R., Dunn, L. K., Gruenloh, S. K., and
Jacobs, E. R. (2008) Role of JNK in network formation of human lung microvascular endothelial
cells. American journal of physiology. Lung cellular and molecular physiology 294, L676-685
112.
Boutros, T., Chevet, E., and Metrakos, P. (2008) Mitogen-activated protein (MAP) kinase/MAP
kinase phosphatase regulation: roles in cell growth, death, and cancer. Pharmacol. Rev. 60, 261310
113.
Muda, M., Boschert, U., Dickinson, R., Martinou, J. C., Martinou, I., Camps, M., Schlegel, W.,
and Arkinstall, S. (1996) MKP-3, a novel cytosolic protein-tyrosine phosphatase that exemplifies
a new class of mitogen-activated protein kinase phosphatase. J. Biol. Chem. 271, 4319-4326
30 114.
Groom, L. A., Sneddon, A. A., Alessi, D. R., Dowd, S., and Keyse, S. M. (1996) Differential
regulation of the MAP, SAP and RK/p38 kinases by Pyst1, a novel cytosolic dual-specificity
phosphatase. EMBO J. 15, 3621-3632
115.
Marchetti, S., Gimond, C., Chambard, J. C., Touboul, T., Roux, D., Pouyssegur, J., and Pages, G.
(2005) Extracellular signal-regulated kinases phosphorylate mitogen-activated protein kinase
phosphatase 3/DUSP6 at serines 159 and 197, two sites critical for its proteasomal degradation.
Mol. Cell. Biol. 25, 854-864
116.
Nemoto, K., Vogt, A., Oguri, T., and Lazo, J. S. (2004) Activation of the Raf-1/MEK/Erk kinase
pathway by a novel Cdc25 inhibitor in human prostate cancer cells. The Prostate 58, 95-102
117.
Peyregne, V. P., Kar, S., Ham, S. W., Wang, M., Wang, Z., and Carr, B. I. (2005) Novel hydroxyl
naphthoquinones with potent Cdc25 antagonizing and growth inhibitory properties. Mol. Cancer
Ther. 4, 595-602
118.
Wang, Z., Zhang, B., Wang, M., and Carr, B. I. (2005) Cdc25A and ERK interaction: EGFRindependent ERK activation by a protein phosphatase Cdc25A inhibitor, compound 5. Journal of
cellular physiology 204, 437-444
119.
Morel, C., Gander, E. S., Herzberg, M., Dubochet, J., and Scherrer, K. (1973) The duck-globin
messenger-ribonucleoprotein complex. Resistance to high ionic strength, particle gel
electrophoresis, composition and visualisation by dark-field electron microscopy. Eur. J.
Biochem. 36, 455-464
120.
Didier, D. K., Schiffenbauer, J., Woulfe, S. L., Zacheis, M., and Schwartz, B. D. (1988)
Characterization of the cDNA encoding a protein binding to the major histocompatibility
complex class II Y box. Proceedings of the National Academy of Sciences of the United States of
America 85, 7322-7326
121.
Eliseeva, I. A., Kim, E. R., Guryanov, S. G., Ovchinnikov, L. P., and Lyabin, D. N. (2011) Ybox-binding protein 1 (YB-1) and its functions. Biochemistry. Biokhimiia 76, 1402-1433
122.
Skabkin, M. A., Liabin, D. N., and Ovchinnikov, L. P. (2006) [Nonspecific and specific
interaction of Y-box binding protein 1 (YB-1) with mRNA and posttranscriptional regulation of
protein synthesis in animal cells]. Molekuliarnaia biologiia 40, 620-633
123.
Evdokimova, V., Tognon, C. E., and Sorensen, P. H. (2012) On translational regulation and
EMT. Semin. Cancer Biol. 22, 437-445
31 124.
Kohno, K., Izumi, H., Uchiumi, T., Ashizuka, M., and Kuwano, M. (2003) The pleiotropic
functions of the Y-box-binding protein, YB-1. Bioessays 25, 691-698
125.
Matsumoto, K., and Wolffe, A. P. (1998) Gene regulation by Y-box proteins: coupling control of
transcription and translation. Trends Cell Biol. 8, 318-323
126.
Morel, C., Kayibanda, B., and Scherrer, K. (1971) Proteins associated with globin messenger
RNA in avian erythroblasts: Isolation and comparison with the proteins bound to nuclear
messenger-likie RNA. FEBS Lett. 18, 84-88
127.
Blobel, G. (1972) Protein tightly bound to globin mRNA. Biochem. Biophys. Res. Commun. 47,
88-95
128.
Evdokimova, V. M., Wei, C. L., Sitikov, A. S., Simonenko, P. N., Lazarev, O. A., Vasilenko, K.
S., Ustinov, V. A., Hershey, J. W., and Ovchinnikov, L. P. (1995) The major protein of
messenger ribonucleoprotein particles in somatic cells is a member of the Y-box binding
transcription factor family. J. Biol. Chem. 270, 3186-3192
129.
Minich, W. B., and Ovchinnikov, L. P. (1992) Role of cytoplasmic mRNP proteins in translation.
Biochimie 74, 477-483
130.
Nekrasov, M. P., Ivshina, M. P., Chernov, K. G., Kovrigina, E. A., Evdokimova, V. M., Thomas,
A. A., Hershey, J. W., and Ovchinnikov, L. P. (2003) The mRNA-binding protein YB-1 (p50)
prevents association of the eukaryotic initiation factor eIF4G with mRNA and inhibits protein
synthesis at the initiation stage. J. Biol. Chem. 278, 13936-13943
131.
Davydova, E. K., Evdokimova, V. M., Ovchinnikov, L. P., and Hershey, J. W. (1997)
Overexpression in COS cells of p50, the major core protein associated with mRNA, results in
translation inhibition. Nucleic Acids Res. 25, 2911-2916
132.
Evdokimova, V. M., Kovrigina, E. A., Nashchekin, D. V., Davydova, E. K., Hershey, J. W., and
Ovchinnikov, L. P. (1998) The major core protein of messenger ribonucleoprotein particles (p50)
promotes initiation of protein biosynthesis in vitro. J. Biol. Chem. 273, 3574-3581
133.
Pisarev, A. V., Skabkin, M. A., Thomas, A. A., Merrick, W. C., Ovchinnikov, L. P., and Shatsky,
I. N. (2002) Positive and negative effects of the major mammalian messenger ribonucleoprotein
p50 on binding of 40 S ribosomal subunits to the initiation codon of beta-globin mRNA. J. Biol.
Chem. 277, 15445-15451
32 134.
Hasegawa, S. L., Doetsch, P. W., Hamilton, K. K., Martin, A. M., Okenquist, S. A., Lenz, J., and
Boss, J. M. (1991) DNA binding properties of YB-1 and dbpA: binding to double-stranded,
single-stranded, and abasic site containing DNAs. Nucleic Acids Res. 19, 4915-4920
135.
Grant, C. E., and Deeley, R. G. (1993) Cloning and characterization of chicken YB-1: regulation
of expression in the liver. Mol. Cell. Biol. 13, 4186-4196
136.
Zasedateleva, O. A., Krylov, A. S., Prokopenko, D. V., Skabkin, M. A., Ovchinnikov, L. P.,
Kolchinsky, A., and Mirzabekov, A. D. (2002) Specificity of mammalian Y-box binding protein
p50 in interaction with ss and ds DNA analyzed with generic oligonucleotide microchip. J. Mol.
Biol. 324, 73-87
137.
Stratford, A. L., Habibi, G., Astanehe, A., Jiang, H., Hu, K., Park, E., Shadeo, A., Buys, T. P.,
Lam, W., Pugh, T., Marra, M., Nielsen, T. O., Klinge, U., Mertens, P. R., Aparicio, S., and Dunn,
S. E. (2007) Epidermal growth factor receptor (EGFR) is transcriptionally induced by the Y-box
binding protein-1 (YB-1) and can be inhibited with Iressa in basal-like breast cancer, providing a
potential target for therapy. Breast Cancer Res 9, R61
138.
Stein, U., Jurchott, K., Walther, W., Bergmann, S., Schlag, P. M., and Royer, H. D. (2001)
Hyperthermia-induced nuclear translocation of transcription factor YB-1 leads to enhanced
expression of multidrug resistance-related ABC transporters. J. Biol. Chem. 276, 28562-28569
139.
Fukada, T., and Tonks, N. K. (2003) Identification of YB-1 as a regulator of PTP1B expression:
implications for regulation of insulin and cytokine signaling. EMBO J. 22, 479-493
140.
Dooley, S., Said, H. M., Gressner, A. M., Floege, J., En-Nia, A., and Mertens, P. R. (2006) Y-box
protein-1 is the crucial mediator of antifibrotic interferon-gamma effects. J. Biol. Chem. 281,
1784-1795
141.
Lasham, A., Moloney, S., Hale, T., Homer, C., Zhang, Y. F., Murison, J. G., Braithwaite, A. W.,
and Watson, J. (2003) The Y-box-binding protein, YB1, is a potential negative regulator of the
p53 tumor suppressor. J. Biol. Chem. 278, 35516-35523
142.
Coles, L. S., Lambrusco, L., Burrows, J., Hunter, J., Diamond, P., Bert, A. G., Vadas, M. A., and
Goodall, G. J. (2005) Phosphorylation of cold shock domain/Y-box proteins by ERK2 and
GSK3beta and repression of the human VEGF promoter. FEBS Lett. 579, 5372-5378
143.
Samuel, S., Twizere, J. C., and Bernstein, L. R. (2005) YB-1 represses AP1-dependent gene
transactivation and interacts with an AP-1 DNA sequence. Biochem. J. 388, 921-928
33 144.
Saji, M., Shong, M., Napolitano, G., Palmer, L. A., Taniguchi, S. I., Ohmori, M., Ohta, M.,
Suzuki, K., Kirshner, S. L., Giuliani, C., Singer, D. S., and Kohn, L. D. (1997) Regulation of
major histocompatibility complex class I gene expression in thyroid cells. Role of the cAMP
response element-like sequence. J. Biol. Chem. 272, 20096-20107
145.
Shibao, K., Takano, H., Nakayama, Y., Okazaki, K., Nagata, N., Izumi, H., Uchiumi, T.,
Kuwano, M., Kohno, K., and Itoh, H. (1999) Enhanced coexpression of YB-1 and DNA
topoisomerase II alpha genes in human colorectal carcinomas. Int. J. Cancer 83, 732-737
146.
Wu, J., Lee, C., Yokom, D., Jiang, H., Cheang, M. C., Yorida, E., Turbin, D., Berquin, I. M.,
Mertens, P. R., Iftner, T., Gilks, C. B., and Dunn, S. E. (2006) Disruption of the Y-box binding
protein-1 results in suppression of the epidermal growth factor receptor and HER-2. Cancer Res.
66, 4872-4879
147.
Fujita, T., Ito, K., Izumi, H., Kimura, M., Sano, M., Nakagomi, H., Maeno, K., Hama, Y., Shingu,
K., Tsuchiya, S., Kohno, K., and Fujimori, M. (2005) Increased nuclear localization of
transcription factor Y-box binding protein 1 accompanied by up-regulation of P-glycoprotein in
breast cancer pretreated with paclitaxel. Clin. Cancer. Res. 11, 8837-8844
148.
Oda, Y., Sakamoto, A., Shinohara, N., Ohga, T., Uchiumi, T., Kohno, K., Tsuneyoshi, M.,
Kuwano, M., and Iwamoto, Y. (1998) Nuclear expression of YB-1 protein correlates with Pglycoprotein expression in human osteosarcoma. Clin. Cancer. Res. 4, 2273-2277
149.
Gessner, C., Woischwill, C., Schumacher, A., Liebers, U., Kuhn, H., Stiehl, P., Jurchott, K.,
Royer, H. D., Witt, C., and Wolff, G. (2004) Nuclear YB-1 expression as a negative prognostic
marker in nonsmall cell lung cancer. Eur. Respir. J. 23, 14-19
150.
Gimenez-Bonafe, P., Fedoruk, M. N., Whitmore, T. G., Akbari, M., Ralph, J. L., Ettinger, S.,
Gleave, M. E., and Nelson, C. C. (2004) YB-1 is upregulated during prostate cancer tumor
progression and increases P-glycoprotein activity. The Prostate 59, 337-349
151.
Gao, Y., Fotovati, A., Lee, C., Wang, M., Cote, G., Guns, E., Toyota, B., Faury, D., Jabado, N.,
and Dunn, S. E. (2009) Inhibition of Y-box binding protein-1 slows the growth of glioblastoma
multiforme and sensitizes to temozolomide independent O6-methylguanine-DNA
methyltransferase. Mol. Cancer Ther. 8, 3276-3284
152.
Astanehe, A., Finkbeiner, M. R., Hojabrpour, P., To, K., Fotovati, A., Shadeo, A., Stratford, A.
L., Lam, W. L., Berquin, I. M., Duronio, V., and Dunn, S. E. (2009) The transcriptional induction
of PIK3CA in tumor cells is dependent on the oncoprotein Y-box binding protein-1. Oncogene
28, 2406-2418
34 153.
Evdokimova, V., Tognon, C., Ng, T., and Sorensen, P. H. (2009) Reduced proliferation and
enhanced migration: two sides of the same coin? Molecular mechanisms of metastatic
progression by YB-1. Cell cycle 8, 2901-2906
154.
Evdokimova, V., Tognon, C., Ng, T., Ruzanov, P., Melnyk, N., Fink, D., Sorokin, A.,
Ovchinnikov, L. P., Davicioni, E., Triche, T. J., and Sorensen, P. H. (2009) Translational
activation of snail1 and other developmentally regulated transcription factors by YB-1 promotes
an epithelial-mesenchymal transition. Cancer Cell 15, 402-415
155.
Mani, S. A., Guo, W., Liao, M. J., Eaton, E. N., Ayyanan, A., Zhou, A. Y., Brooks, M., Reinhard,
F., Zhang, C. C., Shipitsin, M., Campbell, L. L., Polyak, K., Brisken, C., Yang, J., and Weinberg,
R. A. (2008) The epithelial-mesenchymal transition generates cells with properties of stem cells.
Cell 133, 704-715
156.
Mertens, P. R., Alfonso-Jaume, M. A., Steinmann, K., and Lovett, D. H. (1998) A synergistic
interaction of transcription factors AP2 and YB-1 regulates gelatinase A enhancer-dependent
transcription. J. Biol. Chem. 273, 32957-32965
157.
Kelm, R. J., Jr., Elder, P. K., and Getz, M. J. (1999) The single-stranded DNA-binding proteins,
Puralpha, Purbeta, and MSY1 specifically interact with an exon 3-derived mouse vascular smooth
muscle alpha-actin messenger RNA sequence. J. Biol. Chem. 274, 38268-38275
158.
Lasham, A., Lindridge, E., Rudert, F., Onrust, R., and Watson, J. (2000) Regulation of the human
fas promoter by YB-1, Puralpha and AP-1 transcription factors. Gene 252, 1-13
159.
Liu, X., Kelm, R. J., Jr., and Strauch, A. R. (2009) Transforming growth factor beta1-mediated
activation of the smooth muscle alpha-actin gene in human pulmonary myofibroblasts is inhibited
by tumor necrosis factor-alpha via mitogen-activated protein kinase kinase 1-dependent induction
of the Egr-1 transcriptional repressor. Molecular biology of the cell 20, 2174-2185
160.
Evdokimova, V., Ruzanov, P., Anglesio, M. S., Sorokin, A. V., Ovchinnikov, L. P., Buckley, J.,
Triche, T. J., Sonenberg, N., and Sorensen, P. H. (2006) Akt-mediated YB-1 phosphorylation
activates translation of silent mRNA species. Mol. Cell. Biol. 26, 277-292
161.
Sutherland, B. W., Kucab, J., Wu, J., Lee, C., Cheang, M. C., Yorida, E., Turbin, D., Dedhar, S.,
Nelson, C., Pollak, M., Leighton Grimes, H., Miller, K., Badve, S., Huntsman, D., Blake-Gilks,
C., Chen, M., Pallen, C. J., and Dunn, S. E. (2005) Akt phosphorylates the Y-box binding protein
1 at Ser102 located in the cold shock domain and affects the anchorage-independent growth of
breast cancer cells. Oncogene 24, 4281-4292
35 162.
Stratford, A. L., Fry, C. J., Desilets, C., Davies, A. H., Cho, Y. Y., Li, Y., Dong, Z., Berquin, I.
M., Roux, P. P., and Dunn, S. E. (2008) Y-box binding protein-1 serine 102 is a downstream
target of p90 ribosomal S6 kinase in basal-like breast cancer cells. Breast Cancer Res 10, R99
163.
Dephoure, N., Zhou, C., Villen, J., Beausoleil, S. A., Bakalarski, C. E., Elledge, S. J., and Gygi,
S. P. (2008) A quantitative atlas of mitotic phosphorylation. Proceedings of the National
Academy of Sciences of the United States of America 105, 10762-10767
164.
Jackson, R. J., Hellen, C. U., and Pestova, T. V. (2010) The mechanism of eukaryotic translation
initiation and principles of its regulation. Nat. Rev. Mol. Cell Biol. 11, 113-127
165.
Proudfoot, N. (2000) Connecting transcription to messenger RNA processing. Trends Biochem.
Sci. 25, 290-293
166.
Caceres, J. F., and Kornblihtt, A. R. (2002) Alternative splicing: multiple control mechanisms and
involvement in human disease. Trends Genet. 18, 186-193
167.
Kim, V. N., and Dreyfuss, G. (2001) Nuclear mRNA binding proteins couple pre-mRNA splicing
and post-splicing events. Molecules and cells 12, 1-10
168.
Gorlach, M., Burd, C. G., and Dreyfuss, G. (1994) The mRNA poly(A)-binding protein:
localization, abundance, and RNA-binding specificity. Exp. Cell Res. 211, 400-407
169.
Sonenberg, N., and Hinnebusch, A. G. (2009) Regulation of translation initiation in eukaryotes:
mechanisms and biological targets. Cell 136, 731-745
170.
Wells, S. E., Hillner, P. E., Vale, R. D., and Sachs, A. B. (1998) Circularization of mRNA by
eukaryotic translation initiation factors. Mol. Cell 2, 135-140
171.
Pestova, T. V., and Kolupaeva, V. G. (2002) The roles of individual eukaryotic translation
initiation factors in ribosomal scanning and initiation codon selection. Genes Dev. 16, 2906-2922
172.
Pestova, T. V., Lomakin, I. B., Lee, J. H., Choi, S. K., Dever, T. E., and Hellen, C. U. (2000) The
joining of ribosomal subunits in eukaryotes requires eIF5B. Nature 403, 332-335
173.
Rodnina, M. V., and Wintermeyer, W. (2001) Fidelity of aminoacyl-tRNA selection on the
ribosome: kinetic and structural mechanisms. Annu. Rev. Biochem. 70, 415-435
36 174.
Moore, P. B., and Steitz, T. A. (2003) After the ribosome structures: how does peptidyl
transferase work? RNA 9, 155-159
175.
Saini, P., Eyler, D. E., Green, R., and Dever, T. E. (2009) Hypusine-containing protein eIF5A
promotes translation elongation. Nature 459, 118-121
176.
Zhouravleva, G., Frolova, L., Le Goff, X., Le Guellec, R., Inge-Vechtomov, S., Kisselev, L., and
Philippe, M. (1995) Termination of translation in eukaryotes is governed by two interacting
polypeptide chain release factors, eRF1 and eRF3. EMBO J. 14, 4065-4072
177.
Song, H., Mugnier, P., Das, A. K., Webb, H. M., Evans, D. R., Tuite, M. F., Hemmings, B. A.,
and Barford, D. (2000) The crystal structure of human eukaryotic release factor eRF1-mechanism of stop codon recognition and peptidyl-tRNA hydrolysis. Cell 100, 311-321
178.
Kayampilly, P. P., and Menon, K. M. (2007) Follicle-stimulating hormone increases tuberin
phosphorylation and mammalian target of rapamycin signaling through an extracellular signalregulated kinase-dependent pathway in rat granulosa cells. Endocrinology 148, 3950-3957
37 CHAPTER TWO
FSH DEPENDENT REGULATION OF ERK PHOSPHORYLATION BY MKP3(DUSP6)
E. M. Donaubauer, N.C. Law, B. Kyriss, T. Woodiwiss and M. E. Hunzicker – Dunn1
To be submitted to the Journal of Biological Chemistry (JBC) upon completion of current studies
ABSTRACT
Within the ovarian follicle, granulosa cells (GCs) surround and support immature oocytes. FSH
promotes differentiation and proliferation of GCs and is essential for fertility; FSH receptor knockout
mice are infertile. This research focuses on the function and phospho-regulation by FSH of ERK within
GCs. Interestingly, in GCs, only ERK phosphorylation is increased in response to FSH. The upstream
components are constitutively active and their activity is unaffected following FSH treatment.
ERK(Thr202/Tyr204) phosphorylation is PKA dependent, however it requires activity of the upstream
pathway proteins. Treatment of GCs with EGFR inhibitor AG1478, a dominant-negative RAS, and the
MEK inhibitor PD98059 all blocked FSH-dependent ERK(Thr202/Tyr204) phosphorylation. We
hypothesize that FSH via PKA is regulating ERK phosphorylation by inhibiting the activity of a protein
phosphatase that is continually dephosphorylating ERK. Treatment with FSH would inactivate the
phosphatase, and allow for ERK to be phosphorylated, activated and to accumulate within GCs.
Treatment of GCs with different phosphatase inhibitors allowed for elimination of both Ser/Thr
phosphatases and protein tyrosine phosphatases (PTPs), and implicated dual specificity phosphatases
(DUSPs). Our results show that treatment with MKP3(DUSP6) inhibitors increase ERK(Thr202/Tyr204)
phosphorylation in the absence of FSH to levels comparable to ERK phosphorylated in the presence of
FSH. ERK coimmunoprecipitated with MKP3(DUSP6) and treatment of GCs with MKP3(DUSP6)
inhibitors block dephosphorylation of recombinant ERK2-GST. This research aims to better comprehend
38
the regulation of ERK signaling in GCs in response to FSH to gain further understanding of follicular
maturation.
INTRODUCTION
FSH acts selectively on granulosa cells (GCs) contained within ovarian follicles to promote
proliferation and differentiation of GCs to a preovulatory phenotype (reviewed in (1)) as well as meiotic
competency of the enclosed oocyte (2,3). Luteinizing hormone (LH) then initiates meiosis and promotes
ovulation and differentiation of remaining follicular cells into luteal cells. While it is well recognized that
the ERK signaling pathway plays a predominant role in the ovulatory response to LH in preovulatory
GCs (4), the functional significance of the ERK signaling pathway in immature GCs is less well
understood. We recently showed that the ERK signaling pathway in immature GCs is required for the
induction of at least a subset of FSH gene targets that define the mature preovulatory GC, including Inha
(that encodes the  subunit of the hormone inhibin), Egfr (that encodes the epidermal growth factor
receptor [EGFR] required for ovulation), and Cyp19a1 (that encodes the rate limiting enzyme in estrogen
biosynthesis) (5). ERK-dependent gene expression in immature GCs is mediated in part by
phosphorylation of the transcriptional activator Y-box binding protein-1 (YB-1) on Ser102 (5). Based on
the relevance of the ERK signaling pathway to immature GC maturation, we sought to better understand
the mechanism by which FSH activates ERK.
ERK is canonically activated by receptor tyrosine kinases (RTKs). Ligand-dependent activation
of RTKs recruit the RAS guanine exchange factor SOS to the plasma membrane, resulting in RAS and
subsequently RAF activation (reviewed in (6)). The Ser/Thr kinase RAF then phosphorylates/activates the
dual specificity kinase MEK that phosphorylates ERK. ERK then phosphorylates downstream kinases
like ribosomal S6 kinase (RSK-2) (7). Phosphorylation of ERK by MEK on both Thr202 and Tyr204 is
required for ERK activation; hence, inhibition of either phosphorylation by protein tyrosine phosphatases
39
(PTPs), Ser/Thr phosphatases, or dual specificity phosphatases (DUSPs) blocks ERK activation (reviewed
(8)).
