Research Article
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Dynamic trafficking of STAT5 depends on an
unconventional nuclear localization signal
Ha Youn Shin and Nancy C. Reich*
Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, New York 11794, USA
*Author for correspondence ([email protected])
Journal of Cell Science
Accepted 26 April 2013
Journal of Cell Science 126, 3333–3343
ß 2013. Published by The Company of Biologists Ltd
doi: 10.1242/jcs.123042
Summary
Signal transducer and activator of transcription 5 (STAT5) is crucial for physiological processes that include hematopoiesis, liver
metabolism and mammary gland development. However, aberrant continual activity of STAT5 has been causally linked to human
leukemias and solid tumor formation. As a regulated transcription factor, precise cellular localization of STAT5 is essential.
Conventional nuclear localization signals consist of short stretches of basic amino acids. In this study, we provide evidence that STAT5
nuclear import is dependent on an unconventional nuclear localization signal that functions within the conformation of an extensive
coiled-coil domain. Both in vitro binding and in vivo functional assays reveal that STAT5 nuclear import is mediated by the importin-a3/
b1 system independently of STAT5 activation by tyrosine phosphorylation. The integrity of the coiled-coil domain is essential for
STAT5 transcriptional induction of the b-casein gene following prolactin stimulation as well as its ability to synergize with the
glucocorticoid receptor. The glucocorticoid receptor accumulates in the nucleus in response to prolactin and this nuclear import is
dependent on STAT5 nuclear import. STAT5 continually shuttles in and out of the nucleus and live cell imaging demonstrates that
STAT5 nuclear export is mediated by both chromosome region maintenance 1 (Crm1)-dependent and Crm1-independent pathways. A
Crm1-dependent nuclear export signal was identified within the STAT5 N-terminus. These findings provide insight into the fundamental
mechanisms that regulate STAT5 nuclear trafficking and cooperation with the glucocorticoid receptor and provide a basis for clinical
intervention of STAT5 function in disease.
Key words: STAT5, Nuclear import, Nuclear export
Introduction
The rapid response of cells to external stimuli depends on
molecules that can quickly transmit signals from the plasma
membrane into the nucleus. The family of signal transducers and
activators of transcription (STATs) exemplifies signaling
molecules that can sense changes at the plasma membrane and
subsequently redirect gene expression (Darnell et al., 1994; Levy
and Darnell, 2002; Schindler et al., 2007). STATs are activated
by tyrosine phosphorylation in response to Janus kinases (JAKs)
associated with cytokine receptors, and this phosphorylation
promotes a dimer conformation with the ability to bind specific
DNA targets. Proper cellular localization of STATs is thereby
critical for their normal biological functions.
Two of the STAT genes, STAT5a and STAT5b, encode proteins
that are more than 95% identical, and murine gene knockout
studies indicate they have both distinct and redundant functions
(Liu et al., 1995; Liu et al., 1997; Moriggl et al., 1999; Nevalainen
et al., 2000; Udy et al., 1997; Yao et al., 2006). A double knockout
of both STAT5 genes is perinatal lethal, however individual gene
knockouts reveal their preferential role in response to cytokines
such as prolactin, interleukin-2, erythropoietin, and growth
hormone, and consequent effects on biological processes such as
mammary gland development, blood cell differentiation, and liver
metabolism. Loss of function studies confirm the requirement of
STAT5 in normal development, but in contrast, aberrant persistent
activation of STAT5 can promote oncogenesis (Cotarla et al.,
2004; Hantschel et al., 2012; Hayakawa et al., 1998; Schwaller
et al., 2000). Studies in human and murine systems indicate a
causal link between tyrosine phosphorylated STAT5 and
development of leukemias and mammary tumors. Understanding
the mechanisms that regulate STAT5 nuclear trafficking and its
consequent impact on gene expression can provide a basis to
develop methods to target STAT5 activity in human disease.
The transport of proteins between nucleus and cytoplasm is a
tightly regulated process. Movement of molecules into the
nucleus is gated through nuclear pore complexes (NPC) that
allow passive diffusion of small molecules, but restrict passage of
large molecules to those that possess a nuclear localization signal
(NLS) (Chook and Blobel, 2001; Cokol et al., 2000; Görlich and
Kutay, 1999; Macara, 2001; Mattaj and Englmeier, 1998;
Pemberton and Paschal, 2005; Rout and Aitchison, 2001). The
best defined NLSs consist of a monopartite or bipartite stretch of
basic amino acids, particularly lysine or arginine. In classical
transport, the NLS-containing cargo is carried through the NPC
by a dimeric importin-a/importin-b1 transporter complex. The
NLS is recognized directly by one of six characterized importin-a
adapter molecules bound to the importin-b1 protein (Köhler et al.,
1999). Importin-b1 facilitates transport through the NPC. In the
nucleus, importin-b1 binds to Ran-GTP and releases importin-a
and cargo. In this report we identify an unconventional NLS of
STAT5 that is constitutively active, independent of tyrosine
phosphorylation. The NLS function is required for gene induction
by STAT5 and for its ability to cooperate with the glucocorticoid
receptor.
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Nuclear export of macromolecules is also mediated by
transport carriers that recognize a nuclear export signal (NES)
in cargo and facilitate transport through the NPC. The best
characterized NES consensus comprises a leucine-rich
hydrophobic sequence that can be recognized by the exportin
chromosome region maintenance 1 (Crm1) in complex with RanGTP (Fornerod et al., 1997; Ullman et al., 1997; Wen et al.,
1995). Following transport to the cytoplasm, nucleotide exchange
of Ran-GTP to Ran-GDP leads to the dissociation of the exportincargo complex. A specific inhibitor of Crm1, leptomycin B
(LMB), has been a useful tool to determine the role of Crm1 in
export of various proteins (Kudo et al., 1998). Recent studies
have also identified additional exportins, including exportin-4, 6,
7, importin-13, and calreticulin (Güttler and Görlich, 2011;
Pemberton and Paschal, 2005). In this study we demonstrate that
continuous nuclear export of STAT5 is mediated by both Crm1dependent and Crm1-independent mechanisms.
Results
Journal of Cell Science
The coiled-coil domain of STAT5a functions as an
unconventional NLS
Fluorescence imaging of STAT5a tagged with monomeric EGFP
(STAT5a-GFP) was used to investigate the functional NLS in
STAT5. Our previous study indicated the constitutive nuclear
presence of STAT5a-GFP was independent of tyrosine
phosphorylation (Fig. 1A, top) (Iyer and Reich, 2008). In
addition, a small deletion in the coiled-coil domain of STAT5a
(142–149 a.a.) was found to abrogate the NLS function.
