The Pennsylvania State University
The Graduate School
Department of Neural and Behavioral Sciences
ROLES OF PI3-KINASE AND PDK-1
IN ROD PHOTORECEPTOR DIFFERENTIATION
A Thesis in
Anatomy
by
Tiaosi Xing
© 2013 Tiaosi Xing
Submitted in Partial Fulfillment
of the Requirements
for the Degree of
Master of Science
May 2013
The dissertation of Tiaosi Xing was reviewed and approved* by the following:
Colin J. Barnstable
Head of the Department of Neural and Behavioral Sciences
Professor of Neural and Behavioral Sciences
Dissertation Adviser
Chair of Committee
Patricia J. McLaughlin
Professor of Neural and Behavioral Sciences
Director of Graduate Program in Anatomy
Joyce Tombran-Tink
Professor of Neural and Behavioral Sciences
Samuel Shao-Min Zhang
Assistant Professor of Neural and Behavioral Sciences
*Signatures are on file in the Graduate School.
ii
ABSTRACT
The phosphoinositide 3-kinase (PI3-kinase) pathway regulates cell growth,
survival, differentiation and proliferation. In response to insulin growth factor (IGF1), PI3-kinase phosphorylates PIP2 to PIP3. Generation of PIP3 allows the
activation of downstream effector proteins.
Previous experiments suggested that the PI3-kinase class IA/PKCs/STAT3
pathway was linked to rod photoreceptor differentiation. Activation and inhibition of
PI3-kinase class IA increased the number of rods after four days explant culture of
postnatal day1 mouse retinas. Therefore, it was hypothesized that throughout
retinal development there is a change in the expression of regulatory subunits of
PI3-kinases family. Presence of PI3-kinase class IA regulatory subunits was
examined in different ages of mouse retinas. It was found that although p85α, p55α
and p55γ were present in mouse retinas, only tyrosine phosphorylation of p55α
was detected in the retinas after postnatal day 5. These results suggest that p55α
is the essential subunit controlling PI3-kinase class IA activity.
3-phosphoinositide-dependent protein kinase-1 (PDK-1) was identified as one of
the downstream of PI3-kinase. It is a protein serine and threonine kinase required
for protein kinase B (Akt) activation in many tissues. It is also a crucial activator of
multiple protein kinases, including p70 ribosomal S6-kinase (p70S6K1), protein
kinase isoforms (PKCs), and serum and glucocorticoid-inducible kinase (SGK).
We also investigated whether PDK-1 was also involved in the PI3-kinase Class IA
/PKCs/STAT3 pathway. Serine phosphorylation of PDK-1 was detected in the
iii
retina from postnatal day 3. It was found that treatment of retinal explants with
PDK-1 inhibitor (BX795) for four days dramatically increased the number of rod
photoreceptors. On the other hand, pharmacological inhibition of PDK-1 induced a
significant decline of Akt, p70S6K1, PKC-γ threonine phosphorylation and STAT3
tyrosine phosphorylation. The results indicate that PDK-1 activity is one of the
critical in regulating rod photoreceptor differentiation.
iv
TABLE OF CONTENTS
List of Tables
viii
List of Figures
ix
Abbreviations
xii
Acknowledgements
xv
Chapter 1: Background on photoreceptor and intracellular signaling pathway
1.1 Retina and photoreceptor
1
1.2 Photoreceptor differentiation and formation
6
1.3 Rod photoreceptor degeneration disease
8
1.3.1 Different types of rod photoreceptor degeneration
disease
8
1.3.2 Treatment for different types of rod photoreceptor
degeneration disease
1.4 Intracellular signals
9
10
1.4.1 Phosphoinositide 3-kinase signaling in retinal
rod photoreceptor
11
1.4.2 Protein kinase B
13
1.4.3 3-Posphoinositide-dependent kinase 1
14
1.4.4 PKCs regulate the differentiation of rod photoreceptor
through STAT-3
16
Chapter 2: Hypothesis and Specific Aims
19
Chapter 3: Methodology
22
v
3.1 Animals
22
3.2 Reagents
22
3.3 Retinal isolation
25
3.4 Retinal explant culture
25
3.5 Western blot
25
3.6 Immunocytochemistry
25
3.7 Statistical analysis
28
Chapter 4: Results
4.1 p55/p50 is the key functional signal for the development of photoreceptor
among different isoforms of PI3-kinase
29
Role of 3-phosphoinositide-dependent protein kinase-1 in rod
photoreceptor differentiation
40
4.2.1 PDK-1 is expressed in both adult and postnatal day 1
mouse retinas
41
4.2.2 Postnatal day 1 wild type mice develop a higher
amount of rod photoreceptors after four days treatment
with PDK-1 inhibitor
44
4.2.3 The effects of IGF-1, PMA and LY294002 on rod
photoreceptor differentiation were enhanced after PDK-1
inhibition
47
4.2.4 Inhibition of PDK-1 resulted in reduced Akt threonine
phosphorylation, p70S6K1 threonine phosphorylation
vi
and PKC gamma threonine phosphorylation in postnatal day 1
mouse retina
49
4.2.5 Inhibition of PDK-1 inhibited tyrosine 705 phosphorylation
of STAT3
55
Chapter 5: General discussion
57
References
64
vii
LIST OF TABLES
Table 1. Members of the PI3-kinase class I subunits
12
Table 2. Antibodies used for detecting target protein in western blot and
immunohistochemistry
23
Table 3. Reagents used in explant culture
24
Table 4. Members of the PI3-Kinases class I subunits present in the mouse
retinas
39
viii
LIST OF FIGURES
Figure 1. A diagram showing the schematic section of the human eye
1
Figure 2. Simple organization of retina
2
Figure 3. Rod and Cone photoreceptors in mammalian retina
3
Figure 4. A diagram showing the structure of the PDK1 kinase domain
15
Figure 5. PDK-is the hug of diverse kinases by phosphorylating their activation
segment
16
Figure 6. A diagram showing PI3-knase/PDK/PKCs/STAT3 signaling pathway 17
Figure 7. Expression of p85 alpha and p55 alpha regulatory subunits of
PI3-kinase
31
Figure 8. p85 beta is not expressed in either neonatal or adult retinas
32
Figure 9. p55 gamma exists in all age of wild mice and PN28 RD1 retinas
33
Figure 10. p55 gamma is expressed in all age of wild mice retinas
35
Figure 11. Phospho-p55 gamma (Tyr199) found in wild type postnatal day
1, 3, 5, 7, 13, 28(adult) and postnatal day 28 RD1 mouse retinas
37
Figure 12. Tyrosine phosphorylation of p55 at residue 199 are expressed only
on photoreceptor layer of matured mouse retinas
ix
38
Figure 13. PDK-1 and phospho-PDK-1 (Ser241) found in postnatal day and
adult(day 28) mouse retinas
42
Figure 14. Expression of phospho-PDK-1 (Ser241) increased gradually from
postnatal day 1 to adult (day 28) mouse retina
43
Figure 15. Effects of inhibiting of PDK-1 by increasing dose of BX795 in four
days explant culture of P1 wild type mouse retinas
46
Figure 16. The effects of IGF-1, and PMA on rod photoreceptor differentiation are
enhanced after PDK-1 inhibition
48
Figure 17. Inhibition of PDK-1 decreased threonine phosphorylation of p70S6K
at 389 and PKC gamma at 514
51
Figure 18. The level of Akt Thr308 phosphorylation decreased after 30 min
inhibition of PDK-1
53
Figure 19. Ly294002 and BX795 decreased the threonine phosphorylation of Akt
at 308
54
Figure 20. Inhibition of PDK-1 decreased tyrosine 705 phosphorylation of STAT3
56
Figure 21. A diagram showing the relationship of PI3-kinase/PKCs/PDK-1/STAT3
x
signaling pathway
61
xi
ABBREVIATIONS
4α-phorbol 12-myristate 13-Acetate (PMA)
Age-related macular degeneration (AMD)
Bruton’s tyrosine kinase (Btk)
cAMP-dependent protein kinase (PKA)
Ciliary neurotrophic factor (CNTF)
Epithelium growth factor (EGF)
Ganglion cell layer (GCL)
Guanosine disphosphate (GDP)
Inner neuroblastic layer (INBL)
Inner nuclear layer (INL)
Inner nuclear layer (INL)
Inner plexiform layer (IPL)
Inner retinal layer (INL)
Interferon alpha (IGF-α)
Leber congenital amaurosis (LCA)
Outer neuroblastic layer (ONBL)
xii
Outer nuclear layer (ONL)
Outer nuclear layer (ONL)
Outer nuclear retinal layer (ONL)
Outer plexiform layer (OPL)
p70 ribosomal S6-kinase (p70S6K1)
PDK-1 inhibitor (BX795)
Phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2 , PIP2)
Phosphatidylinositol (3,4)-trisphosphate (PI(3,4,5)P3, PIP3)
Phosphoinositide 3-kinase (PI3Ks)
Phosphoinositide-dependent kinase 1(PDK-1)
Photoreceptor-specific guanylate cyclase gene (retGC1)
PI3 kinase pan inhibitor (LY294002)
Pleckstrin homology (PH)
Postnatal (PN)
Proliferating cell nuclear antigen (PCNA)
Protein kinase B (PKB, AKT)
Protein kinase C (PKC)
xiii
Retina multipotent progenitor cells (RPCs)
Retinal pigment epithelium (RPE)
Retinitis pigmentosa (RP)
Serine (Ser)
Serum and glucocorticoid-regulated kinase (SGK)
Signal transducer and activator of transcription 3 (STAT3)
Threonine (Thr)
Tyrosine (Tyr)
xiv
ACKNOWLEDGEMENTS
This study was made possible by funding from the National Institute of Health.