In GCs, FSH activates ERK in a PKA-dependent manner (9-12). We previously reported that the
canonical pathway upstream of ERK is constitutively active in immature GCs but that ERK
phosphorylation/activity is restrained by a PTP (10). We hypothesized that FSH inactivated this
phosphatase, thereby permitting dual phosphorylation of ERK on Tyr202/Thr204 by MEK. We identified
the PTP as a 100 kDa putative member of the PTP-SL (STEP-like) family (13,14) based on the following
criteria: (a) a western blot of GC extracts using rabbit polyclonal anti-PTP-SL antibody (15) revealed a
signal at 100 kDa; (b) an in gel PTP assay detected a signal at 100 kDa in concentrated ovarian extracts;
(c) an anti- PTP-SL reactive signal at 100 kDa was selectively immunoprecipitated with ERK-conjugated
agarose from GC extracts; (d) in GCs loaded with 32Pi, FSH stimulated the phosphorylation of a protein at
100 kDa immunoprecipitated with anti-PTP-SL antibody that was inhibited by the ACG kinase inhibitor
H89 (16); (e) in an ERK-agarose immunoprecipitation, the anti-PTL-SL reactive band at 100 kDa was
reduced by ~ 50% in GCs treated with FSH. Previous results by the Pulido laboratory showed that PTPSL was phosphorylated on Ser231 in a PKA-dependent manner, relieving inhibition of ERK and its
restriction to the cytoplasm and allowing ERK to translocate to the nucleus (17,18).
However, our recent results question our interpretation of our previous results. (a) As we show
below, the pan-PTP inhibitor Na2VO3, which should inhibit PTP-SL (15), does not block FSH-stimulated
ERK phosphorylation. (b) PTP-SL has a MW of ~ 55 kDa, not 100 kDa (19). (c) A second antibody
directed against PTPBR7, which is identical to PTP-SL except for a 127 amino acid insertion in the Nterminus (14), does not detect a signal on western blots of GC extracts at 100 kDa (10). While the gene
that encodes PTP-SL Ptprr is expressed in GCs (based on RNA seq results; Herndon et al., manuscript in
preparation), we thus question whether the PTP activity at 100 kDa that is detected by an anti-PTL-SL
antibody is the phosphatase that constitutively dephosphorylates ERK in the absence of FSH.
40
Results below confirm that the ERK signaling pathway upstream of MEK is constitutively active
in GCs, that ERK but not MEK phosphorylation is PKA-dependent, and that ERK phosphorylation is
prevented in the absence of FSH. FSH-stimulated ERK activation is not inhibited either by inhibitors of
Ser/Thr protein phosphatase PP1 or PP2 or by the pan-PTP inhibitor Na2VO3. Rather, using a panel of
DUSP inhibitors, our results show that inhibitors of DUSP6/MAP kinase phosphatase 3 (MKP3)
selectively enhance phosphorylation of ERK in the absence of FSH equivalent to levels of ERK
phosphorylated in the presence of FSH. ERK co-immunoprecipitates with MKP3(DUSP6);
MKP3(DUSP6) inhibitors block MKP3(DUSP6) phosphatase activity in GC extracts in the absence of
FSH. Together, these results suggest that ERK in GCs is maintained in a dephosphorylated state in the
absence of FSH by MKP3(DUSP6).
EXPERIMENTAL PROCEDURES
Materials - The following were purchased: ovine FSH (oFSH-19) from the National Hormone
and Pituitary Agency of the National Institute of Diabetes and Digestive and Kidney Diseases (Torrence,
CA); human fibronectin from BD Biosciences; anti-phospho–AKT (Ser473; CST 9271), anti-AKT (CST
9272),anti-phospho–ERK (Thr202/Tyr204; CST 9107), anti-ERK (CST 9107), anti-phospho–MEK (Ser217/
Ser220; CST 9121), anti-S6 (CST 2217), anti-phospho-CREB (Ser133; CST 9191), anti-phospho-GAB
(Ser159; CST 3844) and (Tyr452; CST 3881), anti-phospho-GSK3β (Ser9; CST 9336), anti-phospho-myosin
light chain (MLC) (Ser19; CST 3671), anti-GAPDH (CST 5144) from Cell Signaling Technologies; antiSRC homology-2 (SH2) domain-containing tyrosine phosphatase (SHP2) (SC 7384), anti-MKP1 (SC
10769), anti-MKP3 (SC 28902), and anti-phospho-IRS1 (Tyr989; SC 17200) and Protein A/G PLUS
Agarose (SC 2003) from Santa Cruz Biotechnology; anti-RAS (cat. number R02120) from
Transduction Laboratories; tautomycin, okadaic acid, BCI, NSC 663284, active phospho-ERK2-GST (14173) from Calbiochem/EMD Millipore; NSC 87877 from ACROS Organics; Na2VO3 from Fukma.
41
Animals - Sprague-Dawley, CD-outbred rats (breeders from Charles River Laboratories) were
from a breeder colony maintained by our laboratory in a pathogen-free facility at Washington State
University. The facility is maintained in accordance with the Guidelines for the Care and Use of
Laboratory Animals using protocols approved by the Washington State University Animal Care and Use
Committee.
GC Cultures– Immature female rats were primed with subcutaneous injections of 1.5 mg
estradiol-17β (E2) in propylene glycol on days 21-23. Ovaries were collected following three days of
injections. GCs were collected by puncturing individual follicles using 27-gauge needles (20). Cells were
plated on fibronectin-coated plates at a density of ~1x106 cells/mL of serum–free media supplemented
with 1 nM E2, 100 U/mL penicillin (P), and 100 µg/mL streptomycin (S). Indicated treatments were
added to cells ~20 h following plating, and terminated by aspirating media and washing once with PBS
followed by sample collection.
Western Blotting - Total cell extracts were collected by scraping cells in a SDS sample buffer (21)
at 50 µL/1x106 cells, followed by heat denaturation. Equal protein loading was accomplished by plating
equal numbers of cells and collecting in a standardized SDS collection volume. Equal volumes of protein
extract were loaded per gel lane, and equal loading was verified by probing for total SHP2 or total AKT,
as indicated. Proteins were separated by SDS/PAGE and transferred onto either Hybond C-extra or
Protran (Amersham Biosciences) nitrocellulose membrane (22). The membrane was incubated with
primary antibody overnight at 4°C, and antigen–antibody complexes were detected using enhanced
chemiluminescence (ThermoFisher). Westerns were scanned using an Epson Perfection V500 scanner and
Adobe Photoshop CS2 9.0 software with minimal processing and quantified using Quantity One software
(BioRad Laboratories). Experimental densitometric values were divided by load control protein values
and expressed relative to vehicle values. Results were analyzed using GraphPad Prism and significance
was determined using a one way ANOVA with Tukey’s Multiple Comparison Test.
42
Adenoviral Transductions of GCs – Transduction with adenoviruses was done as previously
described (23). Briefly, GCs were plated in 35-mm plates at 1.5 x 106 cells/2 mL in DMEM/F12 +E/PS.
Four h after plating, the indicated adenoviruses were added to the cells. The next morning, the adenovirus
was removed, the cells were washed with PBS, and fresh DMEM/F12 + E/PS was added. The cells were
treated as indicated. Adenoviral optical particle unit (OPU) concentration per milliliter viral stock was
calculated from the A260 OD as described previously (24). Results are expressed as OPU per cell and
based on the number of GCs plated and the volume of virus added. Ad-PKI was kindly provided by
Marco Conti (University of California, San Francisco, CA) (25). Ad-(S17N)-RAS was kindly provided by
Valina Dawson (John Hopkins University School of Medicine, Baltimore, MD) (26).
Immunoprecipitation – Briefly, ~107 cells were scraped into 0.5 mL Immunoprecipitation Lysis
buffer [50 mM Hepes, pH 7.0, 100 mM NaCl, 0.5% Nonidet P-40, 20 mM NaF, 2 mM NaVO3, 2 mM
Na4P2O7, 5 mM EGTA, 5 mM EDTA, 20 mM benzamidine, 10 μg/mL calpain inhibitor III, 50 μg/mL
antipain, 50 μg/mL soybean trypsin inhibitor, 10 μM isomethylbutylxanthine, Halt protease inhibitor
mixture [1 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 0.8 μM aprotinin, 50 μM
bestatin, 15 μM E-64, 20 μM leupeptin, 10 μM pepstatin; (Thermo Fischer Scientific)], sonicated and
clarified by centrifugation at 10, 000 x g for 5 min at 4°C. Following removal of 0.03 mL sample for
input, soluble cell extracts were precleared using species matched preclearing matrix (ExactaCruz
Preclearing Matrix, Santa Cruz Biotechnology) for 60 min at 4°C. Samples were then incubated by
rotating at 4°C overnight with the specified antibodies and Protein A/G PLUS Agarose (Santa Cruz
Biotechnology; SC 2003) or antibody–agarose conjugate along with a species matched IgG antibody
control (Santa Cruz Biotechnology). Following centrifugation, 0.4 mL of the supernatant fraction,
representing the unbound protein fraction, was collected in SDS sample buffer. Agarose beads were
washed 4 times by rotating 1 mL Immunoprecipitation Wash Buffer (20 mM Hepes, pH 7.0, 150 mM
NaCl, 10% glycerol, and 0.1% Triton X-100) followed by centrifugation at 10,000 x g at 4°C. Bound
43
proteins were collected in SDS sample buffer and subjected to SDS/PAGE and western blotting. Inputs
represent ~2%, and bound sample ~98% of total sample.
Whole Cell Lysate Phosphatase Assay – Protocol was adapted from a previous report (27), as
detailed below. Cells were treated as indicated, then collected in 0.2 mL Phosphatase Assay Buffer [10
mM EDTA, 10 mM EGTA, 50 mM HEPES (pH 7.6), 1 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml
leupeptin, 1 mg/ml aprotinin]. Lysates were subjected to five rounds of freeze/thaw, followed aspiration
through a 25-guage needle. Lysates were clarified by centrifugation at 13, 000 x g for 30 min at 4°C. To
assess phosphatase activity, 50 ng of active ERK2-GST (EMD Millipore # 14-173) was added to each
sample, and samples were incubated by rotating for 30 min at 30°C. The reaction was halted by adding
100 µL of 3X STOP, and samples were subjected to SDS/PAGE and western blotting.
Statistics – Results were analyzed using GraphPad Prism and significance was determined using
either a one way ANOVA with Tukey’s Multiple Comparison Test or a one tailed Students t test.
RESULTS
Both canonical signaling to MEK and PKA activity are required for FSH stimulated
phosphorylation of ERK(Thr202/Tyr204). We initially sought to confirm that the canonical ERK signaling
pathway in GCs upstream of MEK is tonically active and independent of FSH. Our previous results
demonstrated an apparent requirement for the tyrosine kinase activity of the EGFR, based on results
showing that the selective EGFR inhibitor AG1478 (28) blocked FSH-stimulated ERK(Thr202/Tyr204)
phosphorylation. However, we did not evaluate the effect of AG1478 on MEK(Ser217/Ser219)
phosphorylation (10). To this end, GCs were pretreated with AG1478 followed by treatment without or
with FSH. Results (Fig. 1A) show (a) that MEK(Ser217/Ser219) phosphorylation is readily detected in
vehicle-treated GCs and is independent of FSH while ERK(Thr202/Tyr204) phosphorylation depends on
FSH, and (b) that AG1478 not only abolishes ERK(Thr202/Tyr204) phosphorylation but also abolishes
MEK(Ser217/Ser219) phosphorylation. AKT(Ser473) phosphorylation serves as a negative control. These
44
results indicated that the signaling pathway that promotes MEK phosphorylation is active in the absence
of FSH and that this pathway requires the tyrosine kinase activity of the ERGR.
While AG1478 primarily inhibits the kinase activity of the EGFR, it is also reported to inhibit
non-kinase targets (29). As the EGFR canonically activates RAS, we utilized two additional approaches
to confirm that the signaling pathway downstream of the EGFR was required for FSH to activate ERK
and was tonically active in the absence of FSH. We transduced GCs with a dominant negative adenoviral
(Ad)-(S17N)-RAS or control Ad-GFP, then treated GCs with vehicle or FSH. Results (Fig. 1B) show that
the dominant negative RAS blocked MEK (Ser217/Ser219) phosphorylation both in the absence and
presence of FSH as well as FSH-stimulated ERK(Thr202/Tyr204) phosphorylation.
CREB(Ser133)
phosphorylation serves as a negative control that is independent of EGFR/RAS signaling. The tyrosine
phosphatase SRC homology-2 (SH2) domain-containing tyrosine phosphatase (SHP2) is also required
for RTK signaling into MEK/ERK and its generally believed to contribute to the activation of RAS (3032). We determined whether SHP2 activity was required for the phosphorylation of MEK(Ser217/Ser219)
by pretreating GCs without or with NSC 87877, a compound that selectively inhibits SHP1 (which is not
expressed in rat GCs, based on RNA-seq results; Herndon et al., manuscript in preparation) and SHP2
(33). NSC 87877 markedly attenuated MEK(Ser217/Ser219) phosphorylation in vehicle- and FSH-treated
GCs and hence similarly reduced FSH-stimulated ERK(Thr202/Tyr204) phosphorylation (Fig. 1C). Taken
together, these results show that the signaling pathway that promotes MEK(Ser217/Ser219) phosphorylation
is tonically active in the absence of FSH and requires the EGFR, RAS and SHP2.
FSH stimulated ERK(Thr202/Tyr204) phosphorylation is also recognized to be dependent on PKA
(9,10). In the following experiment, we sought to confirm that while the PKA inhibitor PKI, that
functions as a PKA catalytic subunit pseudosubstrate (34), blocks FSH-stimulated ERK(Thr202/Tyr204)
phosphorylation, it does not affect MEK(Ser217/Ser219) phosphorylation. As shown in Fig. 2, while
transduction of GCs with Ad-PKI blocked FSH-stimulated ERK(Thr202/Tyr204) phosphorylation,
MEK(Ser217/Ser219) phosphorylation was unaffected. Taken together these results demonstrated that the
45
effect of PKA must be downstream of MEK at the level of ERK phosphorylation. As MEK should readily
phosphorylate ERK yet ERK is not phosphorylated in the absence of FSH, a phosphatase must actively
dephosphorylate ERK in the absence of FSH. It then follows that FSH via PKA must inactivate the ERK
phosphatase.
ERK phosphorylation is not directly regulated by a Ser/Thr phosphatase or PTP. In the following
experiments, we determined whether the phosphatase that dephosphorylates ERK on Thr202 and/or Tyr204
is a Ser/Thr protein phosphatase (PP1 or PP2) or a Tyr phosphatase.
Pretreatment of GCs with PP2
inhibitor okadaic acid (at 200 nM (35-38)) resulted in no change in phosphorylation of
ERK(Thr202/Tyr204) (Fig. 3A). Phosphorylation of GSK3β(Ser9) increased in the okadaic acid alone
treatment, and is used as a positive control. Pretreatment of GCs with the PP1 inhibitor tautomycin (at 1
µM (35-38)) promoted a decrease in both ERK(Thr202/Tyr204) and MEK(Ser217/Ser219) phosphorylations
(Fig. 3B). Blockade of the dephosphorylation of MLC(Ser19) serves as a positive control for tautomycin.
These results indicate that PP1 is necessary for signaling into MEK and acts upstream of MEK, not on
ERK. Pretreatment of GCs with the pan-PTP inhibitor Na2VO3 (39) resulted in an increase in both basal
and FSH-stimulated ERK(Thr202/Tyr204) phosphorylation (Fig. 3C). Concomitantly, MEK(Ser217/Ser219)
phosphorylation also increased with Na2VO3 treatment. The target(s) of the PTP is thus upstream of
MEK, likely at the level of the EGFR, and not at the level of ERK itself. Taken together, these results
suggest that the phosphatase that dephosphorylates ERK in the absence of FSH is neither a Ser/Thr
phospatase nor a PTP.
ERK phosphorylation is regulated by a DUSP. The inability of selective inhibitors of PP1, PP2,
or PTPs to preferentially enhance ERK phosphorylation in the absence of FSH suggested that the relevant
phosphatase must be a member of the family of DUSPs. Indeed, in a recent report, DUSP27 blocked
ERK, but not MEK phosphorylation in a rat luteal cell line in response to prolactin treatment (40).
However, DUSP27 is not expressed in rat GCs, based on both microarray (41) and RNA sequencing
(Herndon et al., manuscript in preparation) results. While 61 DUSPs have been identified (as reviewed in
46
(42)), preantral GCs only express DUSPs 1, 3, 6, 7, 10, 11, 12, 16, 18, 19, and 22, as well as cell division
cycle (CDC) dual specificity phosphatase CDC2A, 25B, and 25C, based on RNA sequencing results
(Herndon et al., manuscript in preparation). Only a subgroup of the DUSPs are classified as MKPs, based
on the presence of a CDC25-like domain (CD2) which confers specificity towards the ThrXTyr motif in
MAPKs (as reviewed in (42)). These include: MKP1(DUSP1), MKP3(DUPS6), MKPX/PYST2(DUSP7),
MKP5(DUSP10), MKP7(DUSP16) and CDC25A-C. Of those, MKP5 and MKP7 are selective for c-Jun
N-terminal kinase and p38 MAPK over ERK (as reviewed in (42)) and were not investigated. Based on
the availability of commercially available selective inhibitors2 and protein detection by Western blotting
in GCs (not shown), in the following experiments we tested the contributions of MKP1(DUSP1),
MKP3(DUSP6) and CDC24A-C to the level of ERK phosphorylation in GCs.
GCs were pretreated without or with NSC 663284, a selective CDC26A, 25B, and 25C inhibitor
that does inhibit MKP1(DUSP1) (43,44). Results (Fig. 4A) show that ERK(Thr202/Tyr204) phosphorylation
in the absence or presence of FSH is not affected (lanes 3 and 4 versus 1 and 2) by this competitive
CDC25 inhibitor. CDC25A-C are thus not functioning as ERK phosphatases in GCs.
GCs were next pretreated without or with BCI, a selective inhibitor of MKP1(DUSP1) and
MKP3(DUSP6) (45). Results show that BCI increased ERK(Thr202/Tyr204) phosphorylation in vehicletreated GCs (lane 3) to levels equivalent to those of FSH-treated cells (lane 2) (Fig. 4B).
MEK(Ser217/Ser219) phosphorylation in the presence of BCI (lanes 3 and 4) was equivalent to that of FSH
treated GCs in the absence of DMSO (lane 2). These results suggest that either MKP1(DUSP1) or
MKP3(DUPS6) may be the relevant ERK DUSP in GCs.
We next sought to verify expression of both MKP1(DUSP1) and MKP3(DUSP6) proteins within
GCs, especially since MKP1(DUSP1) is most commonly induced as an immediate early gene upon RTK
activation of ERK (as reviewed in (42). GCs were treated with vehicle or FSH for the indicated times.
Results show that both MKP1(DUSP1) and MKP3(DUSP6) are readily detected in GCs and total protein
levels do not change within 60 min of FSH treatment (Fig. 4C).
47
To distinguish between MKP1(DUSP1) and MKP3(DUSP6) as regulators of ERK(Thr202/Tyr204)
phosphorylation, GCs were pretreated with the selective MKP1(DUSP1) inhibitor triptolide (46,47).
Treatment with triptolide had no effect on ERK(Thr202/Tyr204) phosphorylation in the absence or presence
of FSH (Fig. 4D, compare lanes 3 and 4 with 1 and 2). Similarly, treatment of GCs with the
MKP1(DUSP1) selective inhibitor chelerythrine did not affect ERK(Thr202/Tyr204) phosphorylation (data
not shown). These results suggest that the relevant ERK DUSP in GCs is likely MKP3(DUSP6) and not
MKP1(DUSP1).
As we were unable to identify a selective MKP3(DUPS6) inhibitor, we pretreated GCs with NSC
295642, a partial MKP3(DUSP6), CDC 25A and 25B inhibitor (48). Results (Fig. 4E) show that NSC
295642 promoted an increase in ERK(Thr202/Tyr204) phosphorylation in vehicle-treated GCs (lane 3)
nearly equivalent to that of FSH-treated cells (lane 2); FSH treatment did not further enhance ERK
phosphorylation in the presence of NSC 295642 (lanes 3 and 4). Because the results from NSC 663284
treatments (see Fig. 4A) allowed us to rule out contributions from CDC25A-C, we can conclude from
these inhibitor studies that MKP3(DUSP6) is likely the DUSP that dephosphorylates ERK(Thr202/Tyr204)
in the absence of FSH. Collectively, these results indicate that MKP3(DUSP6) is dephosphorylating ERK
in untreated GCs, and that FSH promotes inactivation of MKP3(DUSP6) to increase ERK(Thr202/Tyr204)
phosphorylation.
ERK and MKP3(DUSP6) interact within GCs. If MKP3(DUSP6) is dephosphorylating ERK in
the absence of FSH, then MKP3(DUSP6) should bind to ERK in GCs. To determine if MKP3(DUPS6)
and ERK interact, ERK2 was immunoprecipitated from vehicle- and FSH treated GCs. Results (Fig. 5)
show the presence of MKP3(DUPS6) in ERK2 immunoprecipitated samples from both vehicle- and FSHtreated GCs. The identity of the doublet detected by the MKP3 antibody that is most prominent in the
ERK2 immunoprecipitation but is also faintly detectable on the long exposure in input lanes is not known
but might correspond to upshifted bands on MKP3. Unfortunately, the MKP3(DUSP6) antibody does not
48
readily immunoprecipitate MKP3(DUSP6) (not shown), so we could not perform the reverse
immunoprecipitation experiment.
ERK(Thr202/Tyr204) dephosphorylation is dependent on MKP3(DUSP6) activity. In the following
experiments, we sought to demonstrate that MKP3(DUSP6) but not MKP1(DUSP1) is able to
dephosphorylate ERK in vitro. Using the MKP1 and MKP3 inhibitors discussed above, whole GC lysate
phosphatase assays were performed using GST-tagged phospho-ERK2. GCs were pretreated without or
with indicated inhibitors, followed by treatment with vehicle or FSH. Lysates were then assayed for
phosphatase activity, evidenced by their ability to dephosphorylated GST-tagged phospho-ERK2, as
detected by western blotting with phospho- ERK(Thr202/Tyr204) antibody. Pretreatment of GCs with BCI,
the dual MKP1(DUSP1) and MKP3(DUSP6) inhibitor, blocked the DUSP that dephosphorylated
ERK(Thr202/Tyr204) in the absence of FSH (Fig. 6A, compare lanes 3 and 1). Equivalent results were
obtained upon pretreatment of GCs with NSC 295642, the MKP3(DUSP6) inhibitor (Fig. 6B). Taken
together, these results support the conclusion that MKP3(DUSP6) is the DUSP that dephosphorylates
ERK in the absence of FSH.
DISCUSSION
Our results confirm that canonical MEK/ERK signaling upstream of ERK is constitutively active,
and activity of these upstream components is necessary for FSH-dependent ERK(Thr202 and Tyr204)
phosphorylation. FSH-dependent ERK(Thr202 and Tyr204) phosphorylation is dependent on inhibition of a
DUSP, most likely MKP3(DUPS6), based the ability of MKP3(DUPS6) inhibitors to raise ERK(Thr202
and Tyr204) phosphorylation in the absence of FSH to levels comparable to FSH-stimulated ERK(Thr202
and Tyr204) phosphorylation. Additionally, ERK co-immunoprecipitates with MKP3(DUPS6), and
MKP3(DUPS6) inhibitors block dephosphorylation of ERK2-GST. These results indicated that in the
absence of FSH in GCs, ERK is dephosphorylated by MKP3(DUPS6). Our final studies, which are in
progress, are investigating the effect of adenoviral- MKP3(DUPS6) shRNA on the ability of FSH to
49
enhance ERK phosphorylation. The expectation is that successful down-regulation of MKP3(DUPS6)
will mimic the effect of the MKP3(DUPS6) inhibitors and raise ERK phosphorylation in vehicle-treated
cells to levels in cells treated with FSH.