To determine if a peptide corresponding to 142–149 a.a. of
STAT5a was sufficient to function as an NLS, this peptide was
linked to GFP and evaluated for cellular localization. Since a
peptide tagged with GFP is small and can passively diffuse into
the nucleus, it cannot be used to analyze active transport. For this
reason the peptide was linked to a larger protein encoding
glutathione S-transferase (GST) and two tandem repeats of GFP
(GST-2GFP). The GST-2GFP protein alone does not possess an
NLS and does not enter the nucleus (Fig. 1A, bottom panel).
However the monopartite NLS from the well characterized SV40
large T antigen is able to efficiently promote nuclear import of
GST-2GFP, therefore the GST-2GFP protein can be used to study
nuclear trafficking. The STAT5a N-terminus with the coiled-coil
domain (1–330 a.a.) linked to GST-2GFP was found to promote
nuclear import of the fusion protein, although not as prominently
as the T antigen NLS. The STAT5a peptide encoding only 142–
149 a.a. does not confer nuclear localization to GST-2GFP. The
results suggest that a.a. 142–149 are needed for STAT5a nuclear
import, but are not sufficient to function as an NLS.
Since 142–149 a.a. of STAT5a may be part of a larger domain
that serves as an NLS, we evaluated the cellular localization of
various fragments of the STAT5a coiled-coil domain tagged with
GST-2GFP (Iyer and Reich, 2008; Zeng et al., 2002). The crystal
structure of unphosphorylated STAT5a has been solved and
indicates that the STAT5a coiled-coil domain consists of four ahelices (Neculai et al., 2005). Amino acids 142–149 of STAT5a
are located in the first a-helix of the coiled-coil domain. Each ahelix defined by the crystal structure was tested for its
Fig. 1. STAT5a coiled-coil domain and nuclear import. (A) Top: Linear diagram of STAT5a functional domains including the coiled-coil domain (CC),
DNA-binding domain (DBD), Src homology 2 (SH2) domain, transcriptional activation domain (TAD) and specific phosphorylated tyrosine residue 694 (Y694)
(Tan and Nevalainen, 2008). Beneath is a linear depiction of STAT5a 142–149 a.a. deletion construct linked to GFP. Images show cellular localization of STAT5a
full-length (WT) and deletion construct, as visualized by fluorescence microscopy. Bottom: Linear depictions and fluorescence images of STAT5a fragments
linked to GST-2GFP. Numbers correspond to STAT5a amino acids. Images of GST-2GFP and SV40 T antigen NLS linked to GST-2GFP (T-Ag NLS) are
also shown. (B) Diagram of STAT5a coiled-coil domain with four a-helices and constructs used in analyses. Representative fluorescent images are shown.
Bar graphs show statistical analyses of nuclear and cytoplasmic fluorescence intensity of STAT5a proteins expressed in cells. Fluorescence intensity was
quantified in the nucleus and cytoplasm of 10 cells expressing individual constructs by LSM Image Browser program and analyzed statistically by two-tailed tests.
Nuclear fluorescence (black bar) was normalized to the cytoplasmic fluorescence (white bar).
STAT5a nuclear trafficking
contribution to nuclear import and representative images are
shown in Fig. 1B. Fluorescence imaging demonstrates that the
first a-helix (138–190 a.a.) containing a.a. 142–149 is not
sufficient to direct nuclear import. A fragment containing both
the first and second a-helices (138–265 a.a.) also is not sufficient
for nuclear import; however a fragment containing the first,
second and third a-helices (138–305 a.a.) shows nuclear presence
in nearly 50% of the cells in culture. Maximal nuclear import in
all of the cells is only achieved with all four a-helices of the
coiled-coil domain of STAT5a (138–330 a.a.). Fluorescence
intensities of nuclear and cytoplasmic signals were measured
with the LSM Image Browser program and statistical analyses by
two-tailed tests indicate the entire coiled-coil domain of STAT5a
is required for the complete nuclear import function (Fig. 1B,
bottom graph). The data suggest the STAT5a NLS does not
conform to a conventional monopartite or bipartite basic NLS,
but is a larger structural domain.
Journal of Cell Science
Identification of essential residues in the STAT5a NLS
To more closely investigate the nature of the STAT5a NLS, the
sequence of the coiled-coil domain was evaluated for the
presence of classical lysine or arginine-rich basic residues
(Lange et al., 2007). Although amino acids 142–149
(LQINQTFE) in the first a-helix are needed for nuclear import,
they do not include any basic residues. However, since the
charged residue glutamic acid (E) is accessible on the surface of
STAT5a, it may be available for recognition by importins and its
contribution to nuclear import was evaluated. Mutation of
glutamic acid 149 to alanine (E149A) was found to inhibit
nuclear import mediated by the coiled-coil domain of STAT5a in
more than 80% of the population (Fig. 2A). It therefore appears
that E149 plays a major role in STAT5a import.
A potential bipartite basic sequence was identified within the
second a-helix (R241, K242, R257, R258), and its contribution
to STAT5a nuclear import was evaluated by alanine
substitutions. The R241A, K242A/R257A, R258A mutant
disrupted nuclear import in ,30% of the cells. It is not clear
why the impairment was evident in a subpopulation of the
culture, and may indicate this region plays an additive or
auxiliary role. Site directed mutation of basic residues in the
third and fourth a-helices did not identify an essential amino
acid (data not shown), and it is possible that the third and fourth
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a-helices are needed to maintain conformation of the coiled-coil
structure. Comparison of the fourth a-helix of STAT5a with
STAT1 and STAT3 revealed a conserved isoleucine in the
STAT5a and STAT3 proteins, but not in STAT1. Since nuclear
import of STAT5a is independent of tyrosine phosphorylation
similar to STAT3 (Liu et al., 2005), whereas STAT1 is
dependent on tyrosine phosphorylation (McBride et al., 2002),
we evaluated the effect of alanine substitution of this conserved
isoleucine 320 (I320A). The I320A mutant alone had no
apparent effect on nuclear import, however combined with the
other mutations, nuclear import of the coiled-coil domain was
completely inhibited in the total population.
The positions of the residues identified to be crucial for nuclear
import in the STAT5a coiled-coil domain are indicated in the
solved crystal structure of STAT5a (Neculai et al., 2005)
(Fig. 2B). The four a-helices of the coiled-coil are shown in
yellow and the position of critical a.a. are shown in red. E149 and
R241, K242, R257, R258 are exposed on the same surface of the
protein. The side chain of I320 in the fourth a-helix is embedded,
but may interact with third a-helix and contribute to
conformation of the coiled-coil domain.