Special thanks should be given to the committee responsible for mentoring and
assisting in the revisions of the experimental design, this committee included Dr.
Colin J. Barnstable, Dr. Samuel Shao-Min Zhang, Dr. Joyce Tombran-Tink and Dr.
Patricia J. McLaughlin, Their knowledge and research experience were essential in
this process. The Graduate School at the Pennsylvania State College of Medicine
was a perfect setting to ensure academic and research success.
xv
CHAPTER 1:
Background on photoreceptor and intracellular signaling pathway
1.1 Retina and photoreceptors
The vertebrate retina is a thin layer lining between the choroid and sclera of the
eye (Figure 1). It contains light-sensitive photoreceptor cells and several types of
inter-neurons. Light is captured by light-sensitive photoreceptor cells, and initiate
a cascade of chemical and electrical responses that ultimately trigger nerve
impulses. These impulses are then sent through the fibers of the optic nerve to
various visual cortices in the brain where they are interpreted into visual images
(Richard LG et al, 1997)
Figure 1. A diagram showing the schematic section of the human eye
http://webvision.med.utah.edu/book/part-i-foundations/simple-anatomy-of-theretina/
The retina contains a diversity of intrinsic cell types, including rod and cone
photoreceptors, horizontal, bipolar, amacrine cells, ganglion cells and Müller cells
(Dowling et al, 1970). They are organized into a 10-layered structure, beginning
with the pigment epithelium.
Figure 2. Simple organization of retina (Figure adapted from Zhang.S.S.)
Retinal pigment epithelium (PE); outer segment and inner segment of
photoreceptors (OS+IS); outer nuclear layer (ONL); outer plexiform layer (OPL);
2
inner nuclear layer (INL); inner plexiform layer (IPL); ganglion cell layer (GCL);
nerve fiber layer (NFL).
The 10 layers of the retina are as follows:
1. The retinal pigment epithelium is a single layer of cuboidal pigmented cells.
The external side of the cells adjoins the choroid and the internal side forms fine
processes that surround the outer portion of the photoreceptor layer. The
pigment epithelium layer is metabolically involved with the receptors and absorbs
light that has passed through the retina.
2. The rod and cone photoreceptor layer. This layer consists of two types of
photoreceptors: rods and cones. Both rods and cones have outer and inner
segments connected by a cilium. The outer segment of rods is relatively long and
cylindrical, while that of cones is short and tapered. Each type of outer segment
is filled with hundreds of discs, which is formed by infolding of plasma membrane.
In cones, the interior of the discs are continuous with extracellular space,
however, in rods, the folds of the discs pinch off from the membrane and are
entirely intracellular. The major protein constituent of the discs is the visual
pigment, which is called rhodopsin in rods and cone opsin in cones, yet cone
opsin is responsible for low light acuity and color. Rhodopsin is very sensitive to
light, so that rods are much more light-sensitive than cones. The inner segment
is a region containing a prominent number of mitochondria. These mitochondria
supply the energy required for synthesis of visual pigments and light transduction
(Richard LG et al, 1997; John Nolte, 2001).
3
Figure 3. Rod and cone photoreceptors in mammalian retina.
A) A human retinal section showing three neuronal cell layers: outer segment of
photoreceptors (OS); outer nuclear layer (ONL); inner nuclear layer (INL);
ganglion cell layer (GCL). B) Diagram of rod and cone structure. C) Scanning EM
showing the rods and cones.
http://vr-core.wustl.edu/Pages/Chen-Lab-Projects.aspx
3. The outer limiting membrane. It is formed together by the Müller cell processes
and inner segments of the rods and cones.
4. The outer nuclear layer consists of the cell bodies of the rods and cones.
5. The outer plexiform layer. The synaptic terminals of photoreceptors terminate
in this layer, and the rods and cones synapse with dendrites of horizontal
4
cells and different subpopulation of bipolar cells from the inner nuclear layer
(John Nolte, 2001).
6. The inner nuclear layer contains the cell bodies of retinal interneurons, which
include horizontal cells, amacrine cells, and bipolar cells, as well as the intrinsic
glial cell of the retina, the Müller cell.
7. The inner plexiform layer is a relatively thick synaptic layer, which includes the
synapses among bipolar, amacine and ganglion cells.
8. The ganglion cell layer. This layer contains as some displaced amacrine cells
and the cell bodies of the ganglion cells, whose dendrites synapse with bipolar
and amacine cells in the inner plexiform layer while their axons leave the eye as
the optic nerve (John Nolte, 2001).
9. The nerve fiber layer. This layer is the collection of axons of the ganglion cells
together with surrounding astrocytes. They form the optic nerve at the optic
papilla.
10. The inner limiting membrane. It is a thin basal lamina located between retina
and the vitreous body, formed by astrocytes and the proximal ends of the Müller
cells.
There are approximately 5 million cones and 120 million rods in the mature
human retina. Rods account for 70% -80% of the cells in the rodent retina
(Cepko et al, 1996). Rhodopsin in rods is highly sensitive to light, so rods are
5
responsible for vision at dark and they have low spatial acuity. Cones are
responsible for vision at higher light levels; they are capable of perception of
color and have high spatial acuity (John Nolte, 2001). In humans, Cone cells are
concentrated in the fovea, but the density is reduced on the periphery of the
retina.
Rods and cones are the major neurons that are directly sensitive to light in the
retina. They convert light into signals that can stimulate biological processes.
Therefore the photoreceptors, especially the rods, play an extremely important
role in processing visual stimuli.
1.2 Rod Photoreceptor differentiation
In the rodent retina, more than 95% of the retinal ganglion cells, horizontal cells
and cone photoreceptor cells finish cell genesis before embryonic day 17 (E17)
(Cepko, et al, 1996; David et al, 2004). Ganglion cells are first generated,
followed by the production of cone photoreceptors, horizontal cells, and most of
the amacrine cells, bipolar cells, mÜller cells. Most rod photoreceptors are
generated postnatal day one (John Nolte, 2001). Rods take up 70%-80% of the
adult retinal cells. They begin to be born on E15, but the significant cell birth
occurs in the first few postnatal days and is complete by PN5 (Cepko, et al,
1996). Expression of the major visual pigment protein opsin begins at postnatal
day 2 (PN2) (David et al, 2004), so there are different factors between embryonic
and postnatal times affecting the development of rods.
6
All cells of the mammalian retina arise from a pool of multipotent progenitor cells
(RPCs) (Swaroop et al, 2010). Cell extrinsic and intrinsic properties restrict the
progenitor cells to a lineage that gives rise to photoreceptor cells and possibly to
non-photoreceptor cells (Cepko, et al, 1996). It has been verified that homeobox
gene Crx and Otx2 are key molecules that play an important role in the
photoreceptor development (Chen et al, 1997). Crx is activated by Otx2 and is
necessary for the cell fate determination of photoreceptor cell. Another important
molecule, Nrl, functions in deciding the subtypes of cone and rod. Nrl is a basic
leucine zipper transcription factor that is expressed in rod photoreceptors
(Ryosuke et al, 2008). It regulates cell fates of rods through the activation of
Nr2e3, however, how these cell-specific genes interact and to promote the
development of rods and cones are still unclear (Swaroop et al, 2010).
The formation of photoreceptors can be divided into five major steps: 1).
Generation and proliferation of multipotent retinal progenitor cells (RPCs); 2).
limiting the proliferation of multipotent retinal progenitor cells; 3). generation of
cell fate-committed photoreceptor precursors during final mitosis; 4). Specific
photoreceptor gene expression during late morphogenesis; 5). maturation of
photoreceptor cells (Swaroop et al, 2010; David et al, 2004). The generation of
cell fate-committed photoreceptor precursors is a very important step for
photoreceptor cell formation. During this period, transition of RPCs to rod
photoreceptors or other retinal neurons is determined. The extrinsic and intrinsic
factors that induce this transition are not completely understood. The ciliary
neurotrophic factor (CNTF), interferon alpha (IFN-α) and epidermal growth factor
7
(EGF) can all activate signal transducer and activator of transcription 3 (STAT3),
which then allows the proliferation of progenitor cells, thus decreasing the
number of rods (Zhang et al, 2004).
1.3 Retinal degeneration disease
1.3.1 Different types of rod photoreceptor degeneration disease
Millions of people in the world live with various degrees of vision loss because
they have irreversible, degenerative eye disorders such as retinitis pigmentosa
(RP), Leber congenital amaurosis (LCA), age-related macular degeneration
(AMD), diabetic retinopathy, cataracts, or glaucoma. Retinal degenerative
diseases target photoreceptors or the adjacent retinal pigment epithelium (RPE)
(Yiming et al, 2010). Retinitis pigmentosa (RP) is a group of inherited disorders
characterized by progressive peripheral vision loss and night vision difficulties
that can lead to blindness. This blindness is caused by gradually degeneration of
rod and cone photoreceptors. The definite reason why photoreceptors undergo
apoptosis is still unknown (Hartong et al, 2006). LCA is the earliest and most
severe form of the genetically heterogeneous retinal dystrophies responsible for
congenital blindness (Suzanne Y et al, 2012). The common clinical features of LCA
are dysfunction in vision, sensory nystagmus or no light response on
electrophysiology. The pathology shows genetic heterogeneity, for example,
mutations in either the photoreceptor-specific guanylate cyclase gene (retGC1)
8
or the RPE65 gene can cause the disease (Isabelle et al, 1999). Age-related
macular degeneration (AMD) is a group of diseases characterized by loss of
central vision because of death or impairment of the cells in the macula, mainly
cone photoreceptors. The most common risk factors that cause AMD are
increasing age and smoking. Diet, obesity and chronic inflammation can also
contribute to the pathophysiology of AMD (Coleman et al, 2008).