FSH-dependent PKA signaling in GCs has been shown to activate multiple signaling cascades,
but until recently, the mechanisms by which PKA accomplished this regulation of canonical signaling
pathways remained elusive. Our results demonstrate that through regulation of MKP3(DUPS6), FSH is
able regulate ERK(Thr202 and Tyr204) phosphorylation, while the upstream components of the MEK/ERK
signaling cascade remain active, unregulated by FSH treatment (10). Interestingly, our lab recently
published results demonstrating FSH- and PKA-dependent regulation of PP1 to activate PI3K/AKT
signaling in GCs (49). Collectively, these results indicate that FSH via PKA regulates at least two
signaling cascades through regulation of phosphatases, seemingly hijacking these signaling cascades.
MKP3(DUPS6) is a dual specificity phosphatase, and member of the MKP subfamily, whose
substrates are the MAPKs (ERK, p38 MAPK and JNK) (reviewed in (50)). MKP3(DUPS6) has been
shown to have substrate preference for ERK over p38 MAPK and JNK (51). Regulation of
MKP3(DUPS6) is most often placed downstream of ERK signaling (52,53), and is thought to be a
negative feedback mechanism for down-regulating mitogenic signaling. Regulation has also been reported
through induction of Mkp3/Dusp6 mRNA or additional posttranslational modifications of the
MKP3(DUPS6) protein.
Mkp3/Dusp6 is often induced downstream of ERK signaling following ERK stimulation (54), and
requires regulation by the transcription factor Ets1 (53,54). We investigated total MKP3(DUPS6) protein
levels immediately following FSH treatment, and showed that MKP3(DUPS6) protein levels remain
relatively constant, giving no indication of a large change in total protein expression (Fig. 4C). These
results indicate that, in GCs at the time points checked, initial regulation of MKP3(DUPS6) is not at the
transcription level, as protein levels remain stable up to an hour following treatment with FSH.
50
Active ERK phosphorylates MKP3(DUPS6) on Ser159 and Ser197, and these phosphorylations are
reported to promote degradation of MKP3(DUPS6) by the proteosome (55). However, MKP3(DUPS6)
can interact with other proteins, such as casein kinase 2 α (CK2α) and Ca2+/calmodulin-dependent protein
kinase 1 (CAMK1) (56). Additionally, MKP3(DUPS6) can be phosphorylated by CK2α, and CK2α is
reported to inhibit MKP3(DUPS6) phosphatase activity (56). So although it is not common, regulation of
MKP3(DUPS6) activity by phosphorylation in an ERK-independent manner is not unprecedented. It is
also noteworthy that that rat MKP3(DUSP6) also contains one potential PKA phosphorylation motif
(RR/XX/S/T): RSVThr302V. Consistent with our results, we hypothesize that MKP3(DUSP6) is
phosphorylated by PKA on Thr302, resulting in an inhibition of its phosphatase activity.
In conclusion, we have shown that FSH-dependent ERK(Thr202 and Tyr204) phosphorylation is
PKA dependent, as well as being dependent on the constitutively active canonical upstream MEK/ERK
signaling components. In the absence of FSH, ERK is maintained in a dephosphorylated state by a DUSP,
most likely MKP3(DUPS6). ERK2 co-immunoprecipitates with MKP3(DUPS6), and MKP3(DUPS6)
inhibitors are able to raise ERK(Thr202 and Tyr204) phosphorylation in the absence of FSH. These results
indicate that MKP3(DUPS6) dephosphorylates ERK in untreated GCs, and FSH treatment inhibits its
activity.
51
ACKNOWLEDGMENTS: We thank Drs. John H. Nilson and Maria Herndon and Nate Law for many
helpful discussions. This work was supported by National Institutes of Health Grants R01HD065859 and
R01HD62503 (to MEH-D) and National Institute of General Medical Science Training Grant
T32GM083864 (to John H Nilson).
CONFLICT OF INTEREST: The authors declare that they have no conflicts of interest with the
contents of this article.
AUTHOR CONTRIBUTIONS: EMD and MEH-D designed the experiments. EMD, NL, BK, and TW
performed the experiments. EMD and MEH-D analyzed the data and wrote the manuscript.
FOOTNOTES
1
To whom correspondence should be addressed: Dr. Mary E. Hunzicker-Dunn, Biotechnology Life
Sciences Building, Room 202, Washington State University, Pullman WA 99164, Telephone: 509-3355614; Email: [email protected]
2
Commerically available inhibitors for the DUSPs were determined according to Jeffery, K.L., et. al. (57),
and at time of publication, there are no commerically available inhibitors for MKPX/PYST2(DUSP7).
52
REFERENCES
1.
Hunzicker-Dunn, M., and Mayo, K. E. (2015) Gonadotropin Signaling in the Ovary. in KNOBIL
AND NEILL’S PHYSIOLOGY OF REPRODUCTION (Plant, T. M., and Zeleznik, A. J. eds.),
Fourth Ed., Elsevier. pp 895 - 946
2.
Erickson, G. F., and Sorensen, R. A. (1974) In vitro maturation of mouse oocytes isolated from
late, middle, and pre-antral graafian follicles. The Journal of experimental zoology 190, 123-127
3.
Chesnel, F., and Eppig, J. J. (1995) Synthesis and accumulation of p34cdc2 and cyclin B in
mouse oocytes during acquisition of competence to resume meiosis. Mol. Reprod. Dev. 40, 503508
4.
Fan, H. Y., Liu, Z., Shimada, M., Sterneck, E., Johnson, P. F., Hedrick, S. M., and Richards, J. S.
(2009) MAPK3/1 (ERK1/2) in ovarian granulosa cells are essential for female fertility. Science
324, 938-941
5.
Donaubauer, E. M., and Hunzicker-Dunn, M. Extracellular signal-regulated kinase (ERK)–
dependent phosphorylation of Y-Box binding protein 1 (YB1) enhances gene expression in
granulosa cells in response to follicle stimulating hormone (FSH). J. Biol. Chem. in revision
6.
Roskoski, R., Jr. (2012) MEK1/2 dual-specificity protein kinases: structure and regulation.
Biochem. Biophys. Res. Commun. 417, 5-10
7.
Dalby, K. N., Morrice, N., Caudwell, F. B., Avruch, J., and Cohen, P. (1998) Identification of
regulatory phosphorylation sites in mitogen-activated protein kinase (MAPK)-activated protein
kinase-1a/p90rsk that are inducible by MAPK. J. Biol. Chem. 273, 1496-1505
8.
Roskoski, R., Jr. (2012) ERK1/2 MAP kinases: structure, function, and regulation. Pharmacol.
Res. 66, 105-143
9.
Andric, N., and Ascoli, M. (2006) A delayed gonadotropin-dependent and growth factormediated activation of the extracellular signal-regulated kinase 1/2 cascade negatively regulates
aromatase expression in granulosa cells. Mol. Endocrinol. 20, 3308-3320
10.
Cottom, J., Salvador, L. M., Maizels, E. T., Reierstad, S., Park, Y., Carr, D. W., Davare, M. A.,
Hell, J. W., Palmer, S. S., Dent, P., Kawakatsu, H., Ogata, M., and Hunzicker-Dunn, M. (2003)
Follicle-stimulating hormone activates extracellular signal-regulated kinase but not extracellular
signal-regulated kinase kinase through a 100-kDa phosphotyrosine phosphatase. J. Biol. Chem.
278, 7167-7179
53
11.
Das, S., Maizels, E. T., DeManno, D., St Clair, E., Adam, S. A., and Hunzicker-Dunn, M. (1996)
A stimulatory role of cyclic adenosine 3',5'-monophosphate in follicle-stimulating hormoneactivated mitogen-activated protein kinase signaling pathway in rat ovarian granulosa cells.
Endocrinology 137, 967-974
12.
Puri, P., Little-Ihreg, L., Chandran, U., Law, N. C., Hunzicker-Dunn, M., and Zeleznik, A. J.
Protein Kinase A: The Master Kinase of Granulosa Cell Differentiation. Scientific Reports in
revision
13.
Ogata, M., Oh-hora, M., Kosugi, A., and Hamaoka, T. (1999) Inactivation of mitogen-activated
protein kinases by a mammalian tyrosine-specific phosphatase, PTPBR7. Biochem. Biophys. Res.
Commun. 256, 52-56
14.
Van Den Maagdenberg, A. M., Bachner, D., Schepens, J. T., Peters, W., Fransen, J. A., Wieringa,
B., and Hendriks, W. J. (1999) The mouse Ptprr gene encodes two protein tyrosine phosphatases,
PTP-SL and PTPBR7, that display distinct patterns of expression during neural development.
Eur. J. Neurosci. 11, 3832-3844
15.
Pulido, R., Zuniga, A., and Ullrich, A. (1998) PTP-SL and STEP protein tyrosine phosphatases
regulate the activation of the extracellular signal-regulated kinases ERK1 and ERK2 by
association through a kinase interaction motif. EMBO J. 17, 7337-7350
16.
Hidaka, H., Watanabe, M., and Kobayashi, R. (1991) Properties and use of H-series compounds
as protein kinase inhibitors. Methods Enzymol. 201, 328-339
17.
Blanco-Aparicio, C., Torres, J., and Pulido, R. (1999) A novel regulatory mechanism of MAP
kinases activation and nuclear translocation mediated by PKA and the PTP-SL tyrosine
phosphatase. J. Cell Biol. 147, 1129-1136
18.
Zuniga, A., Torres, J., Ubeda, J., and Pulido, R. (1999) Interaction of mitogen-activated protein
kinases with the kinase interaction motif of the tyrosine phosphatase PTP-SL provides substrate
specificity and retains ERK2 in the cytoplasm. J. Biol. Chem. 274, 21900-21907
19.
Noordman, Y. E., Augustus, E. D., Schepens, J. T., Chirivi, R. G., Rios, P., Pulido, R., and
Hendriks, W. J. (2008) Multimerisation of receptor-type protein tyrosine phosphatases PTPBR7
and PTP-SL attenuates enzymatic activity. Biochim. Biophys. Acta 1783, 275-286
20.
Carr, D. W., DeManno, D. A., Atwood, A., Hunzicker-Dunn, M., and Scott, J. D. (1993) Folliclestimulating hormone regulation of A-kinase anchoring proteins in granulosa cells. J. Biol. Chem.
268, 20729-20732
54
21.
Hunzicker-Dunn, M. (1981) Selective activation of rabbit ovarian protein kinase isozymes in
rabbit ovarian follicles and corpora lutea. J. Biol. Chem. 256, 12185-12193
22.
Salvador, L. M., Park, Y., Cottom, J., Maizels, E. T., Jones, J. C., Schillace, R. V., Carr, D. W.,
Cheung, P., Allis, C. D., Jameson, J. L., and Hunzicker-Dunn, M. (2001) Follicle-stimulating
hormone stimulates protein kinase A-mediated histone H3 phosphorylation and acetylation
leading to select gene activation in ovarian granulosa cells. J. Biol. Chem. 276, 40146-40155
23.
Park, Y., Maizels, E. T., Feiger, Z. J., Alam, H., Peters, C. A., Woodruff, T. K., Unterman, T. G.,
Lee, E. J., Jameson, J. L., and Hunzicker-Dunn, M. (2005) Induction of cyclin D2 in rat granulosa
cells requires FSH-dependent relief from FOXO1 repression coupled with positive signals from
Smad. J. Biol. Chem. 280, 9135-9148
24.
Hunzicker-Dunn, M. E., Lopez-Biladeau, B., Law, N. C., Fiedler, S. E., Carr, D. W., and Maizels,
E. T. (2012) PKA and GAB2 play central roles in the FSH signaling pathway to PI3K and AKT
in ovarian granulosa cells. Proceedings of the National Academy of Sciences of the United States
of America 109, E2979-2988
25.
Bruss, M. D., Richter, W., Horner, K., Jin, S. L., and Conti, M. (2008) Critical role of PDE4D in
beta2-adrenoceptor-dependent cAMP signaling in mouse embryonic fibroblasts. J. Biol. Chem.
283, 22430-22442
26.
Gonzalez-Zulueta, M., Feldman, A. B., Klesse, L. J., Kalb, R. G., Dillman, J. F., Parada, L. F.,
Dawson, T. M., and Dawson, V. L. (2000) Requirement for nitric oxide activation of
p21(ras)/extracellular regulated kinase in neuronal ischemic preconditioning. Proceedings of the
National Academy of Sciences of the United States of America 97, 436-441
27.
Woods, D. C., and Johnson, A. L. (2006) Phosphatase activation by epidermal growth factor
family ligands regulates extracellular regulated kinase signaling in undifferentiated hen granulosa
cells. Endocrinology 147, 4931-4940
28.
Levitzki, A., and Gazit, A. (1995) Tyrosine kinase inhibition: an approach to drug development.
Science 267, 1782-1788
29.
Choi, B. H., Choi, J. S., Rhie, D. J., Yoon, S. H., Min, D. S., Jo, Y. H., Kim, M. S., and Hahn, S.
J. (2002) Direct inhibition of the cloned Kv1.5 channel by AG-1478, a tyrosine kinase inhibitor.
Am. J. Physiol. Cell Physiol. 282, C1461-1468
30.
Dance, M., Montagner, A., Salles, J. P., Yart, A., and Raynal, P. (2008) The molecular functions
of Shp2 in the Ras/Mitogen-activated protein kinase (ERK1/2) pathway. Cell. Signal. 20, 453-459
55
31.
De Rocca Serra-Nedelec, A., Edouard, T., Treguer, K., Tajan, M., Araki, T., Dance, M., Mus, M.,
Montagner, A., Tauber, M., Salles, J. P., Valet, P., Neel, B. G., Raynal, P., and Yart, A. (2012)
Noonan syndrome-causing SHP2 mutants inhibit insulin-like growth factor 1 release via growth
hormone-induced ERK hyperactivation, which contributes to short stature. Proceedings of the
National Academy of Sciences of the United States of America 109, 4257-4262
32.
Mashima, R., Hishida, Y., Tezuka, T., and Yamanashi, Y. (2009) The roles of Dok family
adapters in immunoreceptor signaling. Immunol. Rev. 232, 273-285
33.
Chen, L., Sung, S. S., Yip, M. L., Lawrence, H. R., Ren, Y., Guida, W. C., Sebti, S. M.,
Lawrence, N. J., and Wu, J. (2006) Discovery of a novel shp2 protein tyrosine phosphatase
inhibitor. Mol. Pharmacol. 70, 562-570
34.
Kemp, B. E., Cheng, H. C., and Walsh, D. A. (1988) Peptide inhibitors of cAMP-dependent
protein kinase. Methods Enzymol. 159, 173-183
35.
Favre, B., Turowski, P., and Hemmings, B. A. (1997) Differential inhibition and posttranslational
modification of protein phosphatase 1 and 2A in MCF7 cells treated with calyculin-A, okadaic
acid, and tautomycin. J. Biol. Chem. 272, 13856-13863
36.
Resjo, S., Oknianska, A., Zolnierowicz, S., Manganiello, V., and Degerman, E. (1999)
Phosphorylation and activation of phosphodiesterase type 3B (PDE3B) in adipocytes in response
to serine/threonine phosphatase inhibitors: deactivation of PDE3B in vitro by protein phosphatase
type 2A. Biochem. J. 341 ( Pt 3), 839-845
37.
Yan, Y., and Mumby, M. C. (1999) Distinct roles for PP1 and PP2A in phosphorylation of the
retinoblastoma protein. PP2a regulates the activities of G(1) cyclin-dependent kinases. J. Biol.
Chem. 274, 31917-31924
38.
Chatfield, K., and Eastman, A. (2004) Inhibitors of protein phosphatases 1 and 2A differentially
prevent intrinsic and extrinsic apoptosis pathways. Biochem. Biophys. Res. Commun. 323, 13131320
39.
Gordon, J. A. (1991) Use of vanadate as protein-phosphotyrosine phosphatase inhibitor. Methods
Enzymol. 201, 477-482
40.
Devi, Y. S., Seibold, A. M., Shehu, A., Maizels, E., Halperin, J., Le, J., Binart, N., Bao, L., and
Gibori, G. (2011) Inhibition of MAPK by prolactin signaling through the short form of its
receptor in the ovary and decidua: involvement of a novel phosphatase. J. Biol. Chem. 286, 76097618
56
41.
Escamilla-Hernandez, R., Little-Ihrig, L., Orwig, K. E., Yue, J., Chandran, U., and Zeleznik, A. J.
(2008) Constitutively active protein kinase A qualitatively mimics the effects of folliclestimulating hormone on granulosa cell differentiation. Mol. Endocrinol. 22, 1842-1852
42.
Patterson, K. I., Brummer, T., O'Brien, P. M., and Daly, R. J. (2009) Dual-specificity
phosphatases: critical regulators with diverse cellular targets. Biochem. J. 418, 475-489
43.
Vogt, A., McDonald, P. R., Tamewitz, A., Sikorski, R. P., Wipf, P., Skoko, J. J., 3rd, and Lazo, J.
S. (2008) A cell-active inhibitor of mitogen-activated protein kinase phosphatases restores
paclitaxel-induced apoptosis in dexamethasone-protected cancer cells. Mol. Cancer Ther. 7, 330340
44.
Lazo, J. S., Aslan, D. C., Southwick, E. C., Cooley, K. A., Ducruet, A. P., Joo, B., Vogt, A., and
Wipf, P. (2001) Discovery and biological evaluation of a new family of potent inhibitors of the
dual specificity protein phosphatase Cdc25. J. Med. Chem. 44, 4042-4049
45.
Molina, G., Vogt, A., Bakan, A., Dai, W., Queiroz de Oliveira, P., Znosko, W., Smithgall, T. E.,
Bahar, I., Lazo, J. S., Day, B. W., and Tsang, M. (2009) Zebrafish chemical screening reveals an
inhibitor of Dusp6 that expands cardiac cell lineages. Nat. Chem. Biol. 5, 680-687
46.
Zhao, Q., Shepherd, E. G., Manson, M. E., Nelin, L. D., Sorokin, A., and Liu, Y. (2005) The role
of mitogen-activated protein kinase phosphatase-1 in the response of alveolar macrophages to
lipopolysaccharide: attenuation of proinflammatory cytokine biosynthesis via feedback control of
p38. J. Biol. Chem. 280, 8101-8108
47.
Srivastava, N., Sudan, R., and Saha, B. (2011) CD40-modulated dual-specificity phosphatases
MAPK phosphatase (MKP)-1 and MKP-3 reciprocally regulate Leishmania major infection. J.
Immunol. 186, 5863-5872
48.
Vogt, A., Cooley, K. A., Brisson, M., Tarpley, M. G., Wipf, P., and Lazo, J. S. (2003) Cell-active
dual specificity phosphatase inhibitors identified by high-content screening. Chem. Biol. 10, 733742
49.
Law, N. C., and Hunzicker-Dunn, M. E. (2015) Insulin Receptor Substrate 1: The Hub Linking
Follicle-stimulating Hormone to Phosphatidylinositol-3 Kinase Activation. J. Biol. Chem.
50.
Keyse, S. M. (2000) Protein phosphatases and the regulation of mitogen-activated protein kinase
signalling. Curr. Opin. Cell Biol. 12, 186-192
57
51.
Muda, M., Boschert, U., Dickinson, R., Martinou, J. C., Martinou, I., Camps, M., Schlegel, W.,
and Arkinstall, S. (1996) MKP-3, a novel cytosolic protein-tyrosine phosphatase that exemplifies
a new class of mitogen-activated protein kinase phosphatase. J. Biol. Chem. 271, 4319-4326
52.
Eblaghie, M. C., Lunn, J. S., Dickinson, R. J., Munsterberg, A. E., Sanz-Ezquerro, J. J., Farrell, E.
R., Mathers, J., Keyse, S. M., Storey, K., and Tickle, C. (2003) Negative feedback regulation of
FGF signaling levels by Pyst1/MKP3 in chick embryos. Curr. Biol. 13, 1009-1018
53.
Nunes-Xavier, C. E., Tarrega, C., Cejudo-Marin, R., Frijhoff, J., Sandin, A., Ostman, A., and
Pulido, R. (2010) Differential up-regulation of MAP kinase phosphatases MKP3/DUSP6 and
DUSP5 by Ets2 and c-Jun converge in the control of the growth arrest versus proliferation
response of MCF-7 breast cancer cells to phorbol ester. J. Biol. Chem. 285, 26417-26430
54.
Ekerot, M., Stavridis, M. P., Delavaine, L., Mitchell, M. P., Staples, C., Owens, D. M., Keenan, I.
D., Dickinson, R. J., Storey, K. G., and Keyse, S. M. (2008) Negative-feedback regulation of FGF
signalling by DUSP6/MKP-3 is driven by ERK1/2 and mediated by Ets factor binding to a
conserved site within the DUSP6/MKP-3 gene promoter. Biochem. J. 412, 287-298
55.
Marchetti, S., Gimond, C., Chambard, J. C., Touboul, T., Roux, D., Pouyssegur, J., and Pages, G.
(2005) Extracellular signal-regulated kinases phosphorylate mitogen-activated protein kinase
phosphatase 3/DUSP6 at serines 159 and 197, two sites critical for its proteasomal degradation.
Mol. Cell. Biol. 25, 854-864
56.
Castelli, M., Camps, M., Gillieron, C., Leroy, D., Arkinstall, S., Rommel, C., and Nichols, A.
(2004) MAP kinase phosphatase 3 (MKP3) interacts with and is phosphorylated by protein kinase
CK2alpha. J. Biol. Chem. 279, 44731-44739
57.
Jeffrey, K. L., Camps, M., Rommel, C., and Mackay, C. R. (2007) Targeting dual-specificity
phosphatases: manipulating MAP kinase signalling and immune responses. Nat. Rev. Drug
Discov. 6, 391-403
58
FIGURE LEGENDS AND FIGURES
FIGURE 1. Canonical MAPK signaling pathway is active in GCs independent of FSH stimulation and is
necessary for phosphorylation of ERK1/2(Thr202/Tyr204). (A) GCs were treated for 15 min with 250 nM
AG1478, a potent EGFR inhibitor, followed by treatment without (veh) or with 50 ng/mL FSH for 15
min. Samples were heat denatured after collection in SDS sample buffer and proteins were separated by
SDS/PAGE. A blot of whole cell extracts was probed with indicated antibodies. Results are representative
of six independent experiments. (B) GCs were transduced with Ad-GFP or Ad-(S17N)RAS overnight,
followed by treatment without (veh) or with FSH 15 min. Samples were collected as described in A.
Results are representative of four independent experiments. (C) GCs were treated without (DMSO) or
with 20 mM NSC 87877, a SHP2 inhibitor, for 3 h, followed by treatment without (veh) or with FSH for
15 min. Samples were collected as described in A. Results are representative of five independent
experiments. 59
60
FIGURE 2. Phosphorylation of ERK1/2 on Thr202/Tyr204 is dependent on PKA activity. GCs were
transduced overnight with Ad-GFP or Ad-PKI, an inhibitor of PKA. Cells were then treated without (veh)
or with FSH for 30 min. Samples were collected as described in 1A. Results are representative of three
independent experiments. 61
62
FIGURE 3. Effect of Ser/Thr and Tyr phosphatase inhibitors on ERK1/2(Thr202/Tyr204) phosphorylation.
(A) GCs were pretreated without (EtOH) or with 0.2 µM okadaic acid, a PP2 inhibitor, for 1 h, followed
by treatment without (veh) and with FSH for 15 min. Samples were collected as described in 1A. Results
are representative of three independent experiments. (B) GCs were pretreated without (EtOH) or with 1
µM tautomycin, a PP1 inhibitor, for 5.5 h, followed by treatment without (veh) and with FSH for 15 min.
Samples were collected as described in 1A. Results are representative of six independent experiments. (C)
GCs were pretreated without (H2O) or with 50 µM Na2VO3, a tyrosine phosphatase inhibitor, for 12 h,
followed by treatment without (veh) and with FSH for 30 min. Samples were collected as described in 1A.