STAT5a nuclear import is mediated by importin-a/importinb1 system
Active transport of large proteins is facilitated by the importinb1/karyopherin-b1 carrier proteins. Most commonly the NLS
is recognized by adapter proteins of the importin-a family
that dimerize with importin-b1. Importin-b1 mediates transport
through the NPC and the importin-a/importin-b1 dimer thereby
facilitates nuclear import of cargo. There are six characterized
members of the importin-a family that can directly recognize the
NLS (Goldfarb et al., 2004; Köhler et al., 1999). To determine if
any of the importin-a family members physically interact with
STAT5a, in vitro binding assays were performed with importins
and STAT5a (Fig. 3A). Mammalian cells were transfected with
V5 tagged STAT5a and cellular lysates were used as a source
of STAT5a. STAT5a-V5 was immunoprecipitated from the
cell lysates using V5 antibody, and incubated with bacterially
expressed GST tagged importin family members. STAT5a bound
importins were eluted from the beads and analyzed by western
blot using anti-GST antibody. Results show STAT5a binding to
both importin-a3 and importin-a6. Since importin-a3 is
Fig. 2. Identification of residues essential for the STAT5a NLS. (A) Location of alanine substitutions in the STAT5a coiled-coil domain. Below are
fluorescence images of STAT5a coiled-coil domain tagged with GST-2GFP with the single or combined mutations noted above the image. Images represent the
entire population of cells in culture, unless noted as 80% for E149A and 30% for 241A, 242A/257A and 258A. (B) Position of mutated residues in a ribbon
diagram of the crystal structure of STAT5a coiled-coil domain (Protein Data Bank ID code 1Y1U). E149A in the first a-helix (a1), basic residues (R241A,
K242A/R257A, R258A) in the second a-helix (a2) and I320A in the fourth a-helix (a4) are indicated.
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Journal of Cell Science
Fig. 3. STAT5a nuclear import is mediated by importin-a3/b1 system. (A) STAT5a-V5 expressed in COS-1 cells was immunoprecipitated using protein G
agarose beads, and incubated with bacterially expressed GST-importins in vitro. Importins bound to STAT5a were detected by western blot using anti-GST
antibody. The input levels of STAT5a are shown with anti-STAT5a antibody. The lower blot shows 10% of purified GST-importin proteins used in the binding
assay. (B) Left: The effect of pooled siRNAs on endogenous importin-a3 or importin-b1 mRNA levels detected by RT-PCR. Pooled siRNAs to importin-a3 or
importin-b1 reduced the endogenous respective mRNA levels by ,80%. Vimentin siRNA was used as a control siRNA. The level of GAPDH was quantified as an
internal control. Right: The effect of importin-a3 or importin-b1 siRNAs on the cellular localization of STAT5a-GFP. Nuclear accumulation of STAT5a was
inhibited in 10–30% of cultures transiently transfected with importin-a3 or importin-b1 pooled siRNAs, whereas there was no effect with vimentin siRNA.
ubiquitously expressed whereas importin-a6 is restricted to the
testes, importin-a3 appears to be the primary adaptor that
recognizes STAT5a (Köhler et al., 1997; Köhler et al., 1999). To
determine if tyrosine-phosphorylated STAT5a has similar
importin binding features, an in vitro binding assay was
performed with STAT5a isolated from cells treated with
epidermal growth factor (EGF) (supplementary material Fig.
S1). STAT5a was immunoprecipitated from EGF treated cell
lysates, and incubated with GST-importins. STAT5a from EGFtreated cells was found to bind importin-a3, importin-a6, and
importin-b1. The binding to importin-b1 may indicate that
tyrosine-phosphorylated STAT5a has an additional ability to bind
importin-b1.
To confirm the functional role of defined importins in STAT5a
nuclear import, we evaluated the cellular localization of STAT5a
after knockdown of importin-a3 and importin-b1 expression by
RNA interference (RNAi; Fig. 3B). siRNA duplexes corresponding
to importin-a3, importin-b1 or vimentin control were transfected
into cells expressing STAT5a-GFP, and cellular localization of
STAT5a-GFP was evaluated by fluorescence microscopy. The
knockdown efficiency of corresponding siRNAs in cells was
determined by measuring endogenous importin-a3 and importinb1 mRNA levels. There was a significant inhibition of STAT5a
nuclear accumulation if cells were treated with pooled importin-a3
siRNAs, or individual importin-a3 siRNAs (supplementary material
Fig. S2). Treatment of cells with pooled importin-b1 siRNAs also
decreased STAT5a nuclear import whereas there was no effect of
vimentin siRNA. Together with the importin binding assay, results
suggest that importin-a3/importin-b1 heterodimer mediates the
nuclear translocation of STAT5a.
To understand the interface between STAT5a and importin-a3,
specific fragments of importin-a3 able to bind STAT5a were
identified. Importin-as possess an N-terminal importin-b1
binding domain followed by 10 tandem armadillo (ARM)
repeats (Cingolani et al., 1999; Conti et al., 1998; Fontes et al.,
2000; Herold et al., 1998; Kobe, 1999). Each ARM motif consists
of ,40 a.a. folded into three a-helices. Co-crystal structures of
importin-a with conventional NLS peptides indicate a basic NLS
can bind to two regions of ARM repeats 2–4 and 6–8. To define
the domains of importin-a3 able to directly recognize the
unconventional STAT5a NLS, we performed in vitro binding
assays with purified proteins from bacteria. Maltose binding
protein (MBP) tagged to STAT5a 1–330 a.a. was immobilized on
amylose resin and incubated with GST-importin-a3 or GSTimportin-a1 as a control (Fig. 4A). Importins bound to STAT5a
were detected by western blot, and importin-a3 but not importina1, was found to directly bind STAT5a. To further define the
region of importin-a3 that binds STAT5a, in vitro binding assays
were performed with MBP-STAT5a and GST-importin-a3
deletions. The results showed that importin-a3 can bind to
STAT5a through two independent regions, ARM repeats 1–4 and
7–10 (Fig. 4B). Additional deletions of importin-a3 narrowed
binding to ARMs 2–4, but maintained binding to a second
broader region ARMs 7–10 (supplementary material Fig. S3).
From both in vitro binding assays using mammalian and bacterial
expression systems and in vivo functional studies using siRNAs,
nuclear import of STAT5a appears to be mediated by importina3/importin-b1 system.