1.3.2 Treatment for different types of rod photoreceptor degeneration
disease
Effective treatment for retinal degeneration has been widely investigated.
However, there is currently no treatment to reverse cell damage. Most of the
remedial methods focus on nutritional supplements that may slow the process of
cell death. There are two major categories of treatments for retinal diseases. The
first treatment is to prolong the life and functions of the photoreceptor cells, which
can be divided into three main therapies: 1) Gene Therapy, 2) Pharmaceutical
Therapy and 3) Nutritional Therapy. The other one is to replace the dead cells
using stem cell transplantation. Among these treatments, gene therapy and stem
cell transplantation are considered the most effective methods, however, they are
still in clinical trial and very little progress has been reported. In gene therapy a
defective (mutated) gene is replaced by a normal gene, such that an important
protein (e.g., enzyme) is again synthesized and present in the cell. With this, the
photoreceptors function better and live longer (Knut et al, 2011).
9
Stem cells are cells that can divide and differentiate into many different types of
cells in the body. Thus, theoretically, stem cells could be transplanted into the
retinas which photoreceptor cells have died, and stem cells could develop and
replace the degenerative photoreceptor cells (Wong et al, 2010).
There is great potential for gene therapy and stem cell therapy, however, there is
a gap in knowledge concerning proper programming of differentiation of
photoreceptor cells. For example, we need a complete understanding of the
physiological process of cell differentiation from stem cell to photoreceptor cells.
The proper biological signals have to be given to the stem cells so that they will
develop into mature, functional photoreceptor cells – instead of other cell types.
1.4 Intracellular signaling pathway
Both extracellular and intrinsic signals are required for cells to grow, divide and
survive. Members of the insulin-like growth factor (IGF) family stimulate many
types of cells to survive and grow. Receptors for the signals are usually located
at the cell surface and bind the signal molecule. Binding activates the receptor,
which in turn activates one or more intracellular signaling pathways. Intracellular
pathway proteins process the signal inside the receiving cell and distribute it to
the intracellular targets. These targets are generally effector proteins, for
example, metabolic enzymes that function in metabolism, gene targets also
include regulation proteins that alter gene expression, and cytoskeletal proteins
that alter cell shape or movement. A cell response to extracellular signals
10
depends not only on the cell surface receptor but also on the intracellular
machinery that integrates the signal (Bruce et al, 2008). These signaltransduction pathways regulate cell functions resulting in cell genesis,
proliferation, metabolism, and apoptosis.
1.4.1 Phosphoinositide 3-kinase signaling in retinal rod photoreceptor
The PI3-Kinase /PDK-1/AKT pathway plays an important role in the transmission
of proliferative signals from the cell surface receptor. Phosphoinositide 3-kinases
(PI3-kinases) are divided into three classes (I, II, III) (Vanhaesbroeck et al, 1997).
Class I consists of catalytic subunits and regulatory subunits (Geering et al,
2007). Class II PI3-kinases comprise three catalytic domains but no regulatory
domain. Their roles in vivo are still not elucidated. Class III PI3-kinases were
found in the immune cells. It also has a catalytic and regulatory subunit and
converts PIP2 to PIP3 (Neri et al, 2002).
Class I PI3-kinases phosphorylate phosphatidylinositol (3,4)-bisphosphate (PIP2)
to
phosphatidylinositol
(3,4)-trisphosphate
(PIP3),
which
triggers
the
phosphorylation of multiple kinases, including threonine-serine protein kinase B
Akt
(PKB),
phosphoinositide-dependent
kinase
1
(PDK-1),
guanosine
disphosphate (GDP)-GTP exchange factor for Rac and Bruton’s tyrosine kinase
(Btk) (Alessi et al, 1998). Class I PI3-kinases are heterodimers composed of a
regulatory and a catalytic subunit; they are further divided into IA and IB isoforms
depending on their sequence similarity.
11
The PI3-Kinase class IA is activated by G protein-coupled receptors and tyrosine
kinase receptors (Raju et al, 2010; Bruce et al, 2001). There are five variants of
the p85 regulatory subunit. They are p85α, p55α, p50α, p85β, and p55γ. These
variants result from three genes: Pik3r1, Pik3r2 and Pik3r3. p85α, p55α and p50α
are encoded by the same gene Pik3r1, p85β is expressed by genes Pik3r2 and
p55γ for Pik3r3 (Table 1) (Liu D et al, 2009; Geering et al, 2007). There are also
three variants of the p110 catalytic subunit: p110α, β, or δ catalytic subunit. All
three catalytic subunits are expressed by separate genes (Pik3ca for p110α,
Pik3cb for p110 β, and Pik3cd for p110δ respectively (Liu D et al, 2009; Geering
et al, 2007). Class IB PI3Ks is composed of the regulatory p110 and catalytic
p110γ subunits, which are encoded by a single gene each (Liu D et al, 2009).
PI3K
Class
Class I A
Class I B
Isoforms Major
substrate
p110α
PtdIns
(4,5) P2
p110β
p110δ
p85α
p55α
p50α
p85β
p55γ
p110γ
PtdIns
(4,5) P2
p101
p87
Gene
PIK3CA
PIK3CB
PIK3CD
PIK3R1
PIK3R1
PIK3R1
PIK3R2
PIK3R3
PIK3CG
PIK3R5
PIK3R6
Table 1. Members of the PI3-Kinases class I subunits
Activation of PI3-kinases response to insulin growth factor has been observed in
different tissues, however, it is not yet clear which subunits of PI3-kinase are
involved in particular signal transduction in response to various growth factors
12
and hormones (Kessler et al, 2001). It has been demonstrated that Class I PI3kinases are expressed in bovine retinal rod outer segment membranes (Guo et al,
1997), and the phosphoinositides generated by these enzymes are important for
photoreceptor function.
1.4.2 Protein kinase B
Protein kinase B (Akt) is a 57 kDa serine\threonine kinase with a pleckstrin
homology (PH) domain. It is activated when phosphorylated at two residues,
Thr308 and Ser473 (Neri et al, 2002; Martelli et al, 2011; Doreen et al, 2001).
The phosphorylation of Thr308 only occurs when the PH domain binds to PIP3
and PIP2 (Alessi et al, 1998). The binding is proposed to relieve auto-inhibition of
the active site, allowing the upstream signal to phosphorylate Thr308 and cause
the conformation change of the molecule – the key step in Akt activation (Martelli
et al, 2011). After activation, Akt is able to translocate to the nucleus and affect
the activity of transcriptional regulators. As well as target a number of
cytoplasmic molecules that affect the survival of the cell, including GSK-3beta,
BAD and inhibitor kappa B (Martelli et al, 2011).
Thr308 is located in the activation loop of the catalytic domain of Akt, which
shares high homology among different AGC family members. AGC family is a
group of kinases that all share a conserved catalytic kinase domain, including
p70 ribosomal S6-kinase (S6K1) (Pullen et al, 1998; Alessi et al, 1998), serum
and glucocorticoid-regulated kinase (SGK), protein kinase C (PKC) (Le Good et
al, 1998; Newton et al, 2001), and the cAMP-dependent protein kinase (PKA)
13
(Johnson et al, 2001), also possess residues lying in a sequence motif equivalent
to Thr308 whose phosphorylation is required for activation.
1.4.3 3-Phosphoinositide-dependent kinase-1
Phosphoinositide-dependent kinase 1 (PDK1) is a 63 kDa serine/threonine
kinase expressed in many tissues (Casamayor et al, 1999; Alessi et al, 1998;
Yogesh et al, 2010). It has amino-terminal catalytic domain, a linker region and a
PH domain. The active expression of PDK-1 demonstrates that PDK-1 has autophosphorylation activity (Casamayor et al, 1999). Phosphorylation of serine 241
is required for the full activation of PDK-1. The PH domain is capable of linking to
the products of PI3Ks such as PIP3 and PIP2, and inducing the redistribution of
enzymes from the cytosol to the plasma membrane (Vanhaesbroeck, et al, 1997;
Martelli et al, 2011; Vanhaesebroeck et al, 2000; Akio et al, 2009). PDK-1
induces the phosphorylation of Akt when its active site is uncovered by PIP3, so
it was discovered as the upstream kinase for Akt (Lewis 2002; Filippa et al, 2000;
Anderson et al, 1998). PDK-1 is necessary for phosphorylation of AGC members.
Furthermore, it was confirmed to be the upstream kinase for PKC family
members and p70S6 kinase, which function on protein synthesis, cell
proliferation and survival (Alessi et al, 1998; Pullen et al, 1998).
PDK-1 activates the AGC members in a PIP3 independent way (Alessi et al,
1998). The AGC kinases lack PH domains but have hydrophobic motifs that
could serve as substrate-docking sites, which enable PDK-1 to dock and
14
phosphorylation of the hydrophobic motifs (HMs). The phosphorylation of HMs
enhances the ability of PDK1 to interact with their substrates.
On the other hand, PDK-1 is the hub of many kinases and its function is only
mediated in part by the PI3-kinase pathway (Casamayor et al, 1999; Alexandra,
2003). How PDK-1 regulates its intrinsic protein kinase activity by other
mechanisms is still not clear.
Figure 4. A diagram showing the structure of the PDK1 kinase domain.