Results are representative of three independent experiments. 63
64
FIGURE 4. Effect of DUSP inhibitors on ERK1/2(Thr202/Tyr204) phosphorylation. (A) GCs were
pretreated without (DMSO) or with 5 µM NSC 663284, a selective CDC25A, 25B and 25C inhibitor (that
does not inhibit MKP1(DUSP1)), for 30 min, followed by treatment without (veh) and with FSH for 30
min. Samples were collected as described in 1A. Results are representative of three independent
experiments. (B) GCs were pretreated without (DMSO) or with 5 µM BCI, a selective MKP1(DUSP1)
and MKP3(DUSP6) inhibitor, for 30 min, followed by treatment without (veh) and with FSH for 30 min.
Samples were collected as described in 1A. Results are representative of three independent experiments.
(C) GCs were treated without (veh) and with FSH for the indicated times. Samples were collected as
described in 1A. (D) GCs were pretreated without (DMSO) or with 10 µM triptolide, a selective
MKP1(DUSP1) inhibitor, for 1 h, followed by treatment without (veh) and with FSH for 30 min. Samples
were collected as described in 1A. Results are representative of three independent experiments. (E) GCs
were pretreated without (DMSO) or with 5 µM NSC 295642, a selective MKP3(DUSP6) and CDC25A
and 25B inhibitor, for 30 min, followed by treatment without (veh) and with FSH for 30 min. Samples
were collected as described in 1A. Results are representative of three independent experiments. 65
66
FIGURE 5. MKP3(DUSP6) interacts with ERK1/2 in GCs. GCs were treated without (veh) or with FSH
for 15 min, and then collected in Immunoprecipitation Lysis buffer and sonicated. After removal of the
insoluble particles, the soluble fraction was rotated overnight with 20 µg Anti–ERK2 (SC 1647) as
described in Experimental Procedures. Inputs, bound (IP) and unbound (flow – though) samples were
separated by SDS/PAGE and the western blot was probed with the indicated antibodies. Arrows indicate
the protein band of interest. Results are representative of two independent experiments.
67
68
FIGURE 6. Active ERK2-GST dephosphorylation is blocked by MKP3(DUSP6) inhibitors. (A) GCs
were pretreated without (DMSO) or with 5 µM BCI for 60 min, followed by treatment without (veh) and
with FSH for 30 min. Samples were collected in Phosphatase Assay Buffer as described in Experimental
Procedures. Active ERK2-GST was added to samples and incubated for 60 min. Samples were separated
by SDS/PAGE and the western blot was probed with the indicated antibodies. Results are representative
of two independent experiments. (B) GCs were pretreated without (DMSO) or with 5 µM NSC 295642
for 30 min, followed by treatment without (veh) and with FSH for 30 min. Samples were collected as
described in 6A. Results are representative of two independent experiments.
69
70
CHAPTER THREE
ERK–DEPENDENT PHOSPHORYLATION OF Y-BOX BINDING PROTEIN-1 (YB-1)
ENHANCES GENE EXPRESSION IN GRANULOSA CELLS IN RESPONSE TO FSH
E. M. Donaubauer and M. E. Hunzicker – Dunn1
Running Title: Function of phosphorylated YB-1 in ovarian granulosa cells
Submitted to the Journal of Biological Chemistry (JBC) November 2015, currently in revision
ABSTRACT
Within the ovarian follicle, immature oocytes are surrounded and supported by granulosa cells
(GCs). Stimulation of GCs by FSH leads to their proliferation and differentiation, events that are
necessary for fertility. FSH activates multiple signaling pathways to regulate genes necessary for
follicular maturation. Herein, we investigated the role of Y-box binding protein-1 (YB-1) within GCs.
YB-1 is a nucleic acid binding protein that regulates both transcription and translation. Our results show
that FSH promotes an increase in the phosphorylation of YB-1 on Ser102 within 15 min that is maintained
at significantly increased levels until ~8 h post treatment. FSH-stimulated phosphorylation of YB1(Ser102) is prevented by pretreatment of GCs with the PKA-selective inhibitor PKI, the MEK inhibitor
PD98059, or the ribosomal S6 kinase-2 (RSK-2) inhibitor BI-D1870. Phosphorylation of YB-1 on Ser102
is thus PKA, ERK, and RSK-2 dependent. However, pretreatment of GCs with the protein phosphatase 1
(PP1) inhibitor tautomycin increased phosphorylation of YB-1(Ser102) in the absence of FSH; FSH did not
further increase YB-1(Ser102) phosphorylation. This result suggests that the major effect of RSK-2 is to
inhibit PP1 rather than to directly phosphorylate YB-1 on Ser102. YB-1 coimmunoprecipitated with PP1β
catalytic subunit and RSK-2. Transduction of GCs with the dephospho-adenoviral-YB-1(S102A) mutant
prevented the induction by FSH of Egfr, Cyp19a1, and Inha mRNAs. Collectively, our results reveal that
71
phosphorylation of YB-1 on Ser102 via the ERK/RSK-2 signaling pathway is necessary for FSH-mediated
expression of target genes required for maturation of follicles to a preovulatory phenotype.
INTRODUCTION
In the female, fertility requires maturation of the ovarian follicle that contains the oocyte
surrounded by granulosa cells (GCs)2 and theca cells. Follicular maturation is a tightly regulated process
that is initiated by FSH. FSH regulates at least 4000 target genes within GCs whose expression drives
development of the follicle, allowing it to respond to the surge of luteinizing hormone (LH) that promotes
ovulation, oocyte maturation, and formation of the corpus luteum that serves to support the developing
embryo after fertilization and implantation (reviewed in (1,2)).
The mechanism by which FSH signals to regulate gene and protein expression in GCs has been
extensively investigated. FSH binds to its G protein-coupled receptor (GPCR) expressed exclusively on
GCs to activate adenylyl cyclase, raise intracellular cAMP levels, and activate PKA (3-6). PKA then
either directly phosphorylates proteins that regulate transcription or indirectly activates signaling cascades
whose targets regulate primarily transcription and translation. Direct PKA targets in GCs include cAMPresponse binding element protein (CREB) (Ser133) (7), histone H3 (Ser10) (8), and β-catenin (Ser552 and
Ser675) (9). Upon phosphorylation, these direct PKA target proteins participate in the regulation of gene
expression though interactions with DNA and additional transcription factors.
One of the signaling cascades activated by FSH is the MAPK/ERK pathway (8,10), evidenced by
the phosphorylation of ERK (Thr202/Tyr204) within 10 min of FSH treatment (11), and phosphorylation of
its downstream target RSK-2 (Ser380) (8). FSH also activates the PI3K/protein kinase B (AKT) pathway
(12,13). In a PKA–dependent manner, FSH enhances phosphorylation of the pivotal kinase AKT on both
the proximal Thr308 and the distal Ser473 sites within 5 min following FSH treatment (12,14), followed by
the rapid phosphorylation of downstream AKT targets, tuberin and forkhead box-containing protein O1
(FOXO1) on Thr1462 and Ser256, respectively (12,15,16).
72
However, the mechanisms by which these signaling pathways regulate gene expression are less
well understood. One of the more important pathways activated by FSH is the PI3K/AKT pathway, based
on evidence that this pathway is necessary for GC differentiation (9,15,17,18). Yet the only
transcriptional factor regulated by this pathway that has been shown to modulate gene expression in GCs
is FOXO1 (15,18). Our recent results, however, suggest the PI3K/AKT pathway is likely regulating
additional transcriptional modulators, based on the ability of exogenous insulin-like growth factor 1
(IGF1) at concentrations greater than 1 ng/mL to synergize with FSH to enhance gene expression without
further increasing the phosphorylation of FOXO1 (Ser256) (19). Strong evidence in cancer cells identified
YB-1 as an AKT target (20-23) that, upon phosphorylation on Ser102, acts as a transcription factor to
promote expression of genes such as Egfr (21,24-26), a recognized FSH gene target (27).
YB-1 was first described as a major component of the inactive mRNA-protein complex (mRNP)
(28,29). Further studies demonstrated that YB-1 interacts with both RNA and DNA specifically though its
cold shock domain (30,31), and nonspecifically via basic and acidic clusters in its C-terminus (reviewed
in (32)). YB-1 functions in the cytosol to regulate mRNA translation and to stabilize mRNA; in the
nucleus, YB-1 can regulate DNA repair, pre-mRNA splicing, and transcription (reviewed in (32)).
Transcriptional targets for YB-1, identified mostly using cancer cell lines, in addition to Egfr, include
multidrug resistance gene 1 (Mdr1) (33), protein tyrosine phosphatase 1B (PTP1B) (34), cyclin A (Ccna)
(35), and vascular endothelial growth factor (Vegf) genes (36).
Herein, we show that FSH promotes the protracted phosphorylation of YB-1 on Ser102. This
phosphorylation is mimicked by forskolin and inhibited by expression of the selective PKA inhibitor PKI,
indicating its dependence on PKA activity. Phosphorylation of YB-1 on Ser102 is unaffected by the PI3K
inhibitor wortmannin, but is blocked by both the MEK/ERK inhibitor PD98059 and the RSK-2 inhibitor
BI-1870, showing dependence on ERK and downstream RSK-2 signaling. Our results surprisingly
suggest that the major RSK-2 target is not YB-1, rather RSK-2 promotes inactivation of PP1 to enhance
YB-1(Ser102) phosphorylation.
FSH leads to an increase of phospho-YB-1 in the nucleus. Using
73
adenoviral YB-1 phosphorylation mutants, we show that phosphorylation of YB-1 on Ser102 is necessary
for the expression of Cyp19a1, Inha, and Egfr. Together these results suggest that the PKA-dependent
activation of ERK/RSK-2 pathways in GCs in response to FSH, by promoting phosphorylation of YB-1
on Ser102, contributes to the expression of at least three target genes, Cyp19a1, Inha, and Egfr, that are
necessary for follicular maturation. These results also suggest a previously underappreciated role for the
ERK signaling pathway in immature GCs.
EXPERIMENTAL PROCEDURES
Materials - The following were purchased: ovine FSH (oFSH-19) from the National Hormone
and Pituitary Agency of the National Institute of Diabetes and Digestive and Kidney Diseases (Torrence,
CA); recombinant human IGF1 from Atlanta Biologicals; purified mouse EGF, anti–Flag (F3165) from
Sigma–Aldrich; human fibronectin from BD Biosciences; antiphospho-YB-1 (Ser102; CST 2900), antiYB-1 (CST 4202), anti-phospho–AKT (Ser473; CST 9271), anti-phospho–ERK (Thr202/Tyr204; CST 9107),
anti-phospho–RSK-2 (Ser380; CST 9335), and anti–GST (CST 2622) from Cell Signaling Technologies;
anti-SRC homology-2 (SH2) domain-containing tyrosine phosphatase (SHP2) (SC 7384) and Protein A/G
PLUS Agarose (SC 2003) from Santa Cruz Biotechnology; wortmannin, PD98059, tautomycin, okadaic
acid, forskolin, BI-D1870, 8-chlorophenylthiol (CPT)-cAMP and myristoylated (Myr)PKI from
Calbiochem/EMD. 8-CPT-2’-O-methyl (Me)-cAMP was purchased from Life Sciences Institute; TRIzolRNA Lysis Reagent and Fast SYBR Green Master Mix from Life Technologies; qScript from VWR.
Animals - Sprague-Dawley, CD-outbred rats (breeders from Charles River Laboratories) were
from a breeder colony maintained by our laboratory in a pathogen-free facility at Washington State
University. The facility is maintained in accordance with the Guidelines for the Care and Use of
Laboratory Animals using protocols approved by the Washington State University Animal Care and Use
Committee.
74
GC culture and Western Blotting- Immature female rats were primed with subcutaneous
injections of 1.5 mg estradiol-17β (E2) in propylene glycol on days 21-23 to promote growth of preantral
follicles. Ovaries were collected following three days of injections. GCs were collected by puncturing
individual follicles using 27-gauge needles (37). Cells were plated on fibronectin-coated plates at a
density of ~1x106 cells/mL of serum–free media supplemented with 1 nM E2, 100 U/mL penicillin (P),
and 100 µg/mL streptomycin (S) (38). Indicated treatments were added to cells ~20 h following plating,
and terminated by aspirating media and washing once with PBS. For western blotting, total cell extracts
were collected by scraping cells in a SDS sample buffer (5) at 50 µL/1x106 cells, followed by heat
denaturation. Equal protein loading was accomplished by plating equal numbers of cells and collecting in
a standardized SDS collection volume (5). Equal volumes of protein extract were loaded per gel lane, and
equal loading was verified by probing for total SHP2 or total AKT, as indicated. Proteins were separated
by SDS/PAGE and transferred onto either Hybond C-extra or Protran (Amersham Biosciences)
nitrocellulose membrane (8). The membrane was incubated with primary antibody overnight at 4°C, and
antigen–antibody complexes were detected using enhanced chemiluminescence (ThermoFisher).
Westerns were scanned using an Epson Perfection V500 scanner and Adobe Photoshop CS2 9.0 software
with minimal processing and quantified using Quantity One software (BioRad Laboratories).
Experimental densitometric values were divided by load control protein values and expressed relative to
vehicle values. Results were analyzed using GraphPad Prism and significance was determined using
either a one way ANOVA with Tukey’s Multiple Comparison Test or a one tailed Students t test.
Immunoprecipitation- Immunoprecipitations were performed using a modified protocol
previously described (14). Briefly, ~107 cells were scraped into 0.5 mL Immunoprecipitation Lysis buffer
[50 mM Hepes, pH 7.0, 100 mM NaCl, 0.5% Nonidet P-40, 20 mM NaF, 2 mM NaVO3, 2 mM Na4P2O7,
5 mM EGTA, 5 mM EDTA, 20 mM benzamidine, 10 μg/mL calpain inhibitor III, 50 μg/mL antipain, 50
μg/mL soybean trypsin inhibitor, 10 μM isomethylbutylxanthine, Halt protease inhibitor mixture [1 mM
4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride, 0.8 μM aprotinin, 50 μM bestatin, 15 μM E-64,
75
20 μM leupeptin, 10 μM pepstatin; (Thermo Fischer Scientific)], sonicated twice on ice for 45 s, and
clarified by centrifugation at 10, 000 x g for 5 min at 4°C. Following removal of 0.03 mL sample for
input, soluble cell extracts were precleared using species matched preclearing matrix (ExactaCruz
Preclearing Matrix, Santa Cruz Biotechnology) for 60 min at 4°C. Samples were then incubated by
rotating at 4°C overnight with the specified antibodies and Protein A/G PLUS Agarose (Santa Cruz
Biotechnology; SC 2003) or antibody–agarose conjugate along with a species matched IgG antibody
control (Santa Cruz Biotechnology). Following centrifugation at 10,000 x g for 5 min at 4°C, 0.4 mL of
the supernatant fraction, representing the unbound protein fraction, was collected in SDS sample buffer.
Agarose beads were washed by rotating 5 min at 4°C with 1 mL Immunoprecipitation Wash Buffer (20
mM Hepes, pH 7.0, 150 mM NaCl, 10% glycerol, and 0.1% Triton X-100) 4 times. Bound proteins were
collected in SDS sample buffer and subjected to SDS/PAGE and western blotting. Inputs represent ~2%,
and bound sample ~98% of total sample.
Transfection of GCs and Luciferase Assay- Cells were plated on fibronectin-coated plates at a
density of 1x106 cells/mL in OptiMEM + P/S + E2 with expression constructs (500 ng/ 1x106 cells) and
transfected using LipofectAMINE 2000 (Invitrogen) according to manufacturer’s instructions. After 6 h
incubation, the media was removed and fresh DMEM +P/S +E2 was added. Cells were treated as
indicated following ~16 h recovery period. Cells were lysed in 0.1 mL Luciferase Assay Buffer (25 mM
Hepes pH 7.8, 15 mM MgSO4, 4 mM EGTA, 1 mM DTT, 1% Triton X -100). Samples were analyzed for
luciferase activity using a Burthold Technologies Lumat LB 9507 luminometer using the Promega
Luciferase Assay System according to manufacturer’s instructions.
DNA constructs and adenoviral production- The full length human Egfr-promoter luciferase was
kindly provided by Dr. Wenlong Bai (University of South Florida, Tampa, FL) (39). Cyp19a1-promoter
luciferase and Inha–promoter luciferase constructs were previously described (40,41). Full length YB-1Flag was purchased from OriGene (Rockville, MD). YB-1–Flag mutants were obtained using
QuikChange Lightning Site-Directed Mutagenesis Kit from Agilent Technologies (Santa Clara, CA)
76
according to manufacturer’s instructions. The following primers were used: YB-1(S102D) forward 5'
GTC TCT CCA TCT CCT ACA TCG CGA AGG TAC TTC CTG GG 3' reverse 5' CCC AGG AAG
TAC CTT CGC GAT GTA GGA GAT GGA GAG AC 3'; YB-1(S102A) forward 5'CCA GGA AGT
ACC TTC GCG CTG TAG GAG ATG GAG AGA 3' reverse 5' TCT CTC CAT CTC CTA CAG CGC
GAA GGT ACT TCC TGG 3'. YB-1-Flag, YB-1 (S102D)–Flag and YB-1 (S102A)–Flag were cloned into
pShuttleX and then cloned into Adeno –X by Vector Development Laboratory (Baylor College of
Medicine, Houston TX). Adenoviral (Ad)-YB-1(S102D)- Flag was used at (1.4 x 108 pfu/mL) and AdYB-1(S102A) – Flag was used at (1 x 109 pfu/mL).
Cellular Fractionation - Fractionation was performed using a modified protocol previously
described (42). Briefly, cells were treated as indicated, washed once with cold PBS, and collected in 0.5
mL Cytoplasmic Lysis buffer (10 mM HEPES pH 7.4, 10 mM KCl, 0.01mM EDTA, 0.1 mM EGTA, 2
mM DTT, 5 mM Na2VO4, 10 mM NaF, 20 mM sodium β- glycerophosphate, 0.1% NP40, Halt protease
inhibitor mixture) and incubated on ice for 20 min. Nuclei were pelleted though centrifugation at 5,000 x
g 4°C for 10 min, and the supernatant containing the cytoplasmic fraction was removed. The cytoplasmic
fraction was collected in SDS sample buffer and heat denatured. The nuclear pellet was washed with 1.0
mL Cytoplasmic Lysis buffer and repelleted. The supernatant was removed and the nuclear pellet was
resuspended in 0.1 mL SDS sample buffer and heat denatured. Samples were subjected to SDS/PAGE
and western blotting with the indicated antibodies. Cytoplasmic fraction represents 9% of total sample
and nuclear fraction represents 50% of total sample.
Adenoviral transduction of GCs- Transduction of GCs was performed as previously described
(9). Briefly, GCs were plated on fibronectin-coated 6 well plates at a density of 1.5x106 cells/well in
DMEM/F12 +P/S +E2. After allowing the cells to adhere to the plate for ~4 h, the indicated adenoviruses
were added to the media. Following an overnight treatment, cells were allowed ~1 h recovery and then
treated as indicated.
77
RNA Isolation and Real–time PCR- RNA isolation was performed per manufactures instructions.
Briefly, cells were treated as indicated, rinsed once with PBS and collected in 1.0 mL of TRIzol-RNA
Lysis Reagent. After 10 min incubation at room temperature, 0.2 mL chloroform was added and the
samples were gently mixed and incubated for 10 min at room temperature. The phases were resolved by
centrifugation for 15 min at 12, 000 x g at 4°C. The upper aqueous phase was removed, 0.5 mL
isopropanol was added to the lower phase and the sample was gently mixed. The sample was incubated
on ice for >30 min, then the RNA was pelleted upon centrifugation for 15 min at 12, 000 x g at 4°C. The
pellet was washed with 75% EtOH, and then allowed to air dry. RNA concentration was determined using
a NanoDrop 2000. cDNA was generated using qScript per manufacturer’s instructions. Real time-PCR
was performed using Fast SYBR Green Master Mix per manufacturer’s instructions. Primers used were:
Egfr forward 5’ AGA TTG VCAA AGG GCA TGA ACT AC 3’ reverse 5’ ACA TTC CTG GCT GCC
AAG TC 3’; Cyp19a1 forward 5’ CAC GGA TGT TTG ATG GTC TGA 3’ reverse 5’ CTC GGC TTG
CTG ACA AAC C 3’; Inha forward 5’ TGG GAC CGC TGG ATC GTA 3’ reverse 5’ GCA TCC CGC
AGC TAC CAT 3’; Star forward 5’ AAG GAC TGC CCA CCA CAT CTA C 3’ reverse 5’ TCT CGT
TGT CCT TGG CTG GAA 3’; Egr1 forward 5’ CCT TTT GCC TGT GAC ATT TGT G 3’ reverse 5’
TGG TAT GCC TCT TGC GTT CA 3’; Ereg forward 5’ GCT CTG ACA TGG ACG GCT ACT 3’
reverse 5’ CTC GCT CAT GTC CAC CAG GTA 3’; ribosome L19 forward 5’ GTG ACC TGG ATG
CGA AGG A 3’ reverse 5’ GCC TTG TCT GCC TTC AGT TTG 3’.
Statistics – Results were analyzed using GraphPad Prism and significance was determined using
either a one way ANOVA with Tukey’s Multiple Comparison Test or a one tailed Students t test.
RESULTS
FSH signals to stimulate phosphorylation of YB-1(Ser102) through a PKA-dependent pathwayWe initially determined whether YB-1 was expressed in GCs and whether its expression at the protein
level was regulated by FSH. Results (Fig.1A and 1C) show that YB-1 protein, which migrates on SDS-
78
PAGE at 50 kDa, is readily detected in GCs and its expression is not acutely regulated by FSH. We then
determined whether YB-1 was phosphorylated on Ser102 in response to FSH. FSH promoted a relatively
slow increase in YB-1(Ser102) phosphorylation first detected 15 min after treatment (Fig. 1B). YB1(Ser102) phosphorylation peaked 1 h following FSH treatment; this peak level of phosphorylation was
maintained until about 8 h and then declined to undetectable levels by 24 h (Fig. 1C). (The identities of
the faster and slower migrating bands in phospho-YB-1 blots are not known.) Increased phosphorylation
of YB-1(Ser102) was mimicked by the adenylyl cyclase activator forskolin (Fig. 2A). Phosphorylation of
AKT(Ser473) serves as a positive control. YB-1(Ser102) phosphorylation also increased in response to 8CPT–cAMP (Fig. 2B), a cell permeable cAMP analog that activates both exchange proteins activated by
cAMP and PKA (14,43). But 8-CPT-2’-O-Me-cAMP, a cAMP analog that does not activate PKA, (44),
failed to increase YB-1(Ser102) phosphorylation (Fig. 2B). Treatment of GCs with Ad–PKA inhibitor
(PKI), a pseudosubstrate for the catalytic subunit of PKA, led to a loss of FSH-dependent YB-1(Ser102)
phosphorylation [82% ± 17%] (Fig. 2C). Similarly, treatment with Myr–PKI peptide resulted in a loss of
YB-1 phosphorylation mediated by FSH (data not shown). Phosphorylation of AKT(Ser473) has been
shown to be PKA dependent in GCs (14) and serves as a positive control. Taken together, these data
indicate that FSH stimulation results in phosphorylation of YB-1 on Ser102 in a PKA-dependent manner in
GCs.
Phosphorylation of YB-1(Ser102) is dependent on the ERK pathway and not PI3K/AKT pathwayThe phosphorylation of YB-1(Ser102), investigated in various cancer cell lines, is most commonly
mediated by AKT (20-23), although there is evidence that this site can be phosphorylated by RSK-2
downstream of ERK (26,45). Because FSH in a PKA-dependent manner activates both the PI3K/AKT
(14) and ERK signaling pathways (8,10,11,46) in GCs, we investigated both pathways as possible
regulators of YB-1 phosphorylation. Pretreatment of GCs with the PI3K inhibitor wortmannin (47), did
not decrease FSH stimulated YB-1(Ser102) phosphorylation, while AKT(Ser473) phosphorylation was
inhibited significantly [74.2% ± 13.0%] (Fig. 3A). However, pretreatment with the MEK inhibitor
79
PD98059 (47) promoted a near complete inhibition of YB-1(Ser102) phosphorylation [94.2% ± 3.6%] in
the presence of FSH (Fig. 3B), similar to the inhibition of ERK(Thr202/Tyr204) phosphorylation [98.1%
±5.9%] that is used as the positive control. Equivalent results were obtained with a different MEK
inhibitor U0126 (data not shown). Consistent with these results, treatment of GCs with EGF, a known
activator of the MEK/ERK pathway, promoted increased levels of YB-1(Ser102) phosphorylation as well
as RSK-2(Ser380) phosphorylation (Fig. 3C). When GCs were treated with the PI3K/AKT pathway
activator IGF1 or FSH, only cells treated with FSH showed enhanced YB-1(Ser102) phosphorylation (Fig.