STAT5a nuclear import is required for synergy with
glucocorticoid receptor and b-casein gene expression
STAT5a has a primary role in mammary epithelial cell
differentiation and alveologenesis (Liu et al., 1997). The
prolactin (PRL) hormone stimulates the tyrosine phosphorylation
of STAT5a during lactation leading to induction of the b-casein
gene in concert with the glucocorticoid receptor (Groner, 2002;
Happ and Groner, 1993). STAT5a synergizes with the
glucocorticoid receptor (GR) for maximal induction of the bcasein gene (Cella et al., 1998; Kabotyanski et al., 2006; Lechner
et al., 1997; Stöcklin et al., 1996; Stoecklin et al., 1997;
Wyszomierski et al., 1999). The GR is a ligand-dependent
transcription factor that is activated by binding glucocorticoid or
derivatives such as dexamethasone or hydrocortisone (Funder,
1997; Kumar and Thompson, 1999).
To assess the effect of the STAT5a NLS mutation D142–149
on transcriptional induction of the b-casein gene, we evaluated
induction of a luciferase reporter gene regulated by the b-casein
gene promoter (Fig. 5A). STAT5a wild-type or the NLS mutant
D142–149 were expressed in a human breast cell line with the bcasein gene reporter, and the cells were stimulated with PRL and/
or hydrocortisone (HC). PRL activation of wild type STAT5a
induced the b-casein reporter, but activation of the STAT5a NLS
Journal of Cell Science
STAT5a nuclear trafficking
Fig. 4. STAT5a directly binds to two independent regions of importin-a3.
(A) Bacterially expressed MBP-STAT5a(1–330) was immobilized on the
amylose resin and incubated with bacterially purified GST-importin-a3 or
importin-a1 as a control. Importins bound to STAT5a were detected by
western blot using anti-GST antibody. The protein level of STAT5a bound to
resin was examined by Ponceau S Staining. The middle blot shows 10% of
purified importin inputs. (B) Top: Linear depiction of importin-a3 motifs
including the importin-b binding domain (IbB) and ARM repeats; fragments
used in the study are indicated. Bottom: Bacterially expressed MBP-STAT5a
(1–330) was immobilized on amylose resin, and incubated with GSTimportin-a3 truncations. Importin-a3 deletions bound to STAT5a were
identified by western blot using anti-GST antibody. STAT5a bound to resin is
shown in the middle blot and 10% of importin input is shown in the lower blot.
mutant did not result in transcriptional induction. The STAT5a
NLS mutant D142–149 is tyrosine phosphorylated in response to
PRL and can bind DNA in vitro (supplementary material Fig. S4)
(Iyer and Reich, 2008). To assess the effect of the STAT5a NLS
mutant on synergy with the GR, cells expressing STAT5a wildtype or the NLS mutant D142–149 were co-treated with
hydrocortisone (HC). HC treatment alone had no effect on
transcription of the b-casein gene by STAT5a. However costimulation with PRL and HC produced the expected synergistic
induction of b-casein gene activity with wild-type STAT5a. Cells
expressing the STAT5a NLS mutant D142–149 did not respond
to PRL alone or with HC in this assay. The data indicate that
STAT5a nuclear import is required for cooperation with the GR
in transcription of b-casein gene.
Although STAT5a and GR can synergize to induce the gene
expression, the molecular mechanism of their cooperation
remains to be completely understood. A physical interaction of
STAT5a with GR has been documented (Cella et al., 1998;
Stöcklin et al., 1996), and for this reason we investigated the role
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of the STAT5a NLS on interdependent effects of their nuclear
accumulation. Immunofluorescence assays were performed with
human breast epithelial cells expressing HA-tagged STAT5a and
V5-tagged GR. Cells were treated with PRL or HC for activation
of STAT5a or GR, respectively. In the absence of hormone
treatment, STAT5a was evident in the nucleus, and GR was
primarily resident in the cytoplasm (Fig. 5B, top left panels).
After PRL treatment, STAT5a accumulated more prominently in
the nucleus as expected due to its induced ability to bind DNA
(Iyer and Reich, 2008), and GR also was found to accumulate
with STAT5a in the nucleus of a significant percentage of cells.
Treatment of cells with HC led to nuclear accumulation of GR as
well as STAT5a. The data suggest that activated STAT5a can
interact with GR in the cytoplasm and promote the nuclear import
of both transcription factors. Similarly, ligand-bound GR can
interact with STAT5a in the cytoplasm leading to the nuclear
import of GR and STAT5a. Activation of either transcription
factor causes the nuclear accumulation of its associated partner.
To determine the influence of the STAT5a NLS on cellular
localization in response to PRL or HC, the STAT5a NLS mutant
D142–149 was evaluated (Fig. 5B, right panel). In the absence of
hormone treatment, both STAT5a D142–149 and GR resided in
the cytoplasm. PRL stimulation did not change the cytoplasmic
localization of either factor. However, treatment of cells with HC
induced the nuclear accumulation of GR as well as STAT5a
D142–149. This indicates that ligand-activated GR is able to
import STAT5a D142–149 into the nucleus even though STAT5a
D142–149 lacks the independent ability to be imported. Together
the results indicate that regulated nuclear accumulation of GR or
STAT5a can influence the nuclear localization of its associated
factor.
STAT5a has a Crm1-dependent nuclear export signal in the
N-terminal domain
Our studies have shown that STAT5a continually shuttles in and
out of the nucleus (Iyer and Reich, 2008). The nuclear export of
STAT5a can serve both as a mechanism of negative regulation
and as a way to recycle the dephosphorylated STAT5a back to
the cytoplasm. One of the best characterized nuclear export
mechanisms is mediated by Crm1/exportin1 (Fornerod et al.,
1997; Ullman et al., 1997). To evaluate the regulation of STAT5a
nuclear export by Crm1, cells expressing STAT5a-GFP were
treated with the inhibitor leptomycin B (LMB). LMB is an
antifungal antibiotic that directly binds to Crm1 and inhibits its
export function (Kudo et al., 1998). The nuclear accumulation of
STAT5a-GFP dramatically increased in cells treated with
leptomycin B, indicating STAT5a export can be mediated by
Crm1 (Fig. 6).