15
Figure 5. PDK-is the hug of diverse kinases by phosphorylating their activation segment.
1.4.4 PKCs play an important role in the development of rod photoreceptor
Protein kinase C (PKC) is well established as a downstream target of PDK-1 in
many tissues (Vanhaesbroeck et al, 1997; Martelli et al, 2011; Vanhaesebroeck
et al, 2000; Pinzon-Guzman et al, 2011). The PKC family of serine/threonine
kinases consist of many isoforms that are grouped into three classes:
Conventional isoforms (α, βI, βII, and γ) require Ca2+, diacylglycerol (DAG), and a
phospholipid, such as phosphatidylserine, for activation (Newton, 2001); novel
isoforms (δ, ε, η, and θ) require DAG, but do not require Ca2+ for activation;
atypical PKCs (including protein kinase M ζ and ι / λ isoforms) require neither
Ca2+ nor DAG for activation.
In previous experiment, it has been shown that PKCs (βI, γ) are required in rod
photoreceptor differentiation and PKCs function through STAT3 signaling
(Pinzon-Guzman et al, 2011). Signal transducer and activator of transcription
factors (STATs) have been identified as a critical molecular in regulating rod
photoreceptor differentiation (Zhang et al, 2004; Pinzon-Guzman et al, 2011).
Stimulation of STAT3 reduced the number of rods, while activation of PKC
suppressed the phosphorylation of STAT3 and increased the number of rods
(Pinzon-Guzman et al, 2011). Here we investigate the role of PDK-1 as an
upstream regulator of PKCs in rod photoreceptor differentiation.
16
IGF-1
RTK
PIP2
PIP3
PI3K IA
Plasma
membrane
PDK-1
PKC βI,γ
P70 S6K1
Akt-1
STAT3
Nuclear
membrane
Gene expression
Figure 6. A diagram shows the relationship of PI3-Kinase IA/ PDK/PKCs/STAT3
signaling pathway
In summary, PI3-Kinases class IA can be activated by IGF-1, which then
phosphorylates PIP2 to PIP3. PIP3 binds to PDK1 to activate Akt. PDK-1 also
activates AGC kinases p70S6K1, PKCs and other signals. The activation of
these kinases coordinates many downstream proteins to promote cell survival,
growth, protein synthesis, and mitosis (Lewis 2002; Doreen et al, 2001; Yogesh
et al, 2010), however, none of these effects have been examined in retina.
17
The theory that PI3-kinase class IA/ PDK/PKCs/STAT3 signaling pathway
stimulates cells to survive and grow has been previously confirmed (Neri et al,
2002; Vanhaesbroece et al, 1997; Martelli et al, 2011; Pinzon-Guzman et al,
2011; Doreen et al, 2001) Postnatal day one to four is the growth peak of rod
photoreceptors. During these four days, how the PI3-kinase class IA / PDK1/PKCs/STAT3 signaling pathway regulates cell proliferation is still unknown. In
previous experiments, PI3-Kinase class IA and PKCs (βI, γ) have been shown to
be important molecules during rod photoreceptor differentiation (Pinzon-Guzman
et al, 2011). IGF-1 activates the PI3-Kinase class IA/Akt pathway and ultimately
increases the number of rod photoreceptors in postnatal day four retina. On the
other hand, inhibitors of PI3-Kinase class IA also activate the formation of rod
photoreceptors, however, inhibition of PKCs decreases the number of rod
photoreceptors. PDK-1 has been reported many times as the downstream signal
of PI3-Kinase class IA and upstream signal of PKCs. The present project will try
to address the unanswered questions of whether PDK-1 connects the PI3-kinase
class IA / PDK1/PKCs/STAT3 pathway to regulate rod photoreceptor
differentiation.
18
CHAPTER 2
Hypothesis and Specific Aims
2.1
Hypotheses
The Hypotheses of the study are:
1) Throughout the retinal development there is a change in the expression of
regulatory subunits of PI3-kinase class IA family. Previous experiments
suggested that the PI3-kinase class IA/PKCs/STAT3 pathway was linked to rod
photoreceptor differentiation. Activation and inhibition of PI3-kinase class IA
increased the number of rods after four days explant culture of postnatal day1
mouse retinas. We hypothesized that the change of PI3-kinase regulatory
subunits in the developmental retinas are response for the different reaction of
the PI3-kinase.
2) PDK-1 plays an important role in regulating the formation of rod
photoreceptors in neonatal mouse retinas. PDK-1 phosphorylates and activates a
number of kinases in the AGC kinase family, including Akt, PKCs, p70S6K1,
SGK. It also hypothesized that PDK-1 regulates rod photoreceptor differentiation
through PKCs signaling instead of Akt. To test the above hypotheses, the
following aims will be followed.
19
2.2
Specific Aims
Aim 1: To investigate which regulatory subunits of PI3-kinase class IA could play
a role in the development of photoreceptors, we examined the presence and
localization of five isoforms of the regulatory subunits, p85α, p55α, p50 α, p85β,
and p55γ, in the wild type mouse retina from each age group at the ages of
postnatal day1, 3, 7, 13, and 28 (adult). In order to estimate the proportion of
expression of the PI3-Kinase class IA subunits due to rod photoreceptors, RD1
mutant mice were included in the experiment.
Aim 2: Test the hypothesis that PDK-1 is important in rod photoreceptor
differentiation.
Retinas from Postnatal day 1 and day 28 wild type mice will be tested to detect
the expression of PDK-1 and Phospho-PDK-1. When it has been confirmed that
PDK-1 and Phospho-PDK-1 are expressed in both P1 and adult mice, PDK-1
inhibitor will be applied to retinal explant cultures for times ranging from 30
minutes to 96 hours. We use 30 minutes explant culture retina to detect the
activation of Akt as a marker of PDK-1 activity. From previous information we
know that postnatal day 1 to 4 is the growth peak of rod photoreceptors and 95%
of rods are formed before PN5. The 96 hours (PN5) retinas will be used to
observe the formation of rod photoreceptors. It is expected that inhibition of PDK1 would affect the activation of Akt and formation of rod photoreceptors.
Aim 3: Find out the interaction between PDK-1 and PKCs.
20
It’s well established that the PI3-kinase class IA /PDK-1/Akt signaling pathway
stimulates animal cells to survive and grow. However, in neonatal retinas Akt is
suggested not to be involved in the formation of rod photoreceptors. On the other
hand, PKCs are required for rod photoreceptor differentiation (Guzman et al,
2011). We hypothesize that PDK-1 effects the formation of rod photoreceptor
through PKCs instead of Akt. To find out how PDK-1 regulates the activity of
PKCs. We will inhibit PDK-1 and then detect the activity of PKCs and STAT3 in
both adult and PN1 retinas.
21
CHAPTER 3:
Methodology
3.1 Animals
C57BL/6J and retinal degeneration (RD1) mice were purchased from Jackson
laboratory (Bar Harbor, ME, USA). The retinal degeneration 1 (RD1) mouse has
a mutation in the gene for the β-subunit of cGMP phosphodies terase-6 (PDE-6).
Degeneration of photoreceptors in RD1 mice results in a depletion of almost all
rod photoreceptors by postnatal day 21 (Satpal et al, 2008). From each group at
the ages of postnatal day1, 3, 5, 7, 13, and 28, four to six C57BL/6J mice from
each age were using for preparing cytoplasmic protein, tissue sections and
immunohistochemistry. Retinas from C57BL/6J PN1 mice were used for four
hour or four days explant cultures. Adult RD1 mice (older than 28 days) were
used for preparing cytoplasmic protein. All experiments were approved by the
Animal Care and Use Committee of Pennsylvania State University School of
Medicine.
3.2 Reagents
Different antibodies and reagents were applied in the experiment. Rhodopsin is
detected by Ret-P1 monoclonal antibody recognizing an epitope on the N-
22
terminus of the protein (Colin et al, 1980).Other antibodies and reagents are
listed below.
Table 2. Antibodies used for detecting target protein in western blot and
immunohistochemistry
Antibody
Source
Dilution
Catalog No.
Company
Phospho-PKCγ (Thr514)
Rabbit
1:750
Ab5778
abcam
P70 S6 kinase
Rabbit
1:750
9202
Cell signaling
Phosphor-P70S6 kinase
Rabbit
1:1000
9205
Cell signaling
Mouse
1:1000
MA5-15797
Thermo
Rabbit
1:1000
PA 1-14336
Thermo
Rabbit
1:1000
3438
Cell signaling
PDK-1 antibody
Rabbit
1:1000
3062
Cell signaling
Phospho-STAT3 (Tyr 705)
Rabbit
1:500
9131
Cell signaling
Akt 1
Rabbit
1:1000
Sc-5298
Santa Cruz
Phospho-Akt (thr308)
Rabbit
1:1000
2965
Cell signaling
Phospho-Akt (Ser473)
Rabbit
1:1000
PTEN (D4.3)
Rabbit
1:1000
9188
Cell signaling
Phospho-PTEN
Rabbit
1:750
9549
EMD Millipore
Rabbit
1:1000
4228
Cell signaling
Mouse
1:500
Sc-56934
Santa Cruz
Mouse
1:800
60225-1-lg
Protein tech
Mouse
1:2500
A1978
Sigma-Aldrich
Mouse
1:1000
2586
Cell signaling
PDK-1 monoclonal
antibody
Phosphor-PDK-1 pSer241
polyclonal antibody
Phosphor-PDK-1
(Ser241)
Phospho-PI3K
p85(Tyr458)/p55(Tyr199)
PI3-Kinase p85β (T15)
PIK3R1Monoclonal
antibody
Monoclonal Anti-β-Actin
Clone AC-15
PCNA(PC10)
23
Santa Cruz
Table 3. Reagents used in explant culture
Reagent
Insulin like growth factor
1(IGF-1)
PDK-1 inhibitor (BX
795)
PDK-1 inhibitor (GSK
2334470)
PI3Kinase inhibitor
(Ly294002)
PTEN inhibitor
(SF1670)
PTEN inhibitor
Bpv(pten)
Go 7874
Concentratio
Catalog No.