3C). Unlike many other cell models, IGF1 does not activate the ERK pathway in GCs (48). Additionally,
GCs treated with the RSK-2 inhibitor BI-D1870 exhibited a marked inhibition [89.4% ± 5.3%] of YB1(Ser102) phosphorylation in the presence of FSH (Fig. 4). CREB(Ser133) phosphorylation serves as a
negative control. Taken together, these results indicate that FSH-stimulated phosphorylation of YB1(Ser102) is mediated by RSK-2 downstream of ERK.
Phosphorylation of YB-1(Ser102) is dependent on protein phosphatase 1 (PP1) inhibitionEnhanced phosphorylation of YB-1(Ser102) is relatively slow, compared to the phosphorylation of CREB
which is detected within 1 min (7) or of ERK which is detected within 10 min (10) of FSH treatment.
Moreover, YB-1(Ser102) phosphorylation is detected up to 8-12 h (Fig. 1B) while phosphorylations of
both ERK (11) and RSK-2 (8) are undetectable by 2 h post treatment with FSH. We therefore sought to
determine if the inhibition of a Ser/Thr phosphatase contributed to the phosphorylation of YB-1(Ser102).
Pretreatment of GCs with the protein phosphatase 2 (PP2) inhibitor okadaic acid (at 200 nM)
resulted in an increase in YB-1(Ser102) phosphorylation in both vehicle- and FSH-treated GCs (Fig. 5A).
Phosphorylation of RSK-2(Ser380 ) is indicative of RSK-2 activity (49) and provides insight into the
regulation of YB-1(Ser102) phosphorylation in the presence of okadaic acid. RSK-2(Ser380)
phosphorylation was enhanced both in the absence and presence of FSH in response to okadaic acid and
appears to account for the increase in YB-1(Ser102) phosphorylation. These results suggest that okadaic
acid is inhibiting a phosphatase that acts upstream of YB-1 rather than at the level of YB-1.
80
Pretreatment of GCs with the PP1 inhibitor tautomycin (at 1 µM) resulted in an increase in YB1(Ser102) phosphorylation in vehicle-treated cells to levels equivalent to those seen in FSH-treated cells in
the absence of tautomycin, while FSH did not further enhance YB-1(Ser102) phosphorylation (Fig. 5B).
Tautomycin pretreatment did not enhance FSH-stimulated RSK-2(Ser380) phosphorylation. These results
suggest that PP1 promotes the dephosphorylation of YB-1 on Ser102 in the absence of FSH.
If PP1 maintains the dephosphorylation of YB-1(Ser102) in the absence of FSH, then a PP1
catalytic subunit should co-immunoprecipitate with YB-1. As total YB-1 antibody is not
immunoprecipitating, we transiently transfected GCs with a full length Flag-tagged YB-1 plasmid,
allowed ~16 h for cells to recover, treated GCs with vehicle or FSH, then immunoprecipitated Flagtagged YB-1 with anti-Flag antibody. Results (Fig. 5C) show that anti-Flag antibody selectively pulls
down comparable levels of the catalytic subunit PP1β (PP1cβ) in both vehicle- and FSH-treated samples.
(Longer exposures in right panel reveal input and flow through signals for PP1cβ.) Additionally, when the
same experiment was performed and the presence of RSK-2 was investigated, results show that RSK-2
also interacts (directly or indirectly) with Flag-tagged YB-1 in a FSH-independent manner (Fig. 5C).
These results suggest that both PP1cβ and RSK-2 are in a complex with YB-1 in an FSH-independent
manner.
Taken together, these results suggest (i) that PP1 maintains YB-1(Ser102) in a dephosphorylated
state in the absence of FSH, (ii) that FSH signals to inhibit the activity of PP1, preventing the
dephosphorylation of YB-1 at Ser102 and (iii) that RSK-2 inhibits PP1 activity while in complex with YB1. The inability of FSH to significantly enhance YB-1(Ser102) phosphorylation in the presence of
tautomycin suggests that the major effect of PKA via ERK/RSK-2 is to promote inactivation of PP1.
Phosphorylation of YB-1 on Ser102 promotes its nuclear localization- As the phosphorylation of
YB-1 on Ser102 in breast cancer cells is most often linked to its function as a transcriptional activator
(21,23,24,26,33) (and as reviewed in (50)), we asked if phospho-YB-1 was preferentially localized to the
nuclear fraction of GCs.
81
To address this question, GCs were transfected with Flag-tagged YB-1 plasmids that express
wildtype YB-1, the phospho- YB-1(S102D) mimic, and dephospho-YB-1(S102A) mutant. Following a
16 h recovery period, GCs were treated with vehicle or FSH and then fractionated into cytosolic and
nuclear fractions; GAPDH and histone H3 serve as positive controls, respectively. All of the Flag-tagged
constructs were readily detected in the cytoplasmic fraction (Fig. 6A), consistent with the presence of
both phosphorylated YB-1(Ser102) and non-phosphorylated YB-1(Ser102) in this fraction. However, the
phospho-YB-1(S102D) mimic strongly localized to the nuclear fraction compared to the dephospho-YB1(S102A) mutant, consistent with reports in breast and ovarian cancer cells (20,22,26).
Phosphorylated YB-1(Ser102) was also readily detected using the phospho-YB-1(Ser102) antibody
in both the nuclear and cytosolic fractions of non-transfected GCs (Fig. 6B, left panel). However, FSH did
not promote a change in total levels of YB-1 within the nucleus (Fig. 6B, right panel), suggesting that
YB-1 phosphorylated on Ser102 comprises a relatively small fraction of total YB-1.
These results suggest that while the majority (~90%) of total and phosphorylated YB-1 is
localized to the cytoplasmic fraction, phosphorylated YB-1(Ser102) in GCs might have a distinctive
function in the nucleus.
Phospho-YB-1(Ser102) is necessary for expression of select FSH target genes- Based on the
localization of the YB-1(Ser102) phospho-mutant to the nuclear fraction, we hypothesized that YB-1 could
participate in the transcriptional regulation of FSH target genes. While ERK signaling is critical to LHinduced ovulation and luteinization in preovulatory GCs (51), the importance of the ERK signaling
pathway to FSH-stimulated differentiation of GCs is less well understood. We therefore sought to identify
FSH target genes that were regulated by the ERK signaling pathway to identify possible YB-1 gene
targets. Pretreatment of GCs with the MEK inhibitor U0126 resulted in inhibition of FSH-induced Egfr
[85.7% ± 6.2%], Cyp19a1 [96.1% ± 2.65%], Inha [82.5% ± 3.2%] and Ereg [67.7% ± 4.4%] mRNA
levels measured at 48 h post FSH treatment, while Star mRNA levels were not affected (Fig. 7).
82
Treatment with the MEK inhibitor PD98059 showed significant inhibition of FSH-induced expression of
the immediate early gene Egr1 [86.4% ± 3.2%] 1 h post FSH treatment (Fig. 7).
We next transduced GCs with adenoviral phospho- and dephospho-mutants of YB-1 to determine
if select ERK–regulated gene targets were activated by the phosphorylation of YB-1 on Ser102. We
focused on the three ERK-regulated target genes that showed the greatest inhibition by the MEK inhibitor
at the 48 h time point; Star expression was included as a negative control. Transduction of GCs with AdYB-1(S102D) phospho-mutant resulted in a statistically significant FSH-independent increase in Egfr and
Cyp19a1 mRNAs compared to Ad-Empty (E), that was not significantly enhanced further by FSH (Fig.
8A). In contrast, Inha mRNA levels were not different between Ad-E and Ad-YB-1(S102D) treatments.
Transduction of GCs with the dephospho-Ad-YB-1(S102A) mutant prevented the induction by FSH of
Egfr [82.0% ± 9.5%] and Cyp19a1 [88.6% ± 5.5%] mRNAs (Fig. 8B), consistent with results in Fig. 8A.
FSH-stimulated induction of Inha mRNA was also abrogated by the dephospho-Ad-YB-1(S102A) mutant
[90.9% ± 2.9%]. In contrast, FSH-stimulated Star mRNA expression was not reduced by the dephosphoAd-YB-1(S102A) mutant. We interpret these results to suggest that YB-1 phosphorylated on Ser102 is
necessary for the induction of Egfr, Cyp19a1, and Inha mRNA by FSH, but does not participate in the
induction of Star mRNA by FSH. The ability of the Ad-YB-1(S102D) phospho-mutant to enhance Egfr
and Cyp19a1 mRNA levels in the absence of FSH suggests that phosphorylated YB-1(Ser102) is a major
contributor to the induction of these genes.
Finally, GCs transfected with the indicated promoter-luciferase constructs, followed by
transduction with Ad-YB-1(S102A) mutant, promoted a significant (p < 0.05) inhibition of Egfr [64.9% ±
13.2%], Cyp19a1 [59.9% ± 10.0%], and Inha promoter – luciferase [72.5% ± 5.0%] activities (Fig. 8C).
These promoter-luciferase results suggest that phosphorylated YB-1 is acting at the level of the gene
promoters and not at the level of mRNA stability. Collectively, these results indicate that YB-1(Ser102)
phosphorylation is necessary for FSH-mediated induction of Egfr, Cyp19a1, and Inha gene expression.
83
DISCUSSION
Our results show that YB-1 is phosphorylated on Ser102 in response to FSH. However, in contrast
to our original premise, YB-1 in GCs is not an AKT target. Rather, our results reveal that YB-1 is
phosphorylated on Ser102 in response to FSH in a PKA- and ERK/RSK-2-dependent manner, consistent
with previous reports that YB-1(Ser102) is a RSK-2 target (26,45). Unexpectedly, our results suggest that
the major effect of RSK-2 on YB-1(Ser102) phosphorylation is to inactivate PP1 rather than to promote the
direct phosphorylation of YB-1, as discussed below. YB-1 phosphorylated on Ser102 localized to the
nuclear fraction and is required for the ability of FSH to induce key target genes required for follicular
maturation. Together, our results also reveal a previously unidentified role for YB-1 and an unappreciated
role for the ERK/RSK-2 signaling pathway in FSH-dependent GC maturation.
YB-1 is a multifunctional DNA and RNA binding protein and consequently participates in most
DNA- and RNA-dependent cellular functions (30,31). In the cytoplasm, YB-1 is one of two primary
proteins (along with poly-A binding protein) that comprise the mRNP complex (as reviewed in (32)).
Hence, YB-1 is associated with both free nontranslated and polysomal mRNA, and is a major regulator of
translation (52-54). YB-1 also functions as a transcriptional factor to both repress and activate
transcription by binding directly to gene promoters or in complex with other transcriptional factors (as
reviewed in (22,32)). YB-1 is generally viewed as an “oncogenic” transcription factor, based on its
overexpression in breast and lung cancer cells (21,55) and on its gene targets, that include Egfr, Her2,
Pi3kca, and Mdr1 (as reviewed in (32)). While YB-1 can be phosphorylated on a number of Ser and Tyr
residues, phosphorylation of Ser102 within the cold shock domain is most commonly associated with the
regulation of translation and transcription (as reviewed in (32)). Phosphorylation of YB-1 on Ser102
promotes its nuclear localization in ovarian and breast cancer cells (20,22,26) and is required for Egfr
expression in breast cancer cells (21,23,24,26).
Our results show that YB-1 is also phosphorylated on Ser102 in ovarian GCs, and that
phosphorylated YB-1 or a phospho-YB-1(S102D) mutant localizes to the nuclear fraction. Unexpectedly,
84
our results also reveal that YB-1 phosphorylated on Ser102 is necessary for the expression of select FSH
targets that define a mature, preovulatory GC, such as Egfr, Cyp19a1, and Inha. The EGFR is required for
LH-induced ovulation (56); Cyp19a1 encodes aromatase, the rate limiting enzyme in estrogen
biosynthesis (57); and Inha encodes for the inhibin hormone subunit inhibin-α that inhibits FSH
expression by the anterior pituitary (58). While it is well-known that CREB, SF-1(Nr5a1)/LRH-1(Nr5a2),
and GATA-4/6 enhance expression of both Cyp19a1 and Inha (40,59-61), and that FOXO1 (15) and Tcell factor 3 (9) repress expression of both genes, a role for YB-1 in the transcriptional activation of these
genes is novel. To our knowledge, the mechanism by which FSH enhances Egfr expression has not been
previously described.
While YB-1 phosphorylated on Ser102 localizes to the nucleus, the great majority of both
phosphorylated YB-1(Ser102) and total YB-1 is present in the cytoplasmic fraction of GCs (see Fig. 6,
where the cytoplasmic and nuclear fractions represent 9% and 50% of the total fraction, respectively).
Cytoplasmic YB-1 has been localized to both translated polysomal mRNPs and to free untranslated
mRNPs (reviewed in (32)). YB-1 is recognized to stabilize both long- and short-lived mRNAs at high
YB-1/mRNA ratios effectively by burying mRNAs within YB-1 protein complexes (reviewed in (32)). In
NIH3T3 cells, AKT-dependent phosphorylation of YB-1 on Ser102 stimulated Cap-dependent translation
(53). It is thus likely that cytosolic YB-1 is playing a major role in both stabilizing GC mRNA and, upon
phosphorylation, enhancing translation.
FSH-stimulated phosphorylation of YB-1 on Ser102 in GCs is mediated by PKA, ERK, and its
downstream kinase RSK-2. However, to our surprise, our results suggest that the major effect of RSK-2 is
to inhibit the phosphatase activity of PP1. This conclusion is based largely on the inability of FSH to
increase phospho-YB-1(Ser102) signal over that seen in vehicle-treated cells in the presence of the PP1
inhibitor tautomycin (see Fig. 5B). Moreover, the RSK-2 inhibitor BI-D1870 abrogates YB-1(Ser102)
phosphorylation in vehicle- and FSH-treated cells, even though PKA remains active (evidenced by CREB
phosphorylation on Ser133; see Fig.4). Additionally the PP1β catalytic subunit, RSK-2, and YB-1 co-
85
immunoprecipitate in a complex. The presence of phosphorylated YB-1(Ser102) in GCs pretreated with
tautomycin and treated with vehicle (i.e., in the absence of FSH) further suggests that while RSK-2 may
also directly phosphorylate YB-1 in the presence of FSH, RSK-2 is not the kinase that phosphorylates
YB-1 on Ser102 in vehicle-treated cells. We hypothesize that Ser102 may be phosphorylated by the
constitutively active kinase casein kinase 2, as the phosphorylation motif on YB-1 (KYLRS102VGD)
partially aligns with that of casein kinase 2 (S*E/DXE/D) (62).
We do not know the mechanism by which RSK-2 inactivates PP1 activity. The catalytic subunits
of PP1 can associate with a large number of regulatory subunits (up to 200 have been identified), many of
which inhibit the catalytic activity of PP1 (63). Unfortunately, we have not been able to identify the PP1
regulatory subunit inhibited by RSK-2. We hypothesize, however, that RSK-2-dependent phosphorylation
of the PP1 regulatory subunit in GCs promotes its degradation, allowing for the prolonged
phosphorylation of YB-1 that persists up to 8 h post FSH treatment, despite the shorter-lived
phosphorylation of RSK-2 that is undetectable within 2 h post FSH treatment (8).
The prolonged phosphorylation of YB-1 is not observed with other transcriptional targets in GCs,
such as CREB and histone H3, whose phosphorylation has returned to the levels of untreated cells by 4 h
post FSH treatment (8). We do not know the functional significance of this sustained phosphorylation of
YB-1. It is unlikely to be required for transcriptional regulation. The prolonged YB-1 phosphorylation is
more likely to participate either in mRNA stabilization and/or translation.
While Ser102 is the most studied phosphorylation site on YB-1, multiple kinases have been shown
to phosphorylate YB-1. ERK and glycogen synthase kinase 3β are reported to phosphorylate YB-1 on
Thr38 and/or Ser41 and/or Ser45 (36). Indeed, mass–spectrometric analysis has identified additional
phosphorylations on YB-1, including Tyr162, Ser165 and/or Ser167, Ser174 and/or Ser176, and Ser313 and/or
Ser314 (64,65). Therefore, we cannot rule out the possibility that FSH promotes phosphorylation of YB-1
on additional sites by other kinases.
86
In conclusion, we have shown that FSH promotes YB-1(Ser102) phosphorylation through a PKAand ERK/RSK-2-dependent signaling pathway. The major effect of RSK-2 appears to be to inhibit the
Ser/Thr phosphatase PP1 rather than to directly phosphorylate YB-1 on Ser102. YB-1 phosphorylated on
Ser102 is necessary for the expression of FSH-dependent gene targets that are essential for GC maturation.
Taken together, our results not only establish a functional role for YB-1 as a transcriptional activator of
FSH target genes in the GCs, but also reveal an underappreciated role for ERK signaling pathway in the
activation of gene expression by FSH in GCs.
87
ACKNOWLEDGMENTS: We thank Dr. Wenlong Bai for the full length human Egfr-promoter
luciferase. We thank Drs. John H. Nilson and Maria Herndon and Nate Law for many helpful discussions.
This work was supported by National Institutes of Health Grants R01HD065859 and R01HD62503 (to
MEH-D) and National Institute of General Medical Science Training Grant T32GM083864 (to John H
Nilson).
CONFLICT OF INTEREST: The authors declare that they have no conflicts of interest with the
contents of this article3.
AUTHOR CONTRIBUTIONS: EMD and MEH-D designed the experiments. EMD performed the
experiments and analyzed the data. EMD and MEH-D wrote the manuscript.
88
References
1.
Richards, J. S., Fitzpatrick, S. L., Clemens, J. W., Morris, J. K., Alliston, T., and Sirois, J. (1995)
Ovarian cell differentiation: a cascade of multiple hormones, cellular signals, and regulated
genes. Recent progress in hormone research 50, 223-254
2.
Hunzicker-Dunn, M., and Mayo, K. E. (2015) Gonadotropin Signaling in the Ovary. in KNOBIL
AND NEILL’S PHYSIOLOGY OF REPRODUCTION (Plant, T. M., and Zeleznik, A. J. eds.),
Fourth Ed., Elsevier. pp 895 - 946
3.
Hsueh, A. J., Adashi, E. Y., Jones, P. B., and Welsh, T. H., Jr. (1984) Hormonal regulation of the
differentiation of cultured ovarian granulosa cells. Endocr. Rev. 5, 76-127
4.
Mukherjee, A., Park-Sarge, O. K., and Mayo, K. E. (1996) Gonadotropins induce rapid
phosphorylation of the 3',5'-cyclic adenosine monophosphate response element binding protein in
ovarian granulosa cells. Endocrinology 137, 3234-3245
5.
Hunzicker-Dunn, M. (1981) Selective activation of rabbit ovarian protein kinase isozymes in
rabbit ovarian follicles and corpora lutea. J. Biol. Chem. 256, 12185-12193
6.
Sprengel, R., Braun, T., Nikolics, K., Segaloff, D. L., and Seeburg, P. H. (1990) The testicular
receptor for follicle stimulating hormone: structure and functional expression of cloned cDNA.
Mol. Endocrinol. 4, 525-530
7.
Hagiwara, M., Brindle, P., Harootunian, A., Armstrong, R., Rivier, J., Vale, W., Tsien, R., and
Montminy, M. R. (1993) Coupling of hormonal stimulation and transcription via the cyclic AMPresponsive factor CREB is rate limited by nuclear entry of protein kinase A. Mol. Cell. Biol. 13,
4852-4859
8.
Salvador, L. M., Park, Y., Cottom, J., Maizels, E. T., Jones, J. C., Schillace, R. V., Carr, D. W.,
Cheung, P., Allis, C. D., Jameson, J. L., and Hunzicker-Dunn, M. (2001) Follicle-stimulating
hormone stimulates protein kinase A-mediated histone H3 phosphorylation and acetylation
leading to select gene activation in ovarian granulosa cells. J. Biol. Chem. 276, 40146-40155
9.
Law, N. C., Weck, J., Kyriss, B., Nilson, J. H., and Hunzicker-Dunn, M. (2013) Lhcgr Expression
in Granulosa Cells: Roles for PKA-Phosphorylated beta-Catenin, TCF3, and FOXO1. Mol.
Endocrinol. 27, 1295-1310
10.
Das, S., Maizels, E. T., DeManno, D., St Clair, E., Adam, S. A., and Hunzicker-Dunn, M. (1996)
A stimulatory role of cyclic adenosine 3',5'-monophosphate in follicle-stimulating hormoneactivated mitogen-activated protein kinase signaling pathway in rat ovarian granulosa cells.
Endocrinology 137, 967-974
89
11.
Cottom, J., Salvador, L. M., Maizels, E. T., Reierstad, S., Park, Y., Carr, D. W., Davare, M. A.,
Hell, J. W., Palmer, S. S., Dent, P., Kawakatsu, H., Ogata, M., and Hunzicker-Dunn, M. (2003)
Follicle-stimulating hormone activates extracellular signal-regulated kinase but not extracellular
signal-regulated kinase kinase through a 100-kDa phosphotyrosine phosphatase. J. Biol. Chem.
278, 7167-7179
12.
Alam, H., Maizels, E. T., Park, Y., Ghaey, S., Feiger, Z. J., Chandel, N. S., and Hunzicker-Dunn,
M. (2004) Follicle-stimulating hormone activation of hypoxia-inducible factor-1 by the
phosphatidylinositol 3-kinase/AKT/Ras homolog enriched in brain (Rheb)/mammalian target of
rapamycin (mTOR) pathway is necessary for induction of select protein markers of follicular
differentiation. J. Biol. Chem. 279, 19431-19440
13.
Gonzalez-Robayna, I. J., Falender, A. E., Ochsner, S., Firestone, G. L., and Richards, J. S. (2000)
Follicle-Stimulating hormone (FSH) stimulates phosphorylation and activation of protein kinase
B (PKB/Akt) and serum and glucocorticoid-lnduced kinase (Sgk): evidence for A kinaseindependent signaling by FSH in granulosa cells. Mol. Endocrinol. 14, 1283-1300
14.
Hunzicker-Dunn, M. E., Lopez-Biladeau, B., Law, N. C., Fiedler, S. E., Carr, D. W., and Maizels,
E. T. (2012) PKA and GAB2 play central roles in the FSH signaling pathway to PI3K and AKT
in ovarian granulosa cells. Proceedings of the National Academy of Sciences of the United States
of America 109, E2979-2988
15.
Park, Y., Maizels, E. T., Feiger, Z. J., Alam, H., Peters, C. A., Woodruff, T. K., Unterman, T. G.,
Lee, E. J., Jameson, J. L., and Hunzicker-Dunn, M. (2005) Induction of cyclin D2 in rat granulosa
cells requires FSH-dependent relief from FOXO1 repression coupled with positive signals from
Smad. J. Biol. Chem. 280, 9135-9148
16.
Richards, J. S., Sharma, S. C., Falender, A. E., and Lo, Y. H. (2002) Expression of FKHR,
FKHRL1, and AFX genes in the rodent ovary: evidence for regulation by IGF-I, estrogen, and the
gonadotropins. Mol. Endocrinol. 16, 580-599
17.
Zeleznik, A. J., Saxena, D., and Little-Ihrig, L. (2003) Protein kinase B is obligatory for folliclestimulating hormone-induced granulosa cell differentiation. Endocrinology 144, 3985-3994
18.
Liu, Z., Rudd, M. D., Hernandez-Gonzalez, I., Gonzalez-Robayna, I., Fan, H. Y., Zeleznik, A. J.,
and Richards, J. S. (2009) FSH and FOXO1 regulate genes in the sterol/steroid and lipid
biosynthetic pathways in granulosa cells. Mol. Endocrinol. 23, 649-661
19.