The cellular localization of STAT5a fragments tagged with
GFP was analyzed to identify the Crm1-mediated NES in
STAT5a. We found that an N-terminal fragment encoding 1–138
a.a. was sensitive to LMB (Fig. 6). STAT5a 1–138 a.a. localized
in the cytoplasm unless nuclear export was inhibited with LMB
and then it accumulated in the nucleus. The NES consensus that
is recognized by Crm1 is a leucine-rich hydrophobic sequence
(Fornerod et al., 1997; Güttler and Görlich, 2011; Wen et al.,
1995). Alanine substitution of leucine residues in this region of
STAT5a, L119A and L133A, impaired the nuclear export of
STAT5a 1–138 a.a.. To determine if a peptide containing this
sequence was sufficient to mediate export of a protein containing
a characterized NLS, 118–138 a.a was linked to a prominently
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Journal of Cell Science
Fig. 5. STAT5a nuclear import is required for
the transcription of the b-casein promoter and
synergy with GR. (A) T47D human breast cells
were transfected with luciferase reporter
regulated by b-casein gene promoter, bgalactosidase (b-gal) control and STAT5a wildtype or import mutation. After serum starvation,
cells were left untreated (CTRL) or treated with
prolactin (PRL) or/and hydrocortisone (HC) for
16 hours to activate STAT5a or GR. The level of
b-casein gene expression was measured by
luciferase assay and normalized to b-gal activity.
(B) MCF-7 human breast cells were cotransfected with human prolactin receptor, GRV5, HA-STAT5a wild type or import mutation
and treated with PRL or HC.
Immunofluorescence of HA-STAT5a and GR-V5
was detected using either FITC- or Texas Redconjugated secondary antibody, respectively.
Fluorescence intensity measurements are
provided in supplementary material Fig. S4.
Images represent the majority of transfected cells.
nuclear protein corresponding to the NLS of the SV40 large T
antigen tagged with GST-2GFP (NLS-GST-2GFP). The effective
nuclear export function of a.a. 118–138 was evident in its ability to
maintain cytoplasmic residence of the NLS-containing protein
(Fig. 6, lower panels). The NES function was sensitive to LMB,
identifying a functional Crm1-mediated NES in the N-terminus of
STAT5a. The function of this NES was also demonstrated in full
length STAT5a by introduction of the internal deletion of 118–138
a.a. or the targeted mutation L119A/L133A (supplementary
material Fig. S5).
The directional transport of proteins through the NPC is
regulated by a gradient of Ran-GTP. The higher relative levels of
GTP-bound Ran in the nucleus facilitate the binding of Crm1 to the
NES for export, and the GTP to GDP nucleotide exchange in the
cytoplasm allows the dissociation of Crm1 from NES (Cook et al.,
2007; Yudin and Fainzilber, 2009). To investigate Ran requirement
for STAT5a nuclear export, cellular localization of STAT5a was
evaluated in the presence of wild type Ran or a constitutively active
Ran mutant Q69L that remains in a GTP-bound state (Bischoff
et al., 1994; Klebe et al., 1995). Following co-transfection of
STAT5a-YFP with CFP-Ran, cellular localization of STAT5a was
examined. Expression of Ran Q69L influenced STAT5a to become
predominantly cytoplasmic indicating a role of Ran in STAT5a
nuclear trafficking (supplementary material Fig. S6).
Fig. 6. STAT5a has a Crm1-mediated NES in the N-terminal domain.
Diagram of STAT5a functional motifs and the STAT5a fragments. Below are
shown fluorescence images of wild-type STAT5a-GFP in cells with or
without leptomycin B (LMB) treatment; STAT5a (1–138) fragment linked to
two tandem repeats of GFP with or without LMB treatment; L119A/L133A
double mutation in STAT5a (1–138)-GFPGFP; SV40 large T Ag NLS linked
to GST-2GFP (NLS-GST-2GFP); and STAT5a (118–138) linked to GST2GFP in cells treated with or without LMB.
Evidence supporting the existence of an additional
exportin
Photobleaching techniques with live cell imaging can provide
information on the kinetics of protein movement within and
between nuclear and cytoplasmic compartments of the cell. To
assess the kinetics of STAT5a nuclear export, we used cytoplasmic
Fluorescence Loss in Photobleaching (cFLIP). With this technique
STAT5a nuclear trafficking
3339
Journal of Cell Science
To investigate the Crm1-independent NES of STAT5a,
additional fragments of STAT5a were evaluated for their cellular
localization (Fig. 8). The 1–330 a.a. that contains both the Crm1dependent NES and the NLS within the coiled-coil domain has
clear nuclear presence indicating dominance of the STAT5a NLS
at steady state. The carboxyl terminal fragment 331–794 a.a of
STAT5a localized in the cytoplasm, and this could be due to either
the lack of an NLS or the function of an NES. To determine if there
is a functional NES in this domain, fragments of this region were
linked to the NLS of SV40 T antigen (NLS-GST-2GFP). NLSGST-2GFP localizes prominently in the nucleus, but when linked
to STAT5a 331–583 a.a. or 403–474 a.a. of STAT5a, the protein is
exported into the cytoplasm (Fig. 8). The nuclear export mediated
by this sequence is independent of Crm1 since LMB did not have
any effect on export. The results indicate that STAT5a has a Crm1independent NES in the DNA-binding domain as well as a Crm1dependent NES in the N-terminus. It is not clear if DNA binding
may inhibit the function of the NES positioned in the DNAbinding domain.
Fig. 7. Evidence for additional nuclear export signal in STAT5a.
Cytoplasmic fluorescence loss in photobleaching (FLIP). A small region in
the cytoplasm of cells expressing STAT5a-GFP untreated or treated with
LMB was subjected to a continuous high intensity laser. Time-lapse images
capture fluorescence in cytoplasm (C) and nucleus (N). Graphs show loss of
fluorescence intensity (FL%) quantified by LSM Image Browser in
cytoplasmic and nuclear compartments of one photobleached cell plotted
against time. The bar graphs show the half-time (T1/2) of nuclear
fluorescence decay, as calculated by curve-fitting analysis for multiple LMBtreated and untreated cells at baseline of cytoplasmic fluorescence.
a small region in the cytoplasm of cells expressing STAT5a-GFP
was subjected to a continuous high intensity laser (Fig. 7).