Company
50 μg/ml
I 8779
Sigma
100 nM
Tlrl-bx7
InvivoGen
50 nM
4143
Tocris bioscience
50 μM
9901
2 μM
C7316-2s
100 nM
203695
n
Cell signaling
technology
Collagen
Technology
Calbiochem,EMD
Millipore
365252-
Calbiochem,EMD
500UG
Millipore
100 nM
63597-44-4
LC Laboratiries
500 nM
124018
100 nM
4α-Phorbol
12_Myristate 13Acetate
Akt inhibitor VIII
24
Calbiochem,EMD
Millipore
3.3 Retinal isolation
Whole eyes of mice were dissected. For immunohistochemical studies, dissected
eyes with attached pigment epithelium (RPE) were immediately fixed,
cryoprotected and sectioned. The frozen sections were stored at -20 °C for
immunohistochemical studies. Retinas dissected free from sclera and most of the
retinal pigment epithelium layer were used for explant culture. Retinas free of
sclera, most of the retinal pigment epithelium layer, and crystalline lens were
used to make retinal extracts
3.4 Retinal explant culture
Retinas were placed in UltraCulture (CambrexBio Science) serum-free medium
supplemented with gentamycin antibiotic (10 μg/mL). They were cultured
individually in 1 mL of medium in a 24-well culture dish at 37°C in a 5% CO2
(balance air) atmosphere. The reagents (Table 2) are dissolved in the medium
and the medium is changed every other day by replacing 0.5 mL with fresh
medium. The retinas were cultured for 30 min for Western blot analysis and for
four days or eight days for immunohistochemical studies (Zhang et al., 2002).
3.5 Western Blot
Retinas were lysed at 4 °C in buffer containing 1mL EDTA, 1 mL protease
inhibitor cocktail (Thermo scientific), and one phosphatase inhibitor cocktail tablet
(Roche) per 10 mL Cytobuster protein extraction reagent (Novagen). The tissues
25
were frozen and thawed three times to lyse the cells. The supernatant was
collected by microcentrifugation. Protein concentrations were measured by using
the Bio-Rad DC Lowry-based protein assay. Equal amounts (10-20 μg) of protein
were loaded onto polyacrylamide gels (any KD, Bio-Rad), and separated by
standard SDS-PAGE. Proteins were transferred to Immun-BlotTM nitrocellulose
membrane (Bio-Rad), and the membrane blocked with 5% nonfat dry milk in 1%
Tris-buffered saline containing 0.1% Tween 20. Membranes were incubated with
primary antibody overnight at 4 °C, followed by incubation with horseradish
peroxidase-conjugated secondary antibodies for 1-2 h at room temperature.
Proteins were detected by ECL western blot substrate (Thermo Scientific). The
quantitative
image
analysis
was
performed
using
Image
J
1.29
(http://rsb.info.nih.gov/ij/)
3.6 Immunohistochemistry
Explanted retinas and whole eyes were fixed with 4% paraformaldehyde in PBS
for 24 h at 4°C. After three washes with PBS for 10 min, fixed explant retinas
were dehydrated through a series of graded ethanol and embedded in paraffin.
The paraffin tissues were sectioned to 7μm. Whole eyes were dehydrated by
placing in 5% sucrose for 15min and 20% sucrose for 24 hours. They were
embedded in OCT compound, frozen and used to prepare 5-8 μm cryostat
sections. All samples for one experiment were placed on the same microscope
sliders for immunohistochemistry. Antigen retrieval was performed by using
26
sodium citrate buffer (pH 6), and boiling for 45 min. Retinal sections were
blocked by 5% nonfat milk for 1 hour and incubated in the following primary
antibodies overnight at 4 °C (diluted in 2% nonfat milk): PDK-1(Pierce, 1:200),
Phospho-PDK1 (Ser241,1:200), Phosphp-Akt (Thr308,1:200) are from Cell
signaling technology (Beverly,MA); Akt1(B1) (Santa Cruz Biotechnology, Santa
Cruz, CA,1:300 ), proliferating cell nuclear antigen (PCNA,1:1000) from
Sigma(Saint Louis, MI), Ret-P1 monoclonal antibody (1:100) (Colin et al,1980).
Next day the sections were incubated in secondary antibodies conjugated with
Alexa Fluor 488 or Alexa Fluor 594 (Invitrogen, 1:1000) for 3 hours, followed by
incubation with Hoechst 33258 (Fisher Scientific) diluted 1:1000 in 1% PBSTriton for 20 minutes. After several washes in PBS-Triton and PBS, the sliders
were mounted with 0.5% n-propyl gallate in 50:50 glycerol: PBS, 40 μL per slide
and covered with 24x50 mm coverslips. The edges were sealed. Sections were
imaged using an Olympus Fluoview FV1000 confocal microscope. For each set
of experiments, acquisition parameters for each antibody were held constant.
Sections used for cell counting were taken adjacent to the optic nerve and
contained the full extent of the retina from central to peripheral regions. Labeled
rods were counted over the full length of retina and normalized for variations in
tissue size. No correction was made for retinal thickness as all were sectioned by
microtome for 7 μm. Cell counts were obtained for three sections from each
retina, and at least three retinas were studied per treatment.
27
3.7 Statistical analysis.
Statistical analyses were performed using the GraphPad Prism software.
Student's t test (two-tailed, unpaired) was used to compare two groups, and oneway ANOVA (with Newman–Keuls post test) was used to compare more than
two groups.
28
CHAPTER 4
RESULTS
4.1 p55/p50α is the key functional signal for the development of
photoreceptor among different isoforms of PI3-kinase
PI3-kinase regulates a wide range of cellular processes including cell
proliferation, growth, survival, and metabolism. They generate lipid second
messengers that serve as membrane docking sites for the downstream signals
(Doreen et al, 2001; Ryosuke et al, 2008; Geering et al, 2007).
In previous experiment, it was found that p110α was expressed in both adult and
neonatal retinas. However, neither p110 β nor p110γ were expressed in adult or
neonatal retinas, indicating that retinal PI3-kinase activity is due to the PI3Kinase class IA isoform (Pinzon-Guzman et al, 2011). The PI3-kinase class IA is
activated by G-protein-coupled receptors and tyrosine kinase receptors, including
the IGF-1 receptor (Concepción et al, 2002). Previous experiment showed that
the action of IGF-1 on PI3-kinase class IA changed during retina development
(Pinzon-Guzman et al, 2011). In neonatal mice IGF-1 treatment of retinal
explants caused a decreased in Akt activation, suggesting a decrease in PI3kinase class IA activity. In adult mice, however, IGF-1 treatment leads to
increased Akt activation, suggesting an activation of PI3-kinase class IA.
29
In this thesis, we hypothesized that a development change in the regulatory
subunits of PI3-kinase class IA might account for the change in downstream
response and might play an important role in regulating rod development. To
investigate which regulatory subunits could play a role in the development of
photoreceptors, we examined the presence and localization of five isoforms of
the regulatory subunits, p85α, p55α, p50α, p85β, and p55γ, in the mouse retina
from each age group of wild type (WT) mice at the ages of PN1, 3, 7, 13, and 28
(adult). In order to estimate the proportion of expression of the PI3-kinase class
IA subunits due to rod photoreceptors, RD1 mutant mice were included in the
experiment. Homozygous mice for the rd1 mutation have an early onset severe
retinal degeneration. By 21 days after birth virtually all rod photoreceptors in the
retina have died in RD1 mice. Therefore we also examined the expression of
class IA PI3 kinase regulatory subunits from RD1 mice (PN28).
First, a specific antibody that targets the C-terminal SH2 domain of p85 alpha
and p55α/p50α subunits was used to find out whether p55α or p50α subunits
exist in mice retinas. As shown in Figure 7, p85α was expressed in retinas from
all ages of wild type mice, while p55α or p50α subunits were detected only after
PN5. p85α also was expressed in PN28 RD1 mice while p55α or p50α was not.
30
Wild type
PN1
PN3
PN5
PN7
0
PN13
PN28
RD1 0
PN28 0
P85α →
P55α →
Beta actin →
Figure 7. Expression of p85α and p55α regulatory subunit of PI3K. Western
blot of lysates from WT mice at PN1, 3, 5, 7, 13, 28(adult) and PN28 RD1 mouse
retinas with anti-p85/p55α antibody.
Significant differences of expression of p55α or p50α were found among retinas
from different time points in the, which suggest p55α or p50α may be one of the
functional subunits controlling of PI3-kinase class IA activity during retinal
development.
We examined the expression of p85β subunit in the same protein lysates. A
specific antibody against PI3-kinase p85β subunit was applied in western blot.
We found that p85β was not detected in any age of wild types and PN28 RD1
mice retinas. It has been reported that p85β was expressed in the liver and heart
of rodent animals (Hartong et al, 2006). In order to verify the validity of the p85β
antibody, we extracted protein lysates from retina, liver and heart tissues from
31
postnatal day 1 mice and adult mice. The same antibody was applied in the
western blot to detect the expression of p85β in different tissues (Figure 7).