Law, N. C., and Hunzicker-Dunn, M. E. (2015) Insulin Receptor Substrate 1: The Hub Linking
Follicle-stimulating Hormone to Phosphatidylinositol-3 Kinase Activation. J. Biol. Chem.
90
20.
Sutherland, B. W., Kucab, J., Wu, J., Lee, C., Cheang, M. C., Yorida, E., Turbin, D., Dedhar, S.,
Nelson, C., Pollak, M., Leighton Grimes, H., Miller, K., Badve, S., Huntsman, D., Blake-Gilks,
C., Chen, M., Pallen, C. J., and Dunn, S. E. (2005) Akt phosphorylates the Y-box binding protein
1 at Ser102 located in the cold shock domain and affects the anchorage-independent growth of
breast cancer cells. Oncogene 24, 4281-4292
21.
Stratford, A. L., Habibi, G., Astanehe, A., Jiang, H., Hu, K., Park, E., Shadeo, A., Buys, T. P.,
Lam, W., Pugh, T., Marra, M., Nielsen, T. O., Klinge, U., Mertens, P. R., Aparicio, S., and Dunn,
S. E. (2007) Epidermal growth factor receptor (EGFR) is transcriptionally induced by the Y-box
binding protein-1 (YB-1) and can be inhibited with Iressa in basal-like breast cancer, providing a
potential target for therapy. Breast Cancer Res 9, R61
22.
Basaki, Y., Hosoi, F., Oda, Y., Fotovati, A., Maruyama, Y., Oie, S., Ono, M., Izumi, H., Kohno,
K., Sakai, K., Shimoyama, T., Nishio, K., and Kuwano, M. (2007) Akt-dependent nuclear
localization of Y-box-binding protein 1 in acquisition of malignant characteristics by human
ovarian cancer cells. Oncogene 26, 2736-2746
23.
To, K., Zhao, Y., Jiang, H., Hu, K., Wang, M., Wu, J., Lee, C., Yokom, D. W., Stratford, A. L.,
Klinge, U., Mertens, P. R., Chen, C. S., Bally, M., Yapp, D., and Dunn, S. E. (2007) The
phosphoinositide-dependent kinase-1 inhibitor 2-amino-N-[4-[5-(2-phenanthrenyl)-3(trifluoromethyl)-1H-pyrazol-1-yl]phenyl]-ace tamide (OSU-03012) prevents Y-box binding
protein-1 from inducing epidermal growth factor receptor. Mol. Pharmacol. 72, 641-652
24.
Wu, J., Lee, C., Yokom, D., Jiang, H., Cheang, M. C., Yorida, E., Turbin, D., Berquin, I. M.,
Mertens, P. R., Iftner, T., Gilks, C. B., and Dunn, S. E. (2006) Disruption of the Y-box binding
protein-1 results in suppression of the epidermal growth factor receptor and HER-2. Cancer Res.
66, 4872-4879
25.
Liu, X., Su, L., and Liu, X. (2013) Loss of CDH1 up-regulates epidermal growth factor receptor
via phosphorylation of YBX1 in non-small cell lung cancer cells. FEBS Lett. 587, 3995-4000
26.
Stratford, A. L., Fry, C. J., Desilets, C., Davies, A. H., Cho, Y. Y., Li, Y., Dong, Z., Berquin, I.
M., Roux, P. P., and Dunn, S. E. (2008) Y-box binding protein-1 serine 102 is a downstream
target of p90 ribosomal S6 kinase in basal-like breast cancer cells. Breast Cancer Res 10, R99
27.
El-Hayek, S., Demeestere, I., and Clarke, H. J. (2014) Follicle-stimulating hormone regulates
expression and activity of epidermal growth factor receptor in the murine ovarian follicle.
Proceedings of the National Academy of Sciences of the United States of America 111, 1677816783
28.
Blobel, G. (1972) Protein tightly bound to globin mRNA. Biochem. Biophys. Res. Commun. 47,
88-95
91
29.
Morel, C., Kayibanda, B., and Scherrer, K. (1971) Proteins associated with globin messenger
RNA in avian erythroblasts: Isolation and comparison with the proteins bound to nuclear
messenger-likie RNA. FEBS Lett. 18, 84-88
30.
Tafuri, S. R., and Wolffe, A. P. (1992) DNA binding, multimerization, and transcription
stimulation by the Xenopus Y box proteins in vitro. New Biol 4, 349-359
31.
Bouvet, P., Matsumoto, K., and Wolffe, A. P. (1995) Sequence-specific RNA recognition by the
Xenopus Y-box proteins. An essential role for the cold shock domain. J. Biol. Chem. 270, 2829728303
32.
Eliseeva, I. A., Kim, E. R., Guryanov, S. G., Ovchinnikov, L. P., and Lyabin, D. N. (2011) Ybox-binding protein 1 (YB-1) and its functions. Biochemistry. Biokhimiia 76, 1402-1433
33.
Finkbeiner, M. R., Astanehe, A., To, K., Fotovati, A., Davies, A. H., Zhao, Y., Jiang, H.,
Stratford, A. L., Shadeo, A., Boccaccio, C., Comoglio, P., Mertens, P. R., Eirew, P., Raouf, A.,
Eaves, C. J., and Dunn, S. E. (2009) Profiling YB-1 target genes uncovers a new mechanism for
MET receptor regulation in normal and malignant human mammary cells. Oncogene 28, 14211431
34.
Fukada, T., and Tonks, N. K. (2003) Identification of YB-1 as a regulator of PTP1B expression:
implications for regulation of insulin and cytokine signaling. EMBO J. 22, 479-493
35.
Jurchott, K., Bergmann, S., Stein, U., Walther, W., Janz, M., Manni, I., Piaggio, G., Fietze, E.,
Dietel, M., and Royer, H. D. (2003) YB-1 as a cell cycle-regulated transcription factor facilitating
cyclin A and cyclin B1 gene expression. J. Biol. Chem. 278, 27988-27996
36.
Coles, L. S., Lambrusco, L., Burrows, J., Hunter, J., Diamond, P., Bert, A. G., Vadas, M. A., and
Goodall, G. J. (2005) Phosphorylation of cold shock domain/Y-box proteins by ERK2 and
GSK3beta and repression of the human VEGF promoter. FEBS Lett. 579, 5372-5378
37.
Carr, D. W., DeManno, D. A., Atwood, A., Hunzicker-Dunn, M., and Scott, J. D. (1993) Folliclestimulating hormone regulation of A-kinase anchoring proteins in granulosa cells. J. Biol. Chem.
268, 20729-20732
38.
DeManno, D. A., Cottom, J. E., Kline, M. P., Peters, C. A., Maizels, E. T., and Hunzicker-Dunn,
M. (1999) Follicle-stimulating hormone promotes histone H3 phosphorylation on serine-10. Mol.
Endocrinol. 13, 91-105
39.
Shen, Z., Zhang, X., Tang, J., Kasiappan, R., Jinwal, U., Li, P., Hann, S., Nicosia, S. V., Wu, J.,
Zhang, X., and Bai, W. (2011) The coupling of epidermal growth factor receptor down regulation
92
by 1alpha,25-dihydroxyvitamin D3 to the hormone-induced cell cycle arrest at the G1-S
checkpoint in ovarian cancer cells. Mol. Cell. Endocrinol. 338, 58-67
40.
Ito, M., Park, Y., Weck, J., Mayo, K. E., and Jameson, J. L. (2000) Synergistic activation of the
inhibin alpha-promoter by steroidogenic factor-1 and cyclic adenosine 3',5'-monophosphate. Mol.
Endocrinol. 14, 66-81
41.
Pei, L., Dodson, R., Schoderbek, W. E., Maurer, R. A., and Mayo, K. E. (1991) Regulation of the
alpha inhibin gene by cyclic adenosine 3',5'-monophosphate after transfection into rat granulosa
cells. Mol. Endocrinol. 5, 521-534
42.
Sunters, A., Armstrong, V. J., Zaman, G., Kypta, R. M., Kawano, Y., Lanyon, L. E., and Price, J.
S. (2010) Mechano-transduction in osteoblastic cells involves strain-regulated estrogen receptor
alpha-mediated control of insulin-like growth factor (IGF) I receptor sensitivity to Ambient IGF,
leading to phosphatidylinositol 3-kinase/AKT-dependent Wnt/LRP5 receptor-independent
activation of beta-catenin signaling. J. Biol. Chem. 285, 8743-8758
43.
Kawasaki, H., Springett, G. M., Mochizuki, N., Toki, S., Nakaya, M., Matsuda, M., Housman, D.
E., and Graybiel, A. M. (1998) A family of cAMP-binding proteins that directly activate Rap1.
Science 282, 2275-2279
44.
Enserink, J. M., Christensen, A. E., de Rooij, J., van Triest, M., Schwede, F., Genieser, H. G.,
Doskeland, S. O., Blank, J. L., and Bos, J. L. (2002) A novel Epac-specific cAMP analogue
demonstrates independent regulation of Rap1 and ERK. Nat. Cell Biol. 4, 901-906
45.
Reipas, K. M., Law, J. H., Couto, N., Islam, S., Li, Y., Li, H., Cherkasov, A., Jung, K., Cheema,
A. S., Jones, S. J., Hassell, J. A., and Dunn, S. E. (2013) Luteolin is a novel p90 ribosomal S6
kinase (RSK) inhibitor that suppresses Notch4 signaling by blocking the activation of Y-box
binding protein-1 (YB-1). Oncotarget 4, 329-345
46.
Andric, N., and Ascoli, M. (2006) A delayed gonadotropin-dependent and growth factormediated activation of the extracellular signal-regulated kinase 1/2 cascade negatively regulates
aromatase expression in granulosa cells. Mol. Endocrinol. 20, 3308-3320
47.
Bain, J., Plater, L., Elliott, M., Shpiro, N., Hastie, C. J., McLauchlan, H., Klevernic, I., Arthur, J.
S., Alessi, D. R., and Cohen, P. (2007) The selectivity of protein kinase inhibitors: a further
update. Biochem. J. 408, 297-315
48.
Alam, H., Weck, J., Maizels, E., Park, Y., Lee, E. J., Ashcroft, M., and Hunzicker-Dunn, M.
(2009) Role of the phosphatidylinositol-3-kinase and extracellular regulated kinase pathways in
the induction of hypoxia-inducible factor (HIF)-1 activity and the HIF-1 target vascular
93
endothelial growth factor in ovarian granulosa cells in response to follicle-stimulating hormone.
Endocrinology 150, 915-928
49.
Dalby, K. N., Morrice, N., Caudwell, F. B., Avruch, J., and Cohen, P. (1998) Identification of
regulatory phosphorylation sites in mitogen-activated protein kinase (MAPK)-activated protein
kinase-1a/p90rsk that are inducible by MAPK. J. Biol. Chem. 273, 1496-1505
50.
Wu, J., Stratford, A. L., Astanehe, A., and Dunn, S. E. (2007) YB-1 is a Transcription/Translation
Factor that Orchestrates the Oncogenome by Hardwiring Signal Transduction to Gene
Expression. Transl Oncogenomics 2, 49-65
51.
Fan, H. Y., Liu, Z., Shimada, M., Sterneck, E., Johnson, P. F., Hedrick, S. M., and Richards, J. S.
(2009) MAPK3/1 (ERK1/2) in ovarian granulosa cells are essential for female fertility. Science
324, 938-941
52.
Bader, A. G., and Vogt, P. K. (2005) Inhibition of protein synthesis by Y box-binding protein 1
blocks oncogenic cell transformation. Mol. Cell. Biol. 25, 2095-2106
53.
Evdokimova, V., Ruzanov, P., Anglesio, M. S., Sorokin, A. V., Ovchinnikov, L. P., Buckley, J.,
Triche, T. J., Sonenberg, N., and Sorensen, P. H. (2006) Akt-mediated YB-1 phosphorylation
activates translation of silent mRNA species. Mol. Cell. Biol. 26, 277-292
54.
Nekrasov, M. P., Ivshina, M. P., Chernov, K. G., Kovrigina, E. A., Evdokimova, V. M., Thomas,
A. A., Hershey, J. W., and Ovchinnikov, L. P. (2003) The mRNA-binding protein YB-1 (p50)
prevents association of the eukaryotic initiation factor eIF4G with mRNA and inhibits protein
synthesis at the initiation stage. J. Biol. Chem. 278, 13936-13943
55.
Gessner, C., Woischwill, C., Schumacher, A., Liebers, U., Kuhn, H., Stiehl, P., Jurchott, K.,
Royer, H. D., Witt, C., and Wolff, G. (2004) Nuclear YB-1 expression as a negative prognostic
marker in nonsmall cell lung cancer. Eur. Respir. J. 23, 14-19
56.
Hsieh, M., Lee, D., Panigone, S., Horner, K., Chen, R., Theologis, A., Lee, D. C., Threadgill, D.
W., and Conti, M. (2007) Luteinizing hormone-dependent activation of the epidermal growth
factor network is essential for ovulation. Mol. Cell. Biol. 27, 1914-1924
57.
Fisher, C. R., Graves, K. H., Parlow, A. F., and Simpson, E. R. (1998) Characterization of mice
deficient in aromatase (ArKO) because of targeted disruption of the cyp19 gene. Proceedings of
the National Academy of Sciences of the United States of America 95, 6965-6970
58.
Woodruff, T. K., and Mayo, K. E. (1990) Regulation of inhibin synthesis in the rat ovary. Annu.
Rev. Physiol. 52, 807-821
94
59.
Weck, J., and Mayo, K. E. (2006) Switching of NR5A proteins associated with the inhibin alphasubunit gene promoter after activation of the gene in granulosa cells. Mol. Endocrinol. 20, 10901103
60.
Bennett, J., Wu, Y. G., Gossen, J., Zhou, P., and Stocco, C. (2012) Loss of GATA-6 and GATA4 in granulosa cells blocks folliculogenesis, ovulation, and follicle stimulating hormone receptor
expression leading to female infertility. Endocrinology 153, 2474-2485
61.
Carlone, D. L., and Richards, J. S. (1997) Functional interactions, phosphorylation, and levels of
3',5'-cyclic adenosine monophosphate-regulatory element binding protein and steroidogenic
factor-1 mediate hormone-regulated and constitutive expression of aromatase in gonadal cells.
Mol. Endocrinol. 11, 292-304
62.
Ubersax, J. A., and Ferrell, J. E., Jr. (2007) Mechanisms of specificity in protein phosphorylation.
Nat. Rev. Mol. Cell Biol. 8, 530-541
63.
Bollen, M., Peti, W., Ragusa, M. J., and Beullens, M. (2010) The extended PP1 toolkit: designed
to create specificity. Trends Biochem. Sci. 35, 450-458
64.
Dephoure, N., Zhou, C., Villen, J., Beausoleil, S. A., Bakalarski, C. E., Elledge, S. J., and Gygi,
S. P. (2008) A quantitative atlas of mitotic phosphorylation. Proceedings of the National
Academy of Sciences of the United States of America 105, 10762-10767
65.
Olsen, J. V., Blagoev, B., Gnad, F., Macek, B., Kumar, C., Mortensen, P., and Mann, M. (2006)
Global, in vivo, and site-specific phosphorylation dynamics in signaling networks. Cell 127, 635648
95
FOOTNOTES
1
To whom correspondence should be addressed: Dr. Mary E. Hunzicker-Dunn, Biotechnology Life
Sciences Building, Room 202, Washington State University, Pullman WA 99164, Telephone: 509-3355614; Email: [email protected]
2
The abbreviations used are: GCs, granulosa cells; YB-1, Y-box binding protein-1; FSH, follicle
stimulating hormone; PKA, protein kinase A; PKI, PKA inhibitor; RSK-2, ribosomal S6 kinase-2; PP1,
protein phosphatase 1; LH, luteinizing hormone; GPCR, G protein-coupled receptor; CREB, cAMPresponse binding element protein; AKT, protein kinase B; PI3K, phosphatidylinositol-3- kinase; FOXO1,
forkhead box- containing protein O1; IGF1, insulin-like growth factor 1; SHP2, SRC homology-2 (SH2)
domain-containing tyrosine phosphatase; Myr, myristoylated; E2, estradiol-17β; P, penicillin; S,
streptomycin; PP2, protein phosphatase 2; PP1cβ, PP1β catalytic subunit; mRNP, mRNA-protein
complex.
3
The content is solely the responsibility of the authors and does not necessarily represent the official
views of the National Institutes of Health.
96
FIGURE LEGENDS AND FIGURES
FIGURE 1. Treatment of GCs with FSH leads to prolonged phosphorylation of YB-1 on Ser102. (A) GCs
were treated for the indicated times without (v) or with 50 ng/mL FSH (F). Samples were heat denatured
after collection in SDS sample buffer and proteins were separated by SDS/PAGE. A blot of whole cell
extracts was probed with indicated antibodies. Results are representative of three independent
experiments. (B) GCs were treated with FSH for the indicated times and samples were collected as
described in A. Blot is representative of three independent experiments. Quantification results represent
the mean ± SEM of three independent experiments, with (*) representing p < 0.05, based on a 1–way
ANOVA followed by Tukey’s posttest. (C) GCs were treated with FSH (F) for the indicated times, and
samples were collected and analyzed as described in B, with (*) representing p < 0.05, based on a
Students t-test of veh vs FSH.
97
98
FIGURE 2. Phosphorylation of YB-1 on Ser102 is mediated by cAMP and PKA and independent of
exchange proteins activated by cAMP. (A) GCs were treated for 1 h without (veh) or with FSH (lanes 1
and 2) or without (EtOH) or with 10 µM forskolin in 0.25% ethanol/DMEM (lanes 3 and 4). Samples
were collected as described in 1A. Quantification of results represents the mean ± SEM of three
independent experiments, with (*) representing p < 0.01, based on a 1–way ANOVA followed by
Tukey’s posttest. (B) GCs were treated for 1 h without (veh) or with FSH or with 250 µM of either 8pCPT-2’-O-Me-cAMP or 8-CPT-cAMP. Samples were collected as described in 1A. Dotted lines between
lanes represent cropped images. Results are representative of three independent experiments.
Quantification is as described in 2A, with (*) representing p < 0.01, based on a 1–way ANOVA followed
by Tukey’s posttest. (C) GCs were transduced overnight with Ad-Empty (E) (5.8 x 103 OPU/cell) or AdPKI (1.8 x 104 OPU/cell) that expresses the full length PKI protein. Following media replacement, cells
were treated for 2 h without (veh) or with FSH. Samples were collected as described in 1A, and
quantification is as described in 2A, with (*) representing p < 0.01, based on a 1–way ANOVA followed
by Tukey’s posttest. Results are representative of three independent experiments.
99
100
FIGURE 3. FSH- stimulated phosphorylation of YB-1 on Ser102 is via ERK signaling, not PI3K/AKT.
(A) GCs were pretreated without (DMSO) or with 100 nM wortmannin for 1 h followed by treatment
without (veh) or with FSH for 1 h. Results are representative of three independent experiments. Samples
were collected as described in 1A and quantification as described in 1B, with (*) representing p < 0.05,
based on a 1–way ANOVA followed by Tukey’s posttest. (B) GCs were pretreated without (DMSO) or
with 50 µM PD98059 for 1 h followed by treatment without (veh) or with FSH for 1 h. Samples were
collected as described in 1A and quantification is as described in 1B, with (*) representing p < 0.001,
based on a 1–way ANOVA followed by Tukey’s posttest. Results are representative of three independent
experiments.
(C) GCs were treated without (veh) or with FSH, 50 ng/mL EGF or 50 ng/mL IGF1 as
indicated. Samples were collected as described in 1A and quantification is as described in 1B, with (*)
representing p < 0.05 and (**) representing p < 0.01, based on a 1–way ANOVA followed by Tukey’s
posttest. Results are representative of three independent experiments.
101
102
FIGURE 4.
FSH- stimulated phosphorylation of YB-1 on Ser102 is RSK-2 dependent. GCs were
pretreated without or with 10 µM BI–D1870 for 1 h followed by treatment without or with FSH for 1 h.
Samples were collected as described in 1A, and quantification is as described in 1B, with (*) representing
p < 0.001, based on a 1–way ANOVA followed by Tukey’s posttest. Results are representative of three
independent experiments. Dotted lines between lanes represent cropped images.
103
104
FIGURE 5. Prolonged FSH- stimulated phosphorylation of YB-1 on Ser102 is dependent on inhibition of
the Ser/Thr phosphatase PP1. (A) GCs were pretreated without (EtOH) or with 200 nM okadaic acid for 1
h followed by treatment without (veh) or with FSH for 1 h. Samples were collected as described in 1A,
and quantification represents the mean ± SEM of three independent experiments, with (**) representing p
< 0.01 and (*) representing p < 0.05, based on a one tailed Student t test. (B) GCs were pretreated
without (EtOH) or with 1 µM tautomycin for 5.5 h followed by treatment without (veh) or with FSH for 1
h. Samples were collected and quantification is as described in 1B, with (*) representing p < 0.05, based
on a 1–way ANOVA followed by Tukey’s posttest.
EtOH FSH, tautomycin veh, and tautomycin FSH
treated samples are not statically different from each other (ns). Results are representative of three
independent experiments.
(C) GCs were transfected with 500 ng YB-1 – Flag plasmid for 6 h as
described in Experimental Procedures. Following an overnight recovery, GCs were treated without (veh)
or with FSH for 1 h, and then collected in Immunoprecipitation Lysis buffer and sonicated. After removal
of the insoluble particles, the soluble fraction was rotated overnight with 20 µg Anti–Flag (Sigma F3165)
as described in Experimental Procedures. Inputs, bound (IP) and unbound (flow – though) samples were
separated by SDS/PAGE and the western blot was probed with the indicated antibodies. Arrows indicate
the protein band of interest. Dotted lines between lanes represent cropped images. Results are
representative of two independent experiments.
105
106
FIGURE 6. YB-1 and phospho-YB-1(Ser102) are detected in the both the nuclear and cytosolic fractions;
phosphorylation of YB-1 on Ser102 promotes nuclear localization. (A) GCs were transfected with 500 ng
of the indicated plasmid for 6 h as described in Experimental Procedures. Following recovery, GCs were
treated without (veh) or with FSH for 4 h, then collected and separated into cytoplasmic and nuclear
fractions as described in Experimental Procedures. Samples were collected as described in 1A and are
representative of two independent experiments. Quantification of results represents the mean ± SEM of
two independent experiments, with (*) representing p < 0.05 and (**) representation p < 0.001, based on a
1–way ANOVA followed by Tukey’s posttest. (B) GCs were treated without (veh) or with FSH for 4 h,
then collected and separated into cytoplasmic and nuclear fractions as described in Experimental
Procedures. Samples were collected as described in 1A, and western blots were probed with the indicated
antibodies. Results are representative of three independent experiments.
107
108
FIGURE 7. ERK signaling is necessary for FSH-stimulated target gene mRNA expression. For Egfr,
Ereg, Cyp19a1, Inha, and Star, GCs were pretreated without (DMSO) and with 10 µM U0126 for 1 h,
then treated without (veh) or with FSH for 48 h. For Egr1, GCs were pretreated without (DMSO) and
with 50 µM PD98059 for 1 h, then treated without (veh) or with FSH for 1 h. Cells were collected for
RNA isolation and real–time PCR was performed for the genes indicated as described in Experimental
Procedures. Quantification results represent the mean ± SEM of three independent experiments for Egfr
and Ereg, and two independent experiments for Cyp19a1, Inha, Egr1 and Star with (*) representing p <
0.01, (**) representing p < 0.001 and (***) representing p < 0.05, based on a 1–way ANOVA followed
by Tukey’s posttest.