Cytoplasmic fluorescence was rapidly lost due to the bleaching
of STAT5a-GFP as it passed through the path of the laser,
indicating rapid continuous movement of STAT5a within the
cytoplasm. With additional time of photobleaching, fluorescence
was also completely lost from the nucleus by 40 minutes. This
indicates STAT5a-GFP is exported from the nucleus and
photobleached in the cytoplasm. To test the effect of LMB on
the kinetics of STAT5a nuclear export, we performed cFLIP on
cells treated with LMB. With the LMB treatment, the rate of
nuclear export was delayed as expected, but STAT5a-GFP nuclear
fluorescence was still lost by 50 minutes. Curve fitting analyses of
multiple cells were used to calculate the half-time loss of nuclear
fluorescence in the presence or absence of LMB, and LMB was
found to delay the half-time of nuclear fluorescence decay by 1.8fold (Fig. 7, bottom panel). The similar reduced nuclear export of
the STAT5a D118–138 NES mutant is shown in supplementary
material Fig. S7, as well as a comparative nuclear control with TAg-NLS-GST-2GFP protein. Taken together, the live cell imaging
data indicate a role of Crm1 in STAT5a nuclear export, but also
provide evidence for the function of export that is independent of
Crm1 since export occurs in the presence of LMB.
Discussion
STAT factors classically can sense cytokine and growth factor
signals in the cytoplasm and deliver those signals to responsive
genes in the nucleus (Levy and Darnell, 2002; Schindler et al.,
2007). Movement between cytoplasmic and nuclear compartments
is thereby central to their biological function. Although tyrosine
phosphorylation of STATs is known to promote the formation of
dimers that can bind to specific DNA targets, an increasing number
Fig. 8. STAT5a has a Crm1-independent NES in the DNA-binding
domain. Diagram of STAT5a functional motifs and STAT5a constructs.
Below are fluorescence images of STAT5a fragments linked to GFP or to
SV40 large T Ag NLS linked to GST-2GFP (NLS-GST-2GFP) with or
without LMB treatment.
Journal of Cell Science
3340
Journal of Cell Science 126 (15)
of studies indicate unphosphorylated STATs function in nuclear
gene expression (Chatterjee-Kishore et al., 2000; Cui et al., 2007;
Yang et al., 2005; Yang and Stark, 2008). In addition, nuclear
import of STAT3, STAT5, and STAT6 has been shown to be
independent of tyrosine phosphorylation (Chen and Reich, 2010;
Cimica et al., 2011; Iyer and Reich, 2008; Liu et al., 2005; Meyer
and Vinkemeier, 2004; Zeng et al., 2002). For these reasons,
accurate nuclear trafficking is key to the function of both
unphosphorylated and tyrosine phosphorylated STATs.
In this study, we describe the novel finding of an unconventional
NLS in STAT5a within the conformation of an extensive coiledcoil domain. By analyzing deletion mutants, we previously
identified a short stretch of amino acids in the coiled-coil
domain to be required for STAT5a nuclear import (142–149a.a.)
(Iyer and Reich, 2008). However, this sequence was not sufficient
to mediate nuclear import, and so larger fragments of the STAT5a
coiled-coil domain were evaluated (Fig. 1). The results indicated
that all four a-helices of the coiled-coil domain are required for
effective nuclear localization. Although the conformation of this
entire domain appears necessary for STAT5a NLS function,
several residues were identified to be critical by targeted mutation.
Glutamic acid 149 in the first a-helix plays a major role in NLS
function, and lesser roles were found for a bipartite basic sequence
in the second a-helix and an isoleucine 320 in the fourth a-helix
(Fig. 2). The effect of the isoleucine mutation is only evident in
combination with other mutations.
The coiled-coil domain of both unphosphorylated and tyrosine
phosphorylated STAT5a is expected to be accessible for
recognition by importin carrier proteins. The crystal structure of
unphosphorylated STAT5a has been solved, and similar to
unphosphorylated STAT1, STAT5a appears to form an antiparallel homodimer (Mao et al., 2005; Neculai et al., 2005). The
interaction between monomers is stabilized primarily by the bbarrel of the DNA-binding domains, but also by weak hydrogen
bonds with a-helices 1 and 3. Following tyrosine phosphorylation,
STAT5a is expected to form a parallel homodimer via reciprocal
SH2 domain and phosphotyrosine interactions similar to that of
STAT1 and STAT3 (Becker et al., 1998; Chen et al., 1998). The
coiled-coil domain of STAT5a should thereby be accessible in
both unphosphorylated and tyrosine-phosphorylated dimeric forms
for potential interaction with importin transporter proteins.
Detection of in vivo interactions between NLS-containing
proteins and importins is technically challenging due to the
transient interaction of importins with thousands of proteins, and
so to evaluate STAT5a recognition by importins we developed in
vitro binding assays (Fig. 3). Assays with STAT5a from either
mammalian cell lysates or as bacterially purified protein revealed
importin-a3 as a primary binding adaptor (Figs 3, 4). More
importantly, silencing expression of endogenous importin-a3 or
importin-b1 with siRNA caused a significant inhibition of STAT5a
nuclear import, indicating a functional requirement of importin-a3
and importin-b1. Our results do not support a previous suggestion
that STAT5 is imported independent of importin carriers (Marg
et al., 2004). Another study suggested that nuclear import of
tyrosine-phosphorylated STAT5 is mediated by a Rac1 GTPase
activating protein and is inhibited by a dominant negative
N17Rac1 (Kawashima et al., 2009). We have found no evidence
for a negative effect of N17Rac1 on nuclear import of
either unphosphorylated or tyrosine phosphorylated STAT5a
(unpublished observations, NCR).
The requirement of the entire coiled-coil domain of STAT5a for
nuclear import suggests the possibility of a significant interface
between STAT5a and importins. To determine the domain(s) of
importin-a3 that binds STAT5a, we evaluated fragments containing
various ARM repeats (Fig. 4; supplementary material Fig. S3). Two
regions of importin-a3 were found to bind STAT5a independently,
ARMs 2–4 and ARMs 7–10. Co-crystal structures of conventional
single or bipartite basic NLS peptides with importin-a have
demonstrated NLS binding in an antiparallel configuration to a
major site within ARMs 2–4 and a minor site within ARMs 6–8
(Conti et al., 1998; Fontes et al., 2003). Although the STAT5a NLS
is not a conventional basic peptide, it does bind importin-a ARMs
2–4. The second binding site in importin-a appears to be more
extensive and include ARMs 7–10. It is possible that this reflects an
extensive interface between STAT5a and importin-a, or it may
indicate the ARM repeats in this region contribute to the structure of
the binding site and do not directly interact with STAT5a. Future
studies with targeted mutations of asparagine and tryptophan
residues of importin-a known to interact with basic NLS peptides
may provide clarification. Since two independent regions of
importin-a3 can bind STAT5a, it could suggest that one importin
protein can bind two STAT5a proteins either as monomers,
unphosphorylated dimers, or tyrosine-phosphorylated dimers.