PN1
retina
heart
0
liver
Adult
retina
heart
0
liver
100→
p85β
75→
50→
Figure 8. p85β is not expressed in either neonatal or adult retinas. Western
blot of lysates from retina, heart and liver of postnatal day 1 and adult mice with
anti-p85β antibody, n=3.
p85β subunit was only weakly expressed in adult heart. It was not found in
protein lysates from postnatal day1 mouse retina, heart, liver, or adult mouse
retina and liver. Therefore, p85β is not contributing in the development of retinal
photoreceptors. On the other hand, as the Figure 8 shows, there are bands
present in the lysates from mouse heart and liver, located in the area between 50
and 75 KDa. However, what they represent is unknown, and requires further
testing.
32
We next examined the expression of p55γ subunit of PI3-kinase class IA in the
retinas. p55γ is derived from a distinct gene Pi3kr3.
A.
0
Wild type
PN1
PN3
PN5
PN7
PN13
PN28
RD1 0
RD1
0
p55γ→
Beta actin→
B.
.
Figure 9. p55γ exists in all age of wild mice and PN28 RD1 retinas.
(A).Western blot of lysates from WT mice at PN1, 3, 5, 7, 13, 28(adult) and PN28
33
RD1 mouse retinas with anti-p55γ antibody; (B).Quantitative graph of Figure 9A.
*P<0.05 **P<0.01 vs Adult. n=4
As shown in figure 9 A, p55γ was expressed in all age of wild type and RD1 mice
retinas. The amount of p55γ presenting in the retinas increased gradually from
postnatal day 1 to day 28. However, the expression of p55γ in RD1 decreased
when it was compared to the adult mice. (Figure 9 B) The amount of p55γ
increased as the growth of retinal cells, which indicated that p55γ may play an
important role in the signaling pathway for growth factor of insulin family in mice
retina. Subsequently, p55γ may also function on the survival of photoreceptor
cells.
To find out which cell population in the mice retina expressed p55γ, an
immunohistochemistry staining for fresh frozen mice retinas from different age
was performed. Proliferating cell nuclear antigen (PCNA) antibody that labels the
dividing cells and hoechst33258 were applied in the experiment as the p55γ
antibody.
34
Figure 10. p55γ is expressed in all age of wild mice retinas. Expression of
p55 gamma co-labeled with PCNA (Green), hochest33258 (Blue) in the central
and peripheral retina of PN1, 3, 5, 7, 13, 28 (adult) WT mice and PN28 RD1 mice.
ONL, outer nuclear retinal; INL, inner retinal; IPL, inner plexiform layer; ONBL,
outer neuroblastic layer; INBL, inner neuroblastic layer
35
As Figure 10 shows, p55γ is expressed in all age of wild type mice retina. In the
PN1, 3, 5, 7 retinas, p55γ signal was presented in the whole layer. By postnatal
day 13, when the retina approached maturity, p55γ became distributed in the
inner segment of photoreceptors, inner nuclear layer and ganglion cell layer.
We previously demonstrated that p85α, p55/p50α and p55γ were expressed in
mouse retinas. The activity of class IA PI3-kinase stimulation is mediated by
tyrosine kinases (Concepción et al, 2002). p85 subunit inhibited the enzymatic
activity of p110 and prevented it from degradation. Tyrosine phosphorylation of
p85 relived the inhibitory activity of p85. p85 mediated p110 translocation to the
cell membrane, and the downstream signal such as PDK-1 and Akt were
subsequently activated (Anderson et al, 1998).
To investigate the expression level of tyrosine phosphorylation of p85 in the
development of photoreceptor cells, we tested the phosphorylation state of p85
by using antibody against phosphor-p85 at tyrosine 459 combined with p55
splice variant tyrosine phosphorylation at residue 199 on the same retinal lysates.
A.
Wild type
PN1
PN3
PN5
Phospho-p85 (Tyr458)→
Phosoho-p55 (Tyr199)→
Beta actin→
36
PN7
0
PN13
PN28 0
RD1 0
PN28
B.
Figure 11. Phosphorylation of p55α at Tyr199 on matured mouse retinas. (A)
Western blot of retinal lysates from PN1, 3, 5, 7, 13, 28 (adult) WT mice and
PN28 RD1 mice with anti-phosphor-p55 (Tyr199) and anti-phosphor-p85 (Tyr458)
antibody. (B) Quantitative graph of Figure 11A.
The tyrosine phosphorylation of p85 was not found in mouse retinas. However,
the tyrosine phosphorylation of p55 at residue 199 was significantly expressed
after postnatal day 13, while none was found in the postnatal day 1 and day 3
retinas. (Figure 11 A) The amount of phosphorylated p55 Tyr199 in adult retinas
was increased at least five fold compared with day 13. Neither phospho-p55 nor
phospho-p85 was detected in RD1 retinas. (Figure 11 A) In order to examine
which cell population in the mice retinas contains phospho-p50/p55, we
37
performed an immunohistochemistry staining for fresh frozen mice retinas from
various age was performed. The phospho-p85 at Tyr459 combined with p55
Tyr199 antibody, proliferating cell nuclear antigen (PCNA) antibody that labels
the dividing cells and hoechst33258 were applied in this experiment.
38
Figure 12. Tyrosine phosphorylation of p55 at residue 199 is expressed
only on photoreceptor layer of matured mouse retinas. Immunofluorescence
labeling of retinas from wild type PN 1 (A,B,C,D),3 (E,F,G,H),5 (I,J,K,L),7
(M,N,O,P,),13 (Q,R,S,T) and adult mice (U,V,W,X). They are stained with Antiphospho-p55 (Tyr 199) in red, PCNA in green, and hoechst in blue. Arrows point
to the p-p55 labeling in the photoreceptors layers. ONBL, outer neuroblast layer;
INBL, inner neuroblast layer; IPL, inner plexiform layer; ONL, outer nuclear layer;
INL, inner nuclear layer.
These data suggest tyrosine phosphorylation of p55 instead of p85 may be
responsible for the regulation of retinal development. It is known that 95% of the
Rod photoreceptor cells finish cell genesis by postnatal day 5 (Cepko et al, 1996).
The absence of phospho-p55 in PN1, 3 and PN28 RD1 mice indicate that
phospho-p55 may play an inhibitory role in the development of rod photoreceptor.
The expression of regulatory subunits of PI3-kinase is summarized in Table 4.
Wild type
RD1
PN1 PN3 PN5 PN7 PN13 PN28 PN28
p85α
+
+
+
+
+
+
+
p55α
-
-
+
+
+
+
+
p50α
-
-
+
+
+
+
+
p85β
-
-
-
-
-
-
-
p55ᵧ
+
+
++
++
+++
++++
++
39
Table 4. Members of the PI3-Kinases class I subunits present in the mouse
retinas.
4.2 Role of 3-phosphoinositide-dependent protein kinase-1 in rod
photoreceptor differentiation
The 3-phosphoinositide-dependent protein kinase-1 (PDK-1) was originally
identified as a protein serine and threonine kinase that is critical for Akt (protein
kinase B) activation in response to growth factor receptor signaling. PDK-1
interacts with PIP3 the product of PI3-Kinase class IA. It is also a crucial activator
of multiple protein kinases, including p70S6K1, PKC isoforms, and SGK.
Activation of these intrinsic cell signals promotes cell growth, proliferation and
inhibition of pro-apoptotic proteins.
In our previous experiment, it was demonstrated that PI3-kinase class IA / PKCs
/STAT3 pathway was linked to rod photoreceptor differentiation. Thus we chose
to investigate whether PDK-1 was also involved in this pathway.
In this chapter we tested the hypothesis that PDK-1 plays an important role in
regulating the formation of rod photoreceptors in neonatal mouse retina. We
show that pharmacological inhibition of PDK-1 induced a significant decline of
Akt
threonine
phosphorylation,
ribosomal
S6-kinase
(S6K-1)
threonine
phosphorylation and PKC gamma threonine phosphorylation. Furthermore,
treatment of retinal explants with PDK-1 inhibitor (BX795) for four days increased
40
the number of rod photoreceptors. The results indicate that PDK-1 activity is a
critical signal in regulating rod photoreceptor differentiation.
4.2.1 PDK-1 is expressed in both adult and PN1 mouse retinas
In order to find out whether PDK-1 exists in mice retinas, we measured the
expression of PDK-1 by using a specific antibody that targets the endogenous
levels of total PDK1 protein in western blot. It has been reported that serine
phosphorylation of PDK-1 at residue 241 is essential for PDK-1 catalytic activity
(Antonio et al, 1999). Therefore, we also examined the expression of serine 241
phosphorylation of PDK-1 in the same retinal lysates.
**
1.5
1.0
0.5
)
0.0
A
du
lt(
P2
8
P1
relative intensity of PDK-1
A.
41
1.5
1.0
0.5
)
0.0
A
du
lt(
P2
8
P1
Intensity of P-PDK-1 Ser241
B.
Figure 13. Quantitative graph of total PDK-1 and phospho-PDK-1 (Ser241)
found in PN1 and adult (day 28) mouse retinas. Western blot of retinal lysates
from PN1 and adult (day 28) mouse retinas with anti- PDK-1 and phospho-PDK-1
(Ser241) antibody, **p<0.01, n=3.
As shown in Figure 13, PDK-1 and P-PDK-1(Ser241) were expressed in both
wild type postnatal day 1 and adult mouse retinas.
To find out which cell population in the mouse retina expressed serine241
phospho-PDK-1, we performed an immunohistochemistry staining of fresh frozen
mouse retinas from different developing stages. The phospho-PDK-1 (Ser241)
antibody, proliferating cell nuclear antigen (PCNA) antibody that labels the
dividing cells and hoechst33258 were applied in the experiment.