109
110
FIGURE 8. Phospho - YB-1 regulates FSH and ERK dependent-target gene mRNA expression. (A) GCs
were transduced with Ad- YB-1(S102D)-Flag or Ad-Empty overnight. Following media replacement and
>1 h recovery, cells were treated without (veh) and with FSH for 48 h. GCs were collected and analysis as
described in 7, and quantification results represent the mean ± SEM of three independent experiments
with (*) representing p < 0.05 and (**) representing p < 0.01, based on a one tailed Students t test. (B)
GCs were transduced with Ad- YB-1(S102A)-Flag or Ad-Empty overnight. Following media replacement
and >1 h recovery, cells were treated without (veh) and with FSH for 48 h. Cells were collected, and
analysis and quantification is as described in 8A, with (*) representing p < 0.05 and (**) representing p <
0.01, based on a one tailed Students t test. (C) GCs were transfected with the indicated promoter
Luciferase constructs for 6 h as described in the Experimental Procedures, followed by overnight
transduction with Ad- YB-1(S102A) - Flag or Ad- Empty. Following media replacement and >1 h
recovery, cells were treated without (veh) and with FSH for 6 h, then collected. Samples were analyzed
according to Experimental Procedures, and results are expressed as the RLU mean ± SEM of triplicates
and are representative of three independent experiments. (*) represents p < 0.05 and (***) represents p <
0.01, based on a one tailed Students t test.
111
112
FIGURE 9. Proposed signaling pathway by which FSH promotes the PKA- dependent phosphorylation
of YB-1 on Ser102 to enhance expression of select FSH target genes. Results support the schematic model
in which FSH via PKA and ERK leads to an increase in phosphorylation of YB-1 on Ser102. This increase
in YB-1 phosphorylation on Ser102 is dependent on ERK–dependent activation of RSK-2. RSK-2 appears
to enhance phosphorylation of YB-1 on Ser102 primarily by inactivating PP1. YB-1 phosphorylated on
Ser102 is necessary for FSH dependent induction of target genes.
113
114
CHAPTER FOUR
YB-1 INTERACTS WITH INITIATION FACTOR COMPLEX PROTEINS AND mRNA
INTRODUCTION
Follicle stimulating hormone (FSH) stimulation results in maturation of the ovarian follicle from
a preantral to a preovulatory state (1,2). This maturation is characterized by differentiation and
proliferation of the granulosa cells (GCs). This is accomplished through regulation of over 500 target
genes. Additionally, FSH promotes an increase in translation through activation of many essential
translation proteins (reviewed in (3)).
FSH binds to its G protein coupled receptor FSHR that is only expressed on GCs. Activation of
the FSHR results in stimulation of adenylyl cyclase and an increase in intracellular levels of cAMP. The
primary role of cAMP within GCs is to bind the regulatory subunit of protein kinase A (PKA) (4),
releasing the active catalytic subunits. Active PKA can directly phosphorylate multiple targets, such as
cAMP-response binding element protein (CREB) and β-catenin that function as transcriptional activators,
and can activate multiple signaling pathways, such as extracellular regulated kinase (ERK) signaling
cascade (5,6) and phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT) signaling cascade (7,8).
Recent studies from our lab have shown that FSH promotes the PKA-dependent phosphorylation
of insulin receptor substrate 1 (IRS1) on select tyrosine resides to which PI3K binds (7). Binding of the
regulatory subunit of the PI3K heterodimer to phosphorylated tyrosine resides on IRS1 is sufficient to
activate the catalytic activity of PI3K (9). The resulting generation of phosphatidylinositol 3,4,5-tris
phosphate (PIP3) brings AKT to the plasmas membrane, where it is phosphorylated by proline dependent
kinase 1 (PDK1) on Thr308 and by mTORC2(mTOR/Rictor) on Ser473, resulting in full activation of AKT.
Activated AKT then phosphorylates and inactivates the GTPase activating protein (GAP) protein tuberin.
Active tuberin stimulates the conversion of active Ras homolog enriched in brain (RHEB)GTP to inactive
RHEBGDP by enhancing the GTPase activity of RHEB (10). Inactivation of tuberin by AKT results in
115
accumulation of active RHEBGTP that in turn promotes the phosphorylation/activation of mTOR/Raptor
(mTORC1). mTORC1 then stimulates phosphorylation of p70 S6 kinase (p70S6K) and the binding
protein of eIF4E (4EBP1), both of which regulate translation (11). Active p70S6K promotes
phosphorylation of the ribosomal protein S6, a member of the 40S ribosomal subunit complex (12).
Phosphorylation of 4EBP1 results in release of eIF4E, allowing eIF4E to bind the 5’methyl cap of
mRNAs, stimulating assembly of the initiation factor complex and cap-dependent translation (13).
FSH stimulated phosphorylation of tuberin(Thr1462) is blocked by the PI3K inhibitor wortmannin
and is thus PI3K dependent, and occurs within 10 min of treatment. FSH stimulated phosphorylation of
p70S6K(Thr389) and its target S6(Ser235/Ser236) are also PI3K dependent as well as mTOR dependent
based on the inhibition by wortmannin or rapamycin, respectively (14). Through regulation of at least
these two proteins, FSH is able to stimulate translation of mRNAs.
Translation consists of three steps: initiation, elongation and termination. All three steps are
tightly regulated, but initiation of translation is the rate-limiting step overall, and thus is more highly
regulated than elongation and termination (15). Initiation is canonically activated through a capdependent mechanism following necessary processing of pre-mRNA such as removing introns and
addition of the 5’ 7-methylguanylate (m7) cap and the poly-(A) tail. The rate-limiting step of initiation is
eIF4E binding to the 5’ cap, due to limiting amounts of eIF4E protein (16). Following binding of the
scaffolding protein eIF4G and the ATP-dependent RNA helicase eIF4A, the 40S ribosomal subunit is
recruited to the complex and will scan the 5’ untranslated region until the initiation AUG codon is
encountered (17). A stable circular mRNA ring is formed by eIF4G binding to poly(A) binding protein 1
(PABP1), linking together the 5’ cap and the 3’ poly(A) tail (18). Once the initiation AUG codon is
recognized, scanning by the 40S ribosome is arrested, the 60S ribosomal subunit joins the complex and
translation of mRNA begins (19).
In addition to the regulation of translation itself, the availability of mRNAs is also regulated
through interactions with mRNA binding proteins. Y box binding protein-1 (YB-1) is a mRNA binding
116
protein that was first identified as a major component of the cytoplasmic messenger ribonuclear protein
(mRNP) complex (20,21) and has since been shown to be able to both promote and inhibit translation of
specific mRNAs depending on the YB-1/mRNA ratio (22,23). Additionally, YB-1 is able to melt mRNA
secondary structure and to accelerate annealing (22,24).
Herein, we identify YB-1 as an mRNA binding protein in GCs. Using an m7GTP-Sepharose bead
to isolate proteins bound to the 5’ cap, we detected increased YB-1 protein binding to the initiation factor
complex following FSH stimulation. Utilizing Flag-tagged YB-1, we were able to detect an FSHindependent interaction between YB-1 and PABP1, but not between YB-1 and eIF4E. We detected
increased levels of Egr1, but not of Hif1α mRNAs, bound to YB-1 in response to FSH. Together, these
results indicate that in GCs, YB-1 is interacting with other mRNA binding proteins, and in response to
FSH, increases binding to mRNA of at least one immediate early gene induced by FSH, Egr1.
EXPERIMENTAL PROCEDURES
Materials – The following were purchased: ovine FSH (oFSH-19) from the National Hormone
and Pituitary Agency of the National Institute of Diabetes and Digestive and Kidney Diseases (Torrence,
CA); anti–Flag (F3165) from Sigma–Aldrich; human fibronectin from BD Biosciences; antiphospho-YB1 (Ser102; CST 2900), anti-YB-1 (CST 4202), anti-PABP1 (CST 4992), anti-eIF4E (CST 2067); Protein
A/G PLUS Agarose (SC 2003) from Santa Cruz Biotechnology; m7GTP-4B Sepharose beads from GE
Heathcare; TRIzol-RNA Lysis Reagent and Fast SYBR Green Master Mix from Life Technologies;
qScript from VWR. Animals - Sprague-Dawley, CD-outbred rats (breeders from Charles River Laboratories) were
from a breeder colony maintained by our laboratory in a pathogen-free facility at Washington State
University. The facility is maintained in accordance with the Guidelines for the Care and Use of
Laboratory Animals using protocols approved by the Washington State University Animal Care and Use
Committee.
117
GC Cultures and Western blotting – Immature female rats were primed with subcutaneous
injections of 1.5 mg estradiol-17β (E2) in propylene glycol on days 21-23. Ovaries were collected
following three days of injections. GCs were collected by puncturing individual follicles using 27-gauge
needles (25). Cells were plated on fibronectin-coated plates at a density of ~1x106 cells/mL of serum–free
media supplemented with 1 nM E2, 100 U/mL penicillin (P), and 100 µg/mL streptomycin (S). Indicated
treatments were added to cells ~20 h following plating, and terminated by aspirating media and washing
once with PBS . Samples were collected by scrapping cells in a SDS sample buffer (26) at 50 µL/1x106
cells, followed by heat denaturation. Proteins were separated by SDS/PAGE and transferred onto Protran
(Amersham Biosciences) nitrocellulose membrane (27). The membrane was incubated overnight at 4°C
with primary antibody, and enhanced chemiluminescence (ThermoFisher) was used to detect the antigenantibody complexes. Westerns were scanned using an Epson Perfection V500 scanner and Adobe
Photoshop CS2 9.0 software with minimal processing and quantified using Quantity One software
(BioRad Laboratories).
Transfection of GCs - Cells were plated on fibronectin-coated plates at a density of 1x106
cells/mL in OptiMEM + P/S + E2 with expression constructs (250 ng/ 1x106 cells) and transfected using
LipofectAMINE 2000 (Invitrogen) according to manufacturer’s instructions. After 6 h incubation, the
media was removed and fresh DMEM +P/S +E2 was added. Cells were treated as indicated following ~16
h recovery period. Cells collected for immunoprecipitation or immunoprecipitation – RNA isolation.
Immunoprecipitation and Pulldowns – Immunoprecipitations (IP) and pulldowns were performed
as previously described (7,28). Briefly, ~107 cells were scraped into 0.5 mL IP Lysis buffer [50 mM
Hepes, pH 7.0, 100 mM NaCl, 0.5% Nonidet P-40, 20 mM NaF, 2 mM NaVO3, 2 mM Na4P2O7, 5 mM
EGTA, 5 mM EDTA, 20 mM benzamidine, 10 μg/mL calpain inhibitor III, 50 μg/mL antipain, 50 μg/mL
soybean trypsin inhibitor, 10 μM isomethylbutylxanthine, Halt protease inhibitor mixture [1 mM 4-(2aminoethyl) benzenesulfonyl fluoride hydrochloride, 0.8 μM aprotinin, 50 μM bestatin, 15 μM E-64, 20
μM leupeptin, 10 μM pepstatin; (Thermo Fischer Scientific)], sonicated twice on ice for 45 s, and
118
clarified by centrifugation at 10, 000 x g for 5 min at 4°C. Following removal of 0.03 mL sample for
input, soluble cell extracts were precleared using species matched preclearing matrix (ExactaCruz
Preclearing Matrix, Santa Cruz Biotechnology) for >60 min at 4°C if necssary. Samples were incubated
overnight by rotating at 4°C with either (i) the specified antibodies and Protein A/G PLUS Agarose
(Santa Cruz Biotechnology; SC 2003), along with a species matched IgG antibody control (Santa Cruz
Biotechnology) for immunoprecipitations or with (ii) 7-methyl-GTP-Sepharose 4B beads (GE Heathcare)
for pull downs. Following centrifugation at 10,000 x g for 5 min at 4°C, 0.4 mL of the supernatant
fraction, representing the unbound protein fraction, was collected in SDS sample buffer. Beads were
washed 4 times by rotating for 5 min at 4°C with 1 mL IP Wash Buffer (20 mM Hepes, pH 7.0, 150 mM
NaCl, 10% glycerol, and 0.1% Triton X-100). Bound proteins were collected in SDS sample buffer and
subjected to SDS/PAGE and western blotting. Inputs represent ~2%, and bound sample ~98% of total
sample.
Immunoprecipitation-RNA Isolation and Real–time PCR - Protocol was adapted from a previous
report (29), as detailed below. GCs (~107) were scraped into 0.25 mL Polysome Lysis Buffer (50 mM
Tris, pH 7.5, 100 mM KCl, 12 mM MgCl2, 1% Nonidet P-40, 1 mM DTT, 200 U/mL Promega RNasin, 1
mg/mL heparin, 100 µg/mL cycloheximide, Sigma protease inhibitor mixture). Samples were subjected to
six rounds of freeze/thaw to lyse cells. Lysates were clarified by centrifugation at 10, 000 x g for 10 min
at 4°C. Following removal of 0.03 mL for input, samples were incubated overnight by rotating at 4°C
with 10 µg antibody and protein G magnetic beads (Dynabeads; Invitrogen). Following collection of the
immunoprecipitated complexes using a magnetic stand, the beads were washed three times by rotating for
5 min at 4°C with 0.4 mL High Salt Wash (50 mM Tris, pH 7.5, 300 mM KCl, 12 mM MgCl2, 1%
Nonidet P-40, 1 mM DTT, 100 µg/mL cycloheximide). Input and bead (IP) samples were subject to
DNase (Thermo Scientific) treatment per manufacturer’s instructions. Qiagen RLT Buffer (0.07 mL) was
added to samples and RNA was isolated according to manufacturer’s instructions using the RNeasy
MinElute Kit (Qiagen). RNA concentration was determined using a NanoDrop 2000. cDNA was
119
generated using qScript per manufacturer’s instructions. Real time-PCR was performed using Fast SYBR
Green Master Mix per manufacturer’s instructions. Primers used were: Hif1α forward 5’ GAT CAG TTG
TCA CCA TTA GAG AGC A 3’ reverse 5’ TGG GTC TGC TGC AAT CCT GTA 3’; Egr1 forward 5’
CCT TTT GCC TGT GAC ATT TGT G 3’ reverse 5’ TGG TAT GCC TCT TGC GTT CA 3’. RESULTS
YB-1 interacts with 5’cap and initiation factor complex. YB-1 has been described as an mRNA
binding protein, and shown to be a regulator of translation (30-33). We sought to determine if total YB-1
is interacting with the translational protein complex in GCs. In these experiments, we utilized m7GTPsepharose beads that mimic the m7GTP of the 5’ end of mRNA (34,35). The initiation factor proteins will
assemble around m7GTP, allowing for isolation of the initiation factor complex and study of the initiation
complex of translation under different treatment groups. GCs were treated without or with FSH for the
indicated times, and the initiation factor complex was pulled down using m7GTP-Sepharose beads. At all
timepoints checked, FSH promotes an increase in total YB-1 bound to m7GTP (Fig. 1A, right panel),
while total YB-1 remained unchanged (Fig. 1A left panel). YB-1(Ser102) phosphorylation has been shown
to be important for translation in breast cancer cells (30). Although we observed an increase in YB1(Ser102) phosphorylation in total GC extracts at 1 and 4 h in response to FSH (Fig. 1A left panel),
phosphorylated YB-1(Ser102) bound to m7GTP and the initiation factor complex was barely detectable 1 h
and undetectable 4 h post FSH treatment (Fig. 1A right panel). Because eIF4E is the first initiation factor
to directly bind the 5’ cap, eIF4E is used as a positive control for m7GTP sepharose bead pull down. To
verify these results, GCs were transfected with Flag-tagged YB-1, treated with FSH for 60 min, and the
initiation factor complex was pulled down using m7GTP-Sepharose. Using an anti-Flag antibody to
detect Flag-tagged YB-1, results (Fig. 1B) show that FSH promotes an increase in Flag-tagged YB-1
bound to m7GTP. eIF4E was used as a positive control for the m7GTP pull down. Taken together, these
results indicate that FSH promotes and increase in YB-1 associated with m7GTP, the 5’ cap, and the
initiation factor complex.
120
YB-1 binds to PABP1, but not eIF4E. YB-1 has been shown to interact with both the initiation
factor complex proteins (31), as well as with the general mRNA binding proteins (36). Based on these
results, we sought to determine if YB-1 interacts with any other mRNA binding proteins in GCs, and if
binding is FSH dependent. GCs were transfected with Flag-tagged YB-1, then treated without and with
FSH. YB-1 was immunoprecipitated from lysates using anti-Flag antibody. In contrast to the m7GTPSepharose pull down results, eIF4E was not detected bound to YB-1-Flag. Interestingly, PABP1 was
found to selectively bind to YB-1-Flag in both vehicle- and FSH-stimulated samples. These results
indicate that YB-1 and PABP1 interact within GCs independently of FSH.
YB-1 binding to mRNAs increases in response to FSH-stimulation. Because YB-1 has been
identified as an mRNA binding protein (31,37,38), and we’ve shown it binding to PABP1 in GCs, we
sought to determine if YB-1 binding to mRNA increases in response to FSH treatment. GCs were
transfected with Flag-tagged YB-1 and treated with FSH. RNA was isolated from YB-1-Flag
immunoprecipitates, and mRNAs were analyzed using real-time PCR. Protein samples were saved to
verify immunoprecipitation of Flag-YB-1 (Fig. 3A). As shown in Fig. 3B, Egr1 mRNA levels from input
samples increased in response to FSH while HIF1α mRNA levels remained constant, consistent with
previous results (39) and (14), respectively. Results show that YB-1 binds HIF1α and Egr1 mRNAs in
GCs at increased amounts relative to the IgG control. Additionally, FSH promotes an increase in Egr1
mRNA bound to YB-1. These results indicated that in addition to binding PABP1, YB-1 also binds
mRNAs within GCs. In response to FSH, Egr1 mRNA is bound at higher amounts to YB-1.
DISCUSSION
Our results show that YB-1 interacts with components of the initiation factor complex, and
exhibits binding to HIF1α and Egr1 mRNA. These results indicate a possible role of YB-1 in translational
regulation in response to FSH in GCs.
121
YB-1 is a nucleic acid binding protein that has many functions within the cell due to its
interactions with both DNA and RNA (reviewed in (40)). Through interactions with RNA, YB-1 is
associated with both nontranslated (free) and translating (polysomal) mRNA, and is a component of the
mRNP complex along with PABP (22,36,41).
Our results show that FSH promotes an increase in the binding of total YB-1 to the m7GTPSepharose 5’-cap and the initiation factor complex. Surprisingly, however, phosphorylated YB-1(Ser102)
is barely detectable in the m7GTP-Sepharose pull down. This result is contradictory to previous studies
that have reported that YB-1(Ser102) phosphorylation is necessary for YB-1 binding to mRNA (30,31).
However, YB-1 can be phosphorylated on multiple Ser and Tyr residues (30,42), so it is possible that
FSH is promoting phosphorylation of a different residue that increases its binding to the initiation factor
complex.
While it is possible that FSH is promoting phosphorylation of additional sites on YB-1, previous
fractionation results from our lab showed that the majority of YB-1 phosphorylated on Ser102 localizes to
the cytoplasmic fraction (see Chapter 3, (39)). If YB-1 phosphorylated on Ser102 is not interacting with the
initiation factor complex, it is possible that cytosolic YB-1(Ser102) phosphorylation is important for
mRNA stability. At high YB-1/mRNA ratios, YB-1 proteins cover the mRNA strand and block other
proteins from binding, thus both protecting the mRNA from degradation and blocking translation
(through obstructed binding of eIF4E). At low YB-1/mRNA ratios, YB-1 is thought to release portions of
the mRNA, including the 5’cap, perhaps promoting binding of eIF4E (37,41). So it is likely that YB1(Ser102) may be participating in mRNA stability.
This hypothesis is further verified by our YB-1 immunoprecipitation results. We report that the
interaction between YB-1 and PABP1 is FSH-independent, indicating a stable interaction. As the two
main components of mRNPs, YB-1 and PABP interact with each other and with mRNA (36).
Additionally, we do not detect YB-1 binding to eIF4E, indicating that the binding of YB-1 seen in the
m7GTP-Sepharose pull down may be indirect. The initiation factor proteins and mRNA form a stable
122
circular complex (18), so it is possible that YB-1 binding to the mRNA or other proteins in the complex
would be detected with the m7GTP-Sepharose pull down. Perhaps YB-1 proteins bind to the 3’ UTR and
poly(A) tail of mRNA, as the YB-1 immunoprecipitation data suggests. The consistent interaction
between PABP1 and YB-1 indicates YB-1 may be acting on mRNA stability, rather than binding to
eIF4E and acting in the initiation of translation.
Our results also show that in GCs, YB-1 is bound to HIF1α and Egr1 mRNAs, and that FSH
enhances the binding of total YB-1 protein to Egr1 mRNA. As FSH promotes an increase in the
transcription of Egr1 mRNA (see Fig 3B, (43)), we cannot determine if the increased binding of YB-1 to
Egr1 mRNA is only due to the increase in Egr1 mRNA or if FSH is promoting YB-1 to bind more
mRNAs, including Egr1. However, based on the ability of YB-1 versus control IgG to bind increased
levels of HIF1α mRNA in an FSH-independent manner, results indicate that YB-1 selectively binds
mRNAs in GCs.
Cytoplasmic YB-1 has been localized to both nontranslated (free) mRNPs and translating
(polysomal) mRNPs (30). Bound to mRNAs and possibly part of the mRNP complex in GCs, YB-1 might
be facilitating stability of mRNAs and participating in the movement of mRNAs from nontranslated to
polysomal mRNPs. Following an increase in translation in response to FSH, YB-1 containing mRNPs
may move to the polysomal mRNPs and interact with the initiation factor complex proteins.
These preliminary results indicate that YB-1 interacts with the initiation factor complex, and its
binding increases with FSH treatment. Further studies are needed to determine the role that YB-1 plays
following mRNA binding in response to FSH. Many roles have been determined for YB-1 bound to
mRNA (reviewed in (40)), and it is likely that more than one apply to YB-1 binding mRNA in response
to FSH.
123
CONFLICT OF INTEREST: The authors declare that they have no conflicts of interest with the
contents of this article.
AUTHOR CONTRIBUTIONS: EMD designed and performed the experiments. EMD and MEH-D
analyzed the data and wrote the manuscript. 124
REFERENCES
1.
Abel, M. H., Wootton, A. N., Wilkins, V., Huhtaniemi, I., Knight, P. G., and Charlton, H. M.
(2000) The effect of a null mutation in the follicle-stimulating hormone receptor gene on mouse
reproduction. Endocrinology 141, 1795-1803
2.
Richards, J. S., Fitzpatrick, S. L., Clemens, J. W., Morris, J. K., Alliston, T., and Sirois, J. (1995)
Ovarian cell differentiation: a cascade of multiple hormones, cellular signals, and regulated
genes. Recent progress in hormone research 50, 223-254
3.
Hunzicker-Dunn, M., and Mayo, K. E. (2015) Gonadotropin Signaling in the Ovary. in KNOBIL
AND NEILL’S PHYSIOLOGY OF REPRODUCTION (Plant, T. M., and Zeleznik, A. J. eds.),
Fourth Ed., Elsevier. pp 895 - 946
4.
Taylor, S. S. (1989) cAMP-dependent protein kinase. Model for an enzyme family. J. Biol. Chem.
264, 8443-8446
5.
Cottom, J., Salvador, L. M., Maizels, E. T., Reierstad, S., Park, Y., Carr, D. W., Davare, M. A.,
Hell, J. W., Palmer, S. S., Dent, P., Kawakatsu, H., Ogata, M., and Hunzicker-Dunn, M. (2003)
Follicle-stimulating hormone activates extracellular signal-regulated kinase but not extracellular
signal-regulated kinase kinase through a 100-kDa phosphotyrosine phosphatase. J. Biol. Chem.
278, 7167-7179
6.
Fan, H. Y., Liu, Z., Shimada, M., Sterneck, E., Johnson, P. F., Hedrick, S. M., and Richards, J. S.
(2009) MAPK3/1 (ERK1/2) in ovarian granulosa cells are essential for female fertility. Science
324, 938-941
7.
Hunzicker-Dunn, M. E., Lopez-Biladeau, B., Law, N. C., Fiedler, S. E., Carr, D. W., and Maizels,
E. T. (2012) PKA and GAB2 play central roles in the FSH signaling pathway to PI3K and AKT
in ovarian granulosa cells. Proceedings of the National Academy of Sciences of the United States
of America 109, E2979-2988
8.