Solving a co-crystal structure will provide a more definitive
understanding of STAT5-importin interactions.
Functional specificity of STAT5 is in part a consequence
of its ability to synergize with other transcription factors, most
notably the glucocorticoid receptor (GR) (Hennighausen and
Robinson, 2008). In hepatocytes, STAT5b interaction with the
glucocorticoid receptor (GR) contributes to normal growth and
sexual maturation (Engblom et al., 2007). In the mammary gland,
STAT5a interaction with the GR enhances development and
expression of the b-casein gene (Liu et al., 1997; Stöcklin et al.,
1996). We found the STAT5a NLS mutant (142–149 a.a.
deletion) was not able to induce the b-casein gene promoter
with or without synergy by the GR in breast cancer cells.
Although the precise mechanisms of GR transport remain to be
determined, the GR is known to exist in a multimeric chaperone
complex and a conformational change occurs following ligand
binding to expose an NLS (Vandevyver et al., 2012). Previous
studies demonstrated a physical interaction of GR with STAT5a,
and transcriptional enhancement of the b-casein gene by GR was
dependent on STAT5a binding to its DNA target (Stoecklin et al.,
1997; Wyszomierski et al., 1999). However it was not clearly
understood if GR-STAT5a interaction takes place in the nucleus
or in the cytoplasm. Our study demonstrates that ligand-bound
GR can bring the STAT5a NLS mutant (142–149a.a. deletion)
into the nucleus, indicating that these factors can interact in the
cytoplasm to effect partner import (Fig. 5).
Both positive and negative regulation of STAT5 is necessary
to maintain normal physiological processes of blood cell
development, mammary development, and body growth.
Negative regulation of STAT5 is known to be mediated by
tyrosine phosphatases and suppressors of cytokine signaling
(SOCS) (Aoki and Matsuda, 2000; Aoki and Matsuda, 2002;
Chen et al., 2004; Cornish et al., 2003; Hoyt et al., 2007; Martens
et al., 2005). Another possible means of negative regulation is
STAT5 export from the nucleus. Our studies indicate that there
are two regulatory signals in STAT5a that effect export, a Crm1dependent NES in the N-terminus, and a Crm1-independent NES
within the DNA-binding domain (Figs 6–8). Although Crm1 is a
STAT5a nuclear trafficking
common exportin, other exportins and chaperones have been
identified that mediate export of translation and transcription
factors (Güttler and Görlich, 2011; Pemberton and Paschal,
2005). Future studies are needed to identify the non-Crm1
exportin for STAT5a. The continuous nuclear import of STAT5a
may facilitate its rapid response to activators, and continuous
nuclear export may facilitate either recycling of STAT5a or
signal termination.
The critical role of negative modulation subsequent to STAT5
activation is apparent. Accumulating evidence indicates continuous
STAT5 activity promotes leukemias, myeloproliferative disorders,
and solid tumors. Since nuclear trafficking is required for STAT5 to
regulate gene expression, it is a putative target for disease
intervention. Therefore knowledge of the interface between
STAT5 and importins may support the development of import
inhibitors. Although a co-crystal structure of STAT5 with importin
remains to be solved, our results provide a fundamental
understanding of the dynamics and molecular mechanisms of
STAT5a nuclear trafficking.
Materials and Methods
Journal of Cell Science
Cell culture and reagents
HeLa and COS-1 cells were obtained from American Type Culture Collection
(ATCC), and were grown in Dulbecco’s modified Eagle’s medium (DMEM) with
8% fetal bovine serum (FBS). T47D cells (ATCC) were maintained in RPMI with
10% FBS and MCF-7 cells (a gift from Todd Miller, Stony Brook University,
Stony Brook, NY) were cultured in DMEM with 10% FBS. DNA transfections
were performed with TransIT-LT1 transfection reagent (Mirus, Madision, WI)
according to the manufacturer’s instructions. Cells were treated with 10 nM
leptomycin B (LMB; a gift from Barbara Wolff-Winiski, Novartis Research
Institute, Vienna, Austria). After serum starvation, T47D and MCF-7 cells were
stimulated with human recombinant prolactin (PRL; PBL Biomedical
Laboratories, New Brunswick, NJ) and hydrocortisone (HC; Sigma-Aldrich, St.
Louis, MO) at 1 mg/ml each.
Plasmid constructs
Human full-length STAT5a cDNA and deletion mutants generated by polymerase
chain reaction (PCR) were cloned into pcDNA3 (Invitrogen, Carlsbad, CA), pEF1/
V5-His (Invitrogen), pCGN (Addgene, Cambridge, MA), or pMAL-c4X (New
England Biolabs, Ipswitch, MA) to express STAT5a, or STAT5a proteins tagged
with V5, HA or maltose-binding protein (MBP). A monomeric form of enhanced
green fluorescent protein (GFP) (Chen and Reich, 2010), glutathione S-transferase
(GST)-2GFP, or SV40 large T antigen NLS-GST-2GFP (Kalderon et al., 1984; Liu
et al., 2005) were linked to full length or deletion mutants of STAT5a. Sitedirected mutagenesis of STAT5a was performed using pfu Turbo DNA polymerase
(Stratagene, La Jolla, Ca) with targeted oligonucleotides. Human importin-a or b1
deletion constructs lacking the importin-b1 binding (IBB) domain were subcloned
into pGEX-KG for bacterial expression and purification as reported previously
(Liu et al., 2005). The b-casein gene promoter responsive luciferase reporter gene
was a gift from David Waxman (Boston University School of Medicine, Boston,
MA), and the b-galactosidase gene was obtained from Promega. Human full-length
GR (Origene, Rockville, MD) was PCR amplified and cloned into pEF1/V5-His
(Invitrogen) to generate V5 tagged GR.
Confocal microscopy
Cells were plated on glass coverslips and transfected with STAT5a constructs.
After 24 hours of serum starvation, cells were treated with or without LMB for
1 hour. Cells were washed with PBS and fixed with 4% paraformaldehyde for
10 minutes. GFP-tagged protein was visualized with a Zeiss LSM 5 pascal
confocal microscope using a 406 oil objective. GFP was excited at 488 nm using
an argon laser, and emission was collected using a 505–530 nm filter. Images were
captured using Zeiss LSM image browser and presented using Adobe Photoshop.
Bacterial protein expression and purification
Human recombinant importin-a or b1 proteins tagged with GST were bacterially
expressed and purified by binding and elution from glutathione agarose beads
(Sigma) as reported previously (Liu et al., 2005).