42
Figure 14. Expression of phospho-PDK-1 (Ser241) increased gradually from
postnatal day 1 to adult (day 28) mice retina. Expression of serine phosphoPDK-1 at residue 241 (Red) co-labeled with PCNA (Green), hoechst33258 (Blue).
43
Immunofluorescence labeling of retinas from wild type PN 1 (A,B,C,D), 3
(E,F,G,H), 5 (I,J,K,L), 7 (M,N,O,P,), 13 (Q,R,S,T) and 28 (adult) mice (U,V,W,X).
ONL, outer nuclear retinal layer; INL, inner retinal layer; IPL, inner plexiform layer;
ONBL, outer neuroblastic layer; INBL, neuroblastic layer.
Phospho-PDK-1 (Ser241) was weakly expressed in PN1. The expression
increased gradually throughout development of the mouse retina. By PN3 the
fluorescence signal was mainly in the neuroblast layer outside the PCNA labeling.
The fluorescence signal became heavily concentrated at the outer plexiform layer
after
PN13.
This
layer
consists
of
a
dense
network
of
synapses
between dendrites of horizontal cells and bipolar cells from the inner nuclear
layer and photoreceptor cell axon terminals from the outer nuclear layer. The size
and spacing of labeled cell bodies in the inner nuclear layer suggests that much
of this labeling may be due to horizontal cells. This result indicated that PDK-1
may function on signal transduction among photoreceptors and bipolar,
horizontal cells by phosphorylating PDK-1 Ser241.
4.2.2 PN 1 wild type mice develop a higher amount of rod photoreceptors
after four days treatment with PDK-1 inhibitor
To test the hypothesis that PDK-1 involves in development of rod photoreceptor
rather than other cells in the retinas, a PDK-1 inhibitor (BX795) was applied to
the four day explant culture of retinas from PN 1 wild type mice. BX795 is a
44
potential PDK-1 inhibitor (IC50 10 nM-1µM) that works as an ATP competitor and
targets the ATP binding site (Harald et al, 2005). The catalytic activity of PDK-1
functions through transferring the phosphate groups from ATP to a protein
substrate or downstream signal, thus BX795 was able to block the PDK-1
enzyme activity. A dose dependent experiment was performed to find out the IC50
of BX795 on stimulating rod photoreceptor differentiation. P1 retinas were
cultured with increasing doses of BX795 for four days. After four days, retinas
were fixed, embedded, sectioned and labeled with a Ret-P1 monoclonal antibody
that recognizes an epitope on the N terminus of opsin of rod photoreceptor and
hoechst33258 to examine differentiated rod photoreceptors. Quantitative
measurement of rhodopsin was collected to determine the IC50.
A.
Dapi+Rhodopsin
Dapi+Rhodopsin
0μM
Dapi+Rhodopsin
0.1μM
Dapi+Rhodopsin
Rhodopsin
5μM
Dapi+Rhodopsin
Dapi+Rhodopsin Rhodopsin
Rhodopsin
Dapi+Rhodopsin
Rhodopsin
10μM
Rhodopsin
40μM
B.
45
1μM
Rhodopsin
20μM
Intensity of Rods
50
40
30
20
10
0
0
1
2
3
4
5
Log(Bx795)nM
Figure 15. Effects of inhibition of PDK-1 by increasing dose of BX795 in
four days explant culture of P1 wild type mouse retinas. (A).
Immunofluorescence staining of opsin (green) co-labeled with nuclear
counterstain (hoechst33258, blue) for PN1 retinas after four days explant culture
in the presence of 0, 100nM, 1µM, 5µM, 10 µM, 20 µM and 40 µM of BX795. (B).
Quantitative measurement of Figure 15 A. Fluorescent intensity of rhodopsin
from postnatal day 1 retinas after four days explant culture with BX795. Values
were obtained by averaging the fluorescence intensity of three representative
areas each of triplicate retinas.
The intensity of rod photoreceptors reached the maximum after treatment with
1µM BX795, and intensity declined when the dose of BX795 exceeded 1µM. In
addition, the number of rods in mouse retinas treated with greater or equal to 10
µM BX795 was lower than those without treatment of BX795, and rods were
almost abolished after treated with 40 µM BX795. These results suggested that
the IC50 of PDK-1 on stimulating differentiation of rod photoreceptors is around
46
100nM. The effect of BX795 changed to inhibiting the formation of rods at the
dose greater or equal to 10 µM.
4.2.3 The effects of IGF-1, and PMA on rod photoreceptor differentiation
were enhanced after PDK-1 inhibition
In previous experiments, it was demonstrated that 1) IGF-1 promoted rod
photoreceptor differentiation in a PKCs dependent manner; 2) Activation of PKCs
by PMA stimulated the formation of rod photoreceptors; 3) Inhibition of PI3
Kinase by the pan inhibitor LY294002 increased the number of rod
photoreceptors, which was also affected through a PKCs dependent manner. In
order to study how PDK-1 promotes the differentiation of rod photoreceptors, we
treated the P1 WT retinas with 1) IGF-1 (50nM) to activate PI3-kinase and BX795
(100nM) to inhibit PDK-1; 2) PMA (100nM) to activate PKCs and BX795 (100nM)
to block the activity of PDK-1; Retinas were cultured for four days and at least
three retinas were used in each experiment.
47
A.
Control
.
IGF-1
BX795
IGF-1+BX795
B.
Control
PMA
BX795
PMA+BX795
48
Figure 16. The effects of IGF-1 and PMA on rod photoreceptor
differentiation are enhanced after PDK-1 inhibition. (A). Immunofluorescence
staining of opsin (green) co-labeled with nuclear counterstain (hoechst33258,
blue) for postnatal 1 retinas after four days explant culture in the presence of
50nM IGF-1 and 100nM BX795. (B). Immunofluorescence staining of opsin
(green) co-labeled with nuclear counterstain (hoechst33258, blue) for postnatal
day 1 retinas after four days explant culture in the presence of 100nM PMA and
100nM BX795.
As indicated in the Figure 16, retinas treated with IGF-1, and PMA developed a
higher number of rod photoreceptors compared to untreated retinas after four
days explant culture. However, the intensity of rod photoreceptor increased more
when each applied together with BX795. It may implicate that inhibition of PDK-1
has a dominant role in regulating rod photoreceptor differentiation.
4.2.4 Inhibition of PDK-1 resulted in reduced Akt, p70S6K1 and PKC gamma
threonine phosphorylation in PN1 mouse retinas
PDK-1 is shown to be the protein kinase that phosphorylates and activates many
protein kinases from AGC family (PKB, PKC and PKA etc.). It has been verified
that Akt and PKCs are the downstream signals of PDK-1 in many tissues (Le
Good et al, 1998; Newton, 2001). Akt and PKCs have a threonine residue in their
activation loop segment that must be phosphorylated before the kinases can
phosphorylate its substrates. Threonine phosphorylation of AKT at 308 is
49
inhibited by BX795. p70S6K1 is recognized as one of the substrates of PDK-1.
PDK-1 catalyzes the threonine phosphorylation of p70S6K1 at 386 and its
phosphorylation represents the kinase activity of PDK-1 (Alessi et al., 1998).
To test the hypothesis that PDK-1 is the upstream of PKCs, we first carried out
the control experiment measuring the phosphorylation of p70S6K1 under
stimulation of PKCs and inhibition of PDK-1 to examine whether p70S6K1 was
the substrate of PDK-1. PN 1 WT mouse retinas were pre-incubated in the
cultured medium for four hours to allow activated intracellular signals to return to
the baseline (Zhang et al, 2004). After four hours, the retinas were treated
separately with PMA, BX795 for 30min and then collected to extract protein
lysates. Specific antibodies against p-p70S6K1 Thr389, p-PKC gamma Thr514
were used in the western blot.
A.
Control
PMA
p-p70 S6K→
Beta actin →
50
BX795
0
B.
p-p70S6K1
intensity of P--p70S6K
1.0
0.8
0.6
**
0.4
0.2
nM
BX
PM
79
5
A
10
0
10
0n
M
co
nt
ro
l
0.0
C.
Control
P-PKC γ(Thr514) →
PMA
BX795 0
0
Beta actin →
D.
1.5
1.0
**
0.5
10
0
79
5
BX
PM
A
10
0n
M
nM
0.0
co
nt
ro
l
intensity of P--PKC r (thr514)
P-PKC gamma Thr514
4 hours preincubation+30 min treatment
n≥4
51
Figure 17. Inhibition of PDK-1 decreased threonine phosphorylation of
p70S6K1 at 389 and PKCγ at 514. (A,C), Western blot of postnatal day 1 retina
after 30 min treatment of PMA and BX795, with phospho-p70S6K at Thr389 and
phospho-PKCγ at Thr514. (B,D), Quantitative graph of phospho-p70S6K1
(Thr389) and phospho-PKCγ at 514 found in PN1 retina after 30 min treatment of
PMA and BX795.
Retinas treated with BX795 had a decrease of threonine phosphorylation of
p70S6K1 at residue 389, while the level of phospho-p70S6K1 did not change in
PMA treated retinas. Phospho-PKC gamma at Thr514 decreased in BX795
treated retinas, yet increased in PMA treated retinas. Therefore, inhibition of
PDK-1 affected the phosphorylation of p70S6K1 and PKCγ, however, activation
of PKCs did not change the level of p70S6K1, which indicated that PKCγ is the
downstream signal of PDK-1.