Zeleznik, A. J., Saxena, D., and Little-Ihrig, L. (2003) Protein kinase B is obligatory for folliclestimulating hormone-induced granulosa cell differentiation. Endocrinology 144, 3985-3994
9.
Law, N. C., and Hunzicker-Dunn, M. E. (2015) Insulin Receptor Substrate 1: The Hub Linking
Follicle-stimulating Hormone to Phosphatidylinositol-3 Kinase Activation. J. Biol. Chem.
10.
Huang, J., and Manning, B. D. (2008) The TSC1-TSC2 complex: a molecular switchboard
controlling cell growth. Biochem. J. 412, 179-190
125
11.
Hay, N., and Sonenberg, N. (2004) Upstream and downstream of mTOR. Genes Dev. 18, 19261945
12.
Isotani, S., Hara, K., Tokunaga, C., Inoue, H., Avruch, J., and Yonezawa, K. (1999)
Immunopurified mammalian target of rapamycin phosphorylates and activates p70 S6 kinase
alpha in vitro. J. Biol. Chem. 274, 34493-34498
13.
Schalm, S. S., Fingar, D. C., Sabatini, D. M., and Blenis, J. (2003) TOS motif-mediated raptor
binding regulates 4E-BP1 multisite phosphorylation and function. Curr. Biol. 13, 797-806
14.
Alam, H., Maizels, E. T., Park, Y., Ghaey, S., Feiger, Z. J., Chandel, N. S., and Hunzicker-Dunn,
M. (2004) Follicle-stimulating hormone activation of hypoxia-inducible factor-1 by the
phosphatidylinositol 3-kinase/AKT/Ras homolog enriched in brain (Rheb)/mammalian target of
rapamycin (mTOR) pathway is necessary for induction of select protein markers of follicular
differentiation. J. Biol. Chem. 279, 19431-19440
15.
Jackson, R. J., Hellen, C. U., and Pestova, T. V. (2010) The mechanism of eukaryotic translation
initiation and principles of its regulation. Nat. Rev. Mol. Cell Biol. 11, 113-127
16.
von der Haar, T., Gross, J. D., Wagner, G., and McCarthy, J. E. (2004) The mRNA cap-binding
protein eIF4E in post-transcriptional gene expression. Nat. Struct. Mol. Biol. 11, 503-511
17.
Jackson, R. J. (2005) Alternative mechanisms of initiating translation of mammalian mRNAs.
Biochem. Soc. Trans. 33, 1231-1241
18.
Wells, S. E., Hillner, P. E., Vale, R. D., and Sachs, A. B. (1998) Circularization of mRNA by
eukaryotic translation initiation factors. Mol. Cell 2, 135-140
19.
Sonenberg, N., and Hinnebusch, A. G. (2009) Regulation of translation initiation in eukaryotes:
mechanisms and biological targets. Cell 136, 731-745
20.
Blobel, G. (1972) Protein tightly bound to globin mRNA. Biochem. Biophys. Res. Commun. 47,
88-95
21.
Morel, C., Kayibanda, B., and Scherrer, K. (1971) Proteins associated with globin messenger
RNA in avian erythroblasts: Isolation and comparison with the proteins bound to nuclear
messenger-likie RNA. FEBS Lett. 18, 84-88
22.
Evdokimova, V. M., Wei, C. L., Sitikov, A. S., Simonenko, P. N., Lazarev, O. A., Vasilenko, K.
S., Ustinov, V. A., Hershey, J. W., and Ovchinnikov, L. P. (1995) The major protein of
126
messenger ribonucleoprotein particles in somatic cells is a member of the Y-box binding
transcription factor family. J. Biol. Chem. 270, 3186-3192
23.
Evdokimova, V. M., Kovrigina, E. A., Nashchekin, D. V., Davydova, E. K., Hershey, J. W., and
Ovchinnikov, L. P. (1998) The major core protein of messenger ribonucleoprotein particles (p50)
promotes initiation of protein biosynthesis in vitro. J. Biol. Chem. 273, 3574-3581
24.
Skabkin, M. A., Evdokimova, V., Thomas, A. A., and Ovchinnikov, L. P. (2001) The major
messenger ribonucleoprotein particle protein p50 (YB-1) promotes nucleic acid strand annealing.
J. Biol. Chem. 276, 44841-44847
25.
Carr, D. W., DeManno, D. A., Atwood, A., Hunzicker-Dunn, M., and Scott, J. D. (1993) Folliclestimulating hormone regulation of A-kinase anchoring proteins in granulosa cells. J. Biol. Chem.
268, 20729-20732
26.
Hunzicker-Dunn, M. (1981) Selective activation of rabbit ovarian protein kinase isozymes in
rabbit ovarian follicles and corpora lutea. J. Biol. Chem. 256, 12185-12193
27.
Salvador, L. M., Park, Y., Cottom, J., Maizels, E. T., Jones, J. C., Schillace, R. V., Carr, D. W.,
Cheung, P., Allis, C. D., Jameson, J. L., and Hunzicker-Dunn, M. (2001) Follicle-stimulating
hormone stimulates protein kinase A-mediated histone H3 phosphorylation and acetylation
leading to select gene activation in ovarian granulosa cells. J. Biol. Chem. 276, 40146-40155
28.
Law, N. C., and Hunzicker-Dunn, M. E. (2015) Insulin Receptor Substrate 1: the Hub Linking
Follicle-stimulating Hormone to Phosphatidylinositol-3 Kinase Activation J. Biol. Chem. in press
29.
Sanz, E., Yang, L., Su, T., Morris, D. R., McKnight, G. S., and Amieux, P. S. (2009) Cell-typespecific isolation of ribosome-associated mRNA from complex tissues. Proceedings of the
National Academy of Sciences of the United States of America 106, 13939-13944
30.
Evdokimova, V., Ruzanov, P., Anglesio, M. S., Sorokin, A. V., Ovchinnikov, L. P., Buckley, J.,
Triche, T. J., Sonenberg, N., and Sorensen, P. H. (2006) Akt-mediated YB-1 phosphorylation
activates translation of silent mRNA species. Mol. Cell. Biol. 26, 277-292
31.
Evdokimova, V., Ruzanov, P., Imataka, H., Raught, B., Svitkin, Y., Ovchinnikov, L. P., and
Sonenberg, N. (2001) The major mRNA-associated protein YB-1 is a potent 5' cap-dependent
mRNA stabilizer. EMBO J. 20, 5491-5502
127
32.
Skabkin, M. A., Kiselyova, O. I., Chernov, K. G., Sorokin, A. V., Dubrovin, E. V., Yaminsky, I.
V., Vasiliev, V. D., and Ovchinnikov, L. P. (2004) Structural organization of mRNA complexes
with major core mRNP protein YB-1. Nucleic Acids Res. 32, 5621-5635
33.
Svitkin, Y. V., Evdokimova, V. M., Brasey, A., Pestova, T. V., Fantus, D., Yanagiya, A.,
Imataka, H., Skabkin, M. A., Ovchinnikov, L. P., Merrick, W. C., and Sonenberg, N. (2009)
General RNA-binding proteins have a function in poly(A)-binding protein-dependent translation.
EMBO J. 28, 58-68
34.
Sonenberg, N., Rupprecht, K. M., Hecht, S. M., and Shatkin, A. J. (1979) Eukaryotic mRNA cap
binding protein: purification by affinity chromatography on sepharose-coupled m7GDP.
Proceedings of the National Academy of Sciences of the United States of America 76, 4345-4349
35.
Altmann, M., Edery, I., Sonenberg, N., and Trachsel, H. (1985) Purification and characterization
of protein synthesis initiation factor eIF-4E from the yeast Saccharomyces cerevisiae.
Biochemistry 24, 6085-6089
36.
Lyabin, D. N., Eliseeva, I. A., Skabkina, O. V., and Ovchinnikov, L. P. (2011) Interplay between
Y-box-binding protein 1 (YB-1) and poly(A) binding protein (PABP) in specific regulation of
YB-1 mRNA translation. RNA biology 8, 883-892
37.
Davydova, E. K., Evdokimova, V. M., Ovchinnikov, L. P., and Hershey, J. W. (1997)
Overexpression in COS cells of p50, the major core protein associated with mRNA, results in
translation inhibition. Nucleic Acids Res. 25, 2911-2916
38.
Minich, W. B., and Ovchinnikov, L. P. (1992) Role of cytoplasmic mRNP proteins in translation.
Biochimie 74, 477-483
39.
Donaubauer, E. M., and Hunzicker-Dunn, M. Extracellular signal-regulated kinase (ERK)–
dependent phosphorylation of Y-Box binding protein 1 (YB1) enhances gene expression in
granulosa cells in response to follicle stimulating hormone (FSH). J. Biol. Chem. in revision
40.
Eliseeva, I. A., Kim, E. R., Guryanov, S. G., Ovchinnikov, L. P., and Lyabin, D. N. (2011) Ybox-binding protein 1 (YB-1) and its functions. Biochemistry. Biokhimiia 76, 1402-1433
41.
Evdokimova, V. M., and Ovchinnikov, L. P. (1999) Translational regulation by Y-box
transcription factor: involvement of the major mRNA-associated protein, p50. Int J Biochem Cell
Biol 31, 139-149
128
42.
Coles, L. S., Lambrusco, L., Burrows, J., Hunter, J., Diamond, P., Bert, A. G., Vadas, M. A., and
Goodall, G. J. (2005) Phosphorylation of cold shock domain/Y-box proteins by ERK2 and
GSK3beta and repression of the human VEGF promoter. FEBS Lett. 579, 5372-5378
43.
Russell, D. L., Doyle, K. M., Gonzales-Robayna, I., Pipaon, C., and Richards, J. S. (2003) Egr-1
induction in rat granulosa cells by follicle-stimulating hormone and luteinizing hormone:
combinatorial regulation by transcription factors cyclic adenosine 3',5'-monophosphate regulatory
element binding protein, serum response factor, sp1, and early growth response factor-1. Mol.
Endocrinol. 17, 520-533
129
FIGURE LEGENDS AND FIGURES
FIGURE 1. YB-1 interacts with 5’cap and initiation factor complex. (A) GCs were treated without (veh)
or with FSH for the indicated times, and then collected in Immunoprecipitation Lysis buffer and
sonicated. After removal of the insoluble particles, the soluble fraction was rotated overnight with 20 µg
m7GTP-Sepharose 4B beads (GE Healthcare) as described in Experimental Procedures. Inputs, bound
(pulldown) and unbound (flow – though) samples were separated by SDS/PAGE and the western blot was
probed with the indicated antibodies. Arrows indicate the protein band of interest. Blots are representative
of three independent experiments. (B) GCs were transfected with 500 ng YB-1 – Flag plasmid for 6 h as
described in Experimental Procedures. Following an overnight recovery, GCs were treated without (veh)
or with FSH for 60 min, then collected as described in 1A. Blots are representative of two independent
experiments. Dotted lines between lanes represent cropped images. Results are representative of two
independent experiments.
130
131
FIGURE 2. YB-1 binds to PABP1, but not eIF4E. (C) GCs were transfected with 500 ng YB-1 – Flag
plasmid for 6 h as described in Experimental Procedures. Following an overnight recovery, GCs were
treated without (veh) or with FSH for 60 min, and then collected in Immunoprecipitation Lysis buffer and
sonicated. After removal of the insoluble particles, the soluble fraction was rotated overnight with 20 µg
Anti–Flag (Sigma F3165) as described in Experimental Procedures. Inputs, bound (IP) and unbound
(flow – though) samples were separated by SDS/PAGE and the western blot was probed with the
indicated antibodies. Arrows indicate the protein band of interest. Results are representative of two
independent experiments.
132
133
FIGURE 3. YB-1 binding to mRNAs increases in response to FSH-stimulation. GCs were transfected
with 500 ng YB-1 – Flag plasmid for 6 h as described in Experimental Procedures. Following an
overnight recovery, GCs were treated without (veh) or with FSH for 1 h, and then collected in
Immunoprecipitation Lysis buffer and sonicated. After removal of the insoluble particles, the soluble
fraction was rotated overnight with 20 µg Anti–Flag (Sigma F3165). Protein (A) and RNA (inputs are B
and IP are C) were collected from the samples as described in Experimental Procedures. (A) Inputs,
bound (IP) and unbound (flow – though) protein samples were separated by SDS/PAGE and the western
blot was probed with the indicated antibodies. Blot is representative of three independent experiments.
(B/C) Real–time PCR was performed on input (B) and IP (C) samples for the genes indicated as described
in Experimental Procedures. Quantification results represent the mean ± SEM of three independent
experiments with (*) representing p < 0.05 and (**) representing p < 0.01, based on a one tailed Students
t test.
134
135
CHAPTER FIVE
CONCLUSIONS AND FUTURE DIRECTIONS
CONCLUSIONS
In this document, we show that FSH dependent ERK/RSK signaling to downstream target YB-1
is necessary for induction of gene targets that include Egfr, Inha, and Cyp19a1that are required for
follicular maturation.
In GCs, ERK(Thr202/Tyr204) phosphorylation requires both the constitutively active upstream
components of canonical MEK signaling and the FSH-dependent inhibition of a DUSP, most likely
MKP3(DUSP6). FSH-stimulated ERK(Thr202/Tyr204) phosphorylation is PKA-dependent, indicating that
FSH via PKA is inhibiting a phosphatase, allowing for MEK-dependent ERK(Thr202/Tyr204
MKP3(DUSP6) phosphorylation to accumulate. Using multiple inhibitors, our results suggest that
MKP3(DUSP6) is dephosphorylating ERK in the absence of FSH. Following treatment with the
MKP3(DUSP6) inhibitor, NSC 295624, ERK(Thr202/Tyr204) phosphorylation increased in the absence of
FSH to levels comparable of ERK phosphorylated in the presence of FSH. ERK2 co-immunoprecipitates
with MKP3(DUSP6) , and treatment of GCs with MKP3(DUSP6) inhibitors block dephosphorylation of
recombinant ERK2-GST. Current studies aim to show an increase in ERK(Thr202/Tyr204) phosphorylation
in the absence of FSH due to selective knockdown of MKP3(DUSP6) using a MKP3(DUSP6) shRNA
(1).
We next show that YB-1 is present in GCs and, in response to FSH stimulation, is phosphorylated
on Ser102. YB-1(Ser102) phosphorylation is first detectable after approximately 15 min and lasts
approximately 8 hr following FSH treatment. This phosphorylation is both PKA- and ERK- dependent,
based on loss of FSH-dependent YB-1(Ser102) phosphorylation following treatment with selective PKA
and MEK inhibitors, respectively. Treatment with the RSK inhibitor BI-D1870 results in loss of FSH-
136
stimulated YB-1(Ser102) phosphorylation, demonstrating RSK dependence. Thus, we place FSHstimulated YB-1(Ser102) phosphorylation downstream of PKA and ERK/RSK signaling in GCs.
In order to identify YB-1-dependent genes, we first identify Egfr, Inha, Cyp19a1, Ereg, and Egr1
as ERK gene targets using MEK inhibitors. We then identify Egfr, Inha, and Cyp19a1 as YB-1 gene
targets using adenoviral YB-1(Ser102) mutants. Transduction of GCs with the dephospho-mutant AdYB1(S102A) results in a loss of FSH-dependent induction of Egfr, Inha, and Cyp19a1 mRNA. Finally,
GCs transfected with promoter-luciferase constructs, followed by transduction with Ad-YB1(S102A),
show inhibition of Egfr, Cyp19a1, and Inha promoter – luciferase activities. These results indicate that
YB-1(Ser102) interacts with these gene promoters directly. Collectively, these results demonstrate a FSHdependent role for YB-1 phosphorylated on Ser102 in regulating the expression of gene targets necessary
for follicular maturation.
Lastly, we show YB-1 interacts with the initiation factor complex, as it pulls down with m7GTPSepharose beads in an FSH-dependent manner and with PABP in an FSH-independent manner.
Additionally, YB-1 binds to HIF1α and Egr1 mRNA over IgG control, and YB-1 bound to Egr1 mRNA
increases following FSH treatment. Taken together, these data place YB-1 binding to mRNAs and mRNA
binding proteins, indicating a possible role for YB-1 regulating mRNA stability or interacting with
initiation factor complex in GCs in response to FSH.
Collectively, these results demonstrate novel regulation of ERK(Thr202/Tyr204) phosphorylation
and identify novel ERK-dependent gene targets in GCs. Additionally, we identify YB-1(Ser102)
phosphorylation as being FSH- and ERK-dependent, and necessary for induction of FSH-dependent gene
expression that is crucial for follicular maturation.
FUTURE DIRECTIONS
137
For a multitude of reasons, there were questions we were unable to answer within the scope of
this research. I would now like to address questions that I would like to answer on the research presented
and additional questions I believe this research topic can still investigate.
I would like to further examine the PKA-dependence of MKP3(DUSP6) inhibition. Examination
of the protein sequence of MKP3(DUSP6) reveals a potential site for PKA phosphorylation (Thr302). First
co-immunoprecipitation experiments can be performed between PKA and MKP3(DUSP6) to determine if
the two proteins interact. I can also immunoprecipitate MKP3(DUSP6) from GCs treated with vehicle and
FSH, and probe with phospho-PKA substrate antibody to indicate that MKP3(DUSP6) is a PKA target in
GCs. Also, phosphorylation mimic (T302D) and dephospho (T302A)-mutations of this site can be
created, and the ability of the mutant to (i) interact with ERK and (ii) dephosphorylate pERK-GST can be
investigated. Finally, co-immunoprecipitation experiments could be performed between PKA and the
MKP3(DUSP6) mutants to determine if the mutation affects binding.
For YB-1, I would like to determine the regulatory protein that interacts with PP1cβ in GCs in
response to FSH and inhibits dephosphorylation of YB1(Ser102). RNA-seq data from our lab (Herndon et
al., MS in preparation) has identified 26 unique PP1 regulatory proteins that are expressed untreated GCs.
Individually examining each regulatory protein on this list for interactions with PP1 in GCs using coimmunoprecipitation experiments is daunting, because (i) of the large number of candidates, and (ii)
many of these regulatory proteins do not yet have readily available antibodies. I propose performing YB1-Flag immunoprecipitation experiments, and analyzing the pull down sample using mass-spec (PNNL).
Additionally, I would like to further investigate the role of YB-1 as a transcription factor in GCs.
Our results indicate YB-1 is binding to the promoters of Egfr, Cyp19a1, Inha using promoter-luciferase
constructs. I would like to confirm these results through ChIP experiments, and determine the specific
region of DNA that YB-1 is interacting with in these promoters. Furthermore, because these genes have
been shown to be regulated by many different transcription factors in GCs (2-6) and YB-1 has been
138
shown to bind other transcription factors (7,8), I would also like to investigate YB-1 binding to additional
transcription factors.
Finally, I would like to investigate the role of YB-1 in GCs in the regulation of translation in
response to FSH. We identified total YB-1 binding to the initiation factor complex, but not to eIF4E. I
would like determine if YB-1 could be interacting with any other members of the initiation factor
complex, such as eIF4G. More likely, YB-1 is binding directly to the mRNA, as it is a mRNA stabilizing
protein (9,10). I would like to use our adenoviral wild type, (S102A), and (S102D) YB-1 mutants to
further investigate the mRNA’s bound to YB-1 in vehicle and FSH-treated GCs at 1 vs 24 h post
treatment. Also, I would like to perform polyribosome isolations combined with YB-1-Flag, and
investigate the possibilty that YB-1 travels with mRNA as it moves from the monoribosomal (nontranslating) to the polyribosomal (translating) fractions (9). Our preliminary results indicate that
YB1(Ser102) phosphorylation is not important to YB-1 interacting with the translational machinery. I
would like to confirm these results using our adenoviral YB-1 mutants. I would also like to investigate if
FSH is causing the phosphorylation of YB-1 on one or more additional sites by submitting YB-1 from
vehicle and FSH treated cells to phospho-mass spec (PNNL). IF one or more additional sites are
identified Ser to Ala mutations could be created, and the binding of these mutants to mRNA, the initiation
factor complex, or the polyribosome could be investigated. There are many questions left to investigate
with regards to the role of YB-1 in translation in GCs in response to FSH.
In summary, the results presented in this document comprise the first investigation of YB-1 in
GCs, and there are still many questions left to be investigated. YB-1 has been shown to play a role in
many different cellular functions (reviewed in (11)), and investigating its role in GCs in response to FSH
open many opportunities for future investigations. 139
REFERENCES
1.
Wu, Z., Jiao, P., Huang, X., Feng, B., Feng, Y., Yang, S., Hwang, P., Du, J., Nie, Y., Xiao, G.,
and Xu, H. (2010) MAPK phosphatase-3 promotes hepatic gluconeogenesis through
dephosphorylation of forkhead box O1 in mice. J. Clin. Invest. 120, 3901-3911
2.
Ito, M., Park, Y., Weck, J., Mayo, K. E., and Jameson, J. L. (2000) Synergistic activation of the
inhibin alpha-promoter by steroidogenic factor-1 and cyclic adenosine 3',5'-monophosphate. Mol.
Endocrinol. 14, 66-81
3.
Carlone, D. L., and Richards, J. S. (1997) Functional interactions, phosphorylation, and levels of
3',5'-cyclic adenosine monophosphate-regulatory element binding protein and steroidogenic
factor-1 mediate hormone-regulated and constitutive expression of aromatase in gonadal cells.
Mol. Endocrinol. 11, 292-304
4.
Pei, L., Dodson, R., Schoderbek, W. E., Maurer, R. A., and Mayo, K. E. (1991) Regulation of the
alpha inhibin gene by cyclic adenosine 3',5'-monophosphate after transfection into rat granulosa
cells. Mol. Endocrinol. 5, 521-534
5.
Parakh, T. N., Hernandez, J. A., Grammer, J. C., Weck, J., Hunzicker-Dunn, M., Zeleznik, A. J.,
and Nilson, J. H. (2006) Follicle-stimulating hormone/cAMP regulation of aromatase gene
expression requires beta-catenin. Proceedings of the National Academy of Sciences of the United
States of America 103, 12435-12440
6.
Salvador, L. M., Park, Y., Cottom, J., Maizels, E. T., Jones, J. C., Schillace, R. V., Carr, D. W.,
Cheung, P., Allis, C. D., Jameson, J. L., and Hunzicker-Dunn, M. (2001) Follicle-stimulating
hormone stimulates protein kinase A-mediated histone H3 phosphorylation and acetylation
leading to select gene activation in ovarian granulosa cells. J. Biol. Chem. 276, 40146-40155
7.
Mertens, P. R., Alfonso-Jaume, M. A., Steinmann, K., and Lovett, D. H. (1998) A synergistic
interaction of transcription factors AP2 and YB-1 regulates gelatinase A enhancer-dependent
transcription. J. Biol. Chem. 273, 32957-32965
8.
Kelm, R. J., Jr., Elder, P. K., and Getz, M. J. (1999) The single-stranded DNA-binding proteins,
Puralpha, Purbeta, and MSY1 specifically interact with an exon 3-derived mouse vascular smooth
muscle alpha-actin messenger RNA sequence. J. Biol. Chem. 274, 38268-38275
9.
Evdokimova, V., Ruzanov, P., Anglesio, M. S., Sorokin, A. V., Ovchinnikov, L. P., Buckley, J.,
Triche, T. J., Sonenberg, N., and Sorensen, P. H. (2006) Akt-mediated YB-1 phosphorylation
activates translation of silent mRNA species. Mol. Cell. Biol. 26, 277-292
140
10.
Evdokimova, V., Ruzanov, P., Imataka, H., Raught, B., Svitkin, Y., Ovchinnikov, L. P., and
Sonenberg, N. (2001) The major mRNA-associated protein YB-1 is a potent 5' cap-dependent
mRNA stabilizer. EMBO J. 20, 5491-5502
11.
Eliseeva, I. A., Kim, E. R., Guryanov, S. G., Ovchinnikov, L. P., and Lyabin, D. N. (2011) Ybox-binding protein 1 (YB-1) and its functions. Biochemistry. Biokhimiia 76, 1402-1433
141