In vitro importin binding assay
COS-1 cells expressing STAT5a-V5 were lysed with cold lysis buffer [50 mM
Tris-HCl pH 8.2, 5 mM EDTA, 280 mM NaCl, 0.5% Nonidet P-40, 1 mM PMSF,
3341
16 mammalian protease inhibitor cocktails (Sigma)], and 500 mg of cellular
proteins were used for each assay. STAT5a-V5 was captured with anti-V5
antibody, immobilized to protein G beads, and incubated with 15 mg of purified
GST-importin-a or b1 proteins. Immunocomplexes were eluted with SDS sample
buffer and separated on 10% SDS-PAGE. Proteins were transferred to
nitrocellulose membrane (Pierce Biotechnology, Rockford, IL), and detected
with anti-GST and anti-STAT5a for western blots. Bacterially expressed MBPtagged STAT5a was immobilized on amylose resin (New England Biolabs) in cold
Column Buffer [20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, and 1 mM DTT,
1 mM PMSF, and 16 bacterial protease inhibitor cocktails (Sigma)] with 0.1%
CHAPS (Sigma). Purified GST-importin-a3 and deletions were incubated with
STAT5a protein immobilized beads and bound protein complexes were detected
by western blot using anti-GST antibody. The amount of STAT5a bound to resin
was examined by Ponceau S staining.
Antibodies
Western blots were performed using rabbit anti-STAT5a antibody (sc-1081,
Santa Cruz Biotechnology, Santa Cruz, CA) and anti-GST antibody (sc-33613,
Santa Cruz Biotechnology). Alexa Fluor 680-labeled (A21109, Invitrogen) or
horseradish peroxidase-conjugated (NA934V, Amersham Bioscience, Piscataway,
NJ) anti-rabbit IgG was used as secondary antibody for western blots. Reactive
signals were detected with an enhanced chemiluminescence system or Odyssey
infrared imaging system (Li-COR Bioscience, Lincoln, NE). Two micrograms of
anti-V5 antibody (R960-25, Invitrogen) was used for immunoprecipitation. For the
immunofluorescence assay, anti-HA (Santa Cruz) and anti-V5 (Santa Cruz) were
used as primary antibodies. Anti-rabbit conjugated with FITC (Molecular Probes,
Carlsbad, CA) and anti-mouse conjugated with Texas Red (Molecular Probes)
were used as secondary antibodies.
RNA interference
Short interfering RNAs (siRNA) specific for human importin-a or b1 (Qiagen Inc.,
Valencia, CA) were transfected with X-tremeGENE siRNA transfection reagent
(Roche, Indianapolis, IN). Vimentin siRNA was used as a negative control. After
24 hours of siRNA transfection, cells were transfected with STAT5a-GFP.
Cellular localization of STAT5a-GFP was evaluated after 24 hours by confocal
microscopy. Isolation of total RNA was performed with TRIzol reagent
(Invitrogen), and cDNA was synthesized with M-MLV reverse transcriptase
(Promega, Madison, WI). RT-PCR was carried out using specific primers for
importin-a3, b1 or GAPDH as an internal control. Primer sequences for importina3 and b1 were reported previously (Chen and Reich, 2010; Liu et al., 2005).
ImageJ software was used for quantification of endogenous importin-a3 or
importin-b1 levels.
Luciferase reporter assay
T47D human breast cells that express endogenous glucocorcoid receptor and
prolactin receptor (PRL-R) were co-transfected with b-casein-luciferase, bgalactosidase (Promega), and pcDNA3-STAT5a wild type or import mutant.
After 24 hours of serum starvation, cells were treated with 1 mg/ml of prolactin
(PRL) or/and hydrocortisone (HC) for 16 hours. Firefly luciferase (Promega) and
luminescent b-galactosidase (Clontech, Mountain View, CA) were measured
according to the manufacturer’s instructions. Firefly luciferase values were
normalized to luminescent b-galactosidase values to eliminate variations of
transfection efficiency.
Immunofluorescence assay
MCF-7 human breast cells were seeded on the glass coverslips and co-transfected
with GR-V5, hPRL-R, HA-STAT5a or HA-STAT5a(D142–149). After 24 hours of
serum starvation, cells were treated with or without PRL or HC for an hour. Cells
were rinsed with cold PBS and fixed with 4% paraformaldehyde. Following the
permeabilization in 0.5% Triton X-100, cells were blocked in 3% BSA, incubated
with primary antibodies for 3 hours followed by secondary antibodies conjugated
with FITC or Texas Red. Immunofluorescence of cells was visualized by a Zeiss
LSM 5 pascal confocal microscope using a 406 oil objective. Images were
obtained using Zeiss LSM image browser program and presented using Adobe
Photoshop.
Live cell imaging
HeLa cells were seeded on 35 mm glass-bottom tissue culture dishes (Mattek
Corporation, Ashland, MA), and transfected with STAT5a-GFP constructs. After
serum starvation, cells were treated with or without LMB for an hour. During live
cell imaging, cells were maintained at 37 ˚C and 5% CO2 using the Zeiss
Tempcontrol 37-2 Digital and CTI Controller 3700. Fluorescence loss in
photobleaching (FLIP) with STAT5a-GFP was performed by bleaching the
region of interest (ROI) in the cytoplasm every 60 seconds with 100% power of an
argon laser at 488 nm for 50 minutes. The time series images were obtained with
the Zeiss LSM 510 META NLO two-photon laser scanning microscope system
using a 636 oil objective. The excitation wavelength of GFP was 488 nm and
3342
Journal of Cell Science 126 (15)
emission was detected with a 505 nm filter. Images were obtained using LSM
image browser and presented with Adobe Photoshop. Nuclear and cytoplasmic
fluorescence intensities of target cells were quantified using LSM Image Browser
and graphically plotted using GraphPad Prism software.
Acknowledgements
We thank the current and past members of the laboratory for their
support and helpful advice, especially Janaki Iyer, Hui-Chen Chen,
and Velasco Cimica. We appreciate the support of Guo-Wei Tian and
Vitaly Citovsky with imaging analyses.
Author contributions
H.Y.S. and N.C.R. designed experiments and wrote the manuscript.
H.Y.S. performed the experiments.
Funding
These studies were supported by the National Institutes of Health
(NIH) [grant numbers R56AI095268, RO1CA122910]; a Carol M.
Baldwin Breast Cancer Research Award; and a Walk-for-Beauty
Foundation Research Award (to N.C.R.). Deposited in PMC for
release after 12 months.
Journal of Cell Science
Supplementary material available online at
http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.123042/-/DC1
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