We next examined the threonine phosphorylation of Akt at 308. PI3-kinase class
IA triggers the activation of Akt through PDK-1.Phosphoinositides produced by
PI3-kinase class IA bind directly to the PH domain and induce a conformational
change of Akt, which enables the activation loop of Akt to be phosphorylated at
Thr308 by PDK-1 (Filippa et al, 2000; Anderson et al, 1998). The Level of Thr308
Akt phosphorylation was used to evaluate the effects of BX795 on the intrinsic
cellular signals. A dose dependent experiment was performed to determine the
IC50 of BX795 by 30min retinal explant culture.
52
A.
0
10nM
250nM 1µM
2.5µM
10µM
0
p-Akt Thr 308 →
Beta actin →
B.
Intensity of Akt T308
1.0
0.8
0.6
0.4
0.2
0.0
0
1
2
3
4
5
Log BX795(µM)
Figure 18. The level of Akt thr308 phosphorylation decreased after 30 min
inhibition of PDK-1.
(A), The detection of Akt Thr308 phosphorylation in Western blot. Lysates were
extracted from PN 1 retinas after 30min explant culture in the presence of 0,
10nM, 250nM, 1µM, 2.5 µM, and 10 µM of BX795. (B), Quantitative
measurement of Figure 18 B. n=3.
53
Thr308 phosphorylation of Akt decreased gradually under the inhibition of PDK-1.
As Figure 18 A shows, the IC50 of BX795 inhibiting Akt activity is around 1µM.
Akt is known as a downstream signal of PI3-kinase that is involved in cell
proliferation and survival. The PI3-kinase pan inhibitor LY294002 was used in
comparison with the PDK-1 inhibitor. PN1 WT mice retinas were pre-incubated in
culture medium for four hours. The retinas were then cultured for 30min in the
presence of LY294002 and BX795 separately. Phosphorylation of Akt at Thr308
was measured by western blot.
Phosphorylation of Akt at Thr308 decreased by 50% of the level in the retina
after treatment with LY294002 (n=3, p<0.001) and BX795 (n=3, p<0.01). In
addition, PDK-1 inhibited retinas showed increased levels of Akt phosphorylation
at Thr308 when compared to retinas treated with the PI3-kinase pan inhibitor.
relative intensity of P-Akt
1.5
1.0
**
***
0.5
uM
B
X7
9
5
1
50
uM
LY
co
nt
ro
l
0.0
Figure 19. LY294002 and BX795 decreased the threonine phosphorylation
of Akt at 308. Quantitative graph of phospho-Akt (Thr308) found in postnatal day
54
1 retina after 30min treatment of LY294002 and BX795. **P <0.01, *** P<0.001
vs control
4.2.5 Inhibition of PDK-1 inhibited tyrosine 705 phosphorylation of STAT3
Activation of STAT3 inhibits differentiation of rod photoreceptors (Zhang et al,
2004). Previous experiments have verified that activation of PKCs enhanced the
differentiation of rod photoreceptor through reduction of the tyrosine 705
phosphorylation of STAT3. We hypothesized that the inhibition of PDK-1
stimulates the differentiation of rod photoreceptors through inhibition of STAT3.
The tyrosine 705 phosphorylation of STAT3 was examined by using lysates
prepared from retinal explant cultured for 30 min with BX795. At the same time,
lysates from retinas treated with PMA were used as the control group.
As the Figure 20 shows, inhibition of PDK-1 decreased the tyrosine 705
phosphorylation of STAT3 compared to untreated retinas (n=3, P<0.001).
55
A.
B.
Control
PMA
BX795
0
P-STAT3 Y705 →
Beta actin →
Figure 20. Inhibition of PDK-1 decreased tyrosine 705 phosphorylation of
STAT3. (A), Quantitative graph of phospho-STAT3 (Y705) found in PN1 retina
after 30min treatment of PMA and BX795. (B), Western blot of PN1 retina after
30min treatment of PMA and BX795 with the antibody for tyrosine 705
phosphorylation of STAT3, *P<0.01 **P<0.001 vs control, n=3.
56
CHAPTER 5
General discussion
We showed previously that the PI3-kinase class IA/PKCs/STAT3 pathway is
linked to rod photoreceptor differentiation (Pinzon-Guzman et al, 2011). IGF-1
was used as an effective extracellular signal that triggers the development of rod
photoreceptor by activating PKCs. STAT3 is the downstream target of PKCs and
is inhibited by active PKCs in PN1 mouse retinas. STAT3 inhibition initiates
differentiation of retinal multipotent stem cells to functional rod photoreceptors.
In this study, we observed significant differences among retinas from different
time points in the expression of p55 or p50 alpha. We also observed the level of
p55 gamma increased with age of retinal cells. These observations lead to the
hypothesis that throughout the retinal development there is a change in the
expression of regulatory subunits of PI3-kinase class IA family.
Class IA PI3-kinases are considered to be a pivotal factor in mediating proper
growth factor signaling to metabolism, proliferation and differentiation. p85α,
p55α, p50α, p85β, p55γ have been identified as the five isoforms of the
regulatory subunits of PI3-kinase class IA. (Geering et al, 2007). Activation of
PI3-kinase class IA is caused by a phosphorylated receptor tyrosine kinase (RTK)
binding to the p85α N-terminal SH2 domain: Binding of the regulatory subunit to
tyrosine-phosphorylated proteins activates the p110 catalytic subunits. The
catalytic subunits phosphorylate the lipid domain PIP2 to PIP3. Generation of PIP3
allows the activation of downstream effector proteins (Concepción et al, 2002).
57
Evidence
has
been
presented
that
rods
use
PI3-kinases-generated
phosphoinositides for photoreceptor function (Ivana et al, 2011), however, it is
not clear which of these isoforms are involved in photoreceptor differentiation. It
has been demonstrated that among the catalytic subunits only p110α was
expressed in both adult and neonatal retinas in previous experiments. The
current study was focused on examining the presence and localization of five
isoforms of the regulatory subunits, p85α, p55α, p50α, p85β, p55γ, in the retinas
from PN1, 3, 5, 7, 13, 28 (adult) WT mice and PN28 RD1 mice.
Our data shown that p85α was expressed in retinas from all ages of wild type
mice, while p55α or p50α subunits were detected only after PN5. p85α was also
expressed in RD1 mice while p55α or p50α was not. It is known that p55α or
p50α alpha are the splice variants from the same gene of p85α (Pik3r1) (Geering
et al, 2007), and the tyrosine phosphorylation of regulatory subunits of PI3-kinase
releases the inhibitory activity of p85α (Concepción et al, 2002). We have also
examined the level of tyrosine phosphorylation of p55 and p50α in different age
of retina cells. The tyrosine phosphorylation of p85 was not found in the mouse
retinas. However, the amount of tyrosine phosphorylation of p55 at residue 199
increased significantly after PN 13, while none was detected in the PN1 and PN3
retinas. Moreover, neither phospho-p85 nor phospho-p55 was detected in RD1
retinas. Phosphorylation of p55 at Tyr199 was expressed only on photoreceptor
layer of matured mouse retinas (Figure 12). Therefore it is possible that in mouse
retinal cells p55α or p50α are the functional subunits controlling of PI3-kinase
activity during retinal photoreceptor development. In previous experiment, IGF-1
58
did not induce activation of the regulatory subunits of PI3-kinase in neonatal
mouse retinas, which might have been due to the absence of the p55α or p50α
subunits. On the other hand, we examined the expression of p85β and p55γ in
different age of mouse retinas. p85β was not present in the mice retinas while
p55γ expressed in all age of wild type and RD1 mice retinas. The amount of p55γ
present in the retinas increased gradually from postnatal day 1 to day 28,
indicating that p55γ might also play an important role in the signaling pathway for
growth
factor
of
insulin
family
in
mouse
retinas.
Meanwhile,
immunohistochemical staining using anti-p55γ antibody revealed that p55γ signal
was presented in the whole layer of PN1, 3, 5, 7 retinas. By 13 day, when the
retinas approached maturity, p55γ became distributed in the inner segment of
photoreceptors, inner nuclear layer and ganglion cell layer. These results
suggest that p55γ may also function in supporting the survival of photoreceptor
and other retinal cells. However, the functional roles of p55/p50α and p55γ in rod
photoreceptor development and the mechanism of how they affect the formation
of rods under different treatments are still unknown. It is a question that needs to
be addressed in future experiments.
PI3-kinase catalyzes PIP2 to PIP3, which interacts with the PH domain of Akt and
PDK-1. PDK-1 interacts with different downstream signals, such as Akt, PKCs
and p70S6K1. It is reported that PKCs (βI, γ) are required in rod photoreceptor
differentiation and PKCs function through STAT3 signaling (Pinzon-Guzman et al,
2011). In our current study, we have investigated the role of PDK-1 in the
differentiation of rod photoreceptor and demonstrated that PDK-1 activity is one
59
of the critical signals regulating rod photoreceptor differentiation. PDK-1 and
serine phosphorylated PDK-1 at 241 was found in both adult and postnatal day 1
mouse retinas.
It is known that serine phosphorylated of PDK-1 at 241 is
required for its full activation (Casamayor et al, 1999). The level of phospho-PDK1 (Ser241) gradually increased in the central region of the most outer layer of the
outer retina (Figure 14), indicating that phospho-PDK-1 could be involved in the
rod photoreceptor development at the early stage. The fluorescence signal
became heavily concentrated at the outer plexiform layer after PN13. This result
indicates that PDK-1 might be in a position to transduce signals among
developing photoreceptors, bipolar and horizontal cells. How the PDK-1 functions
in the rod photoreceptor differentiation was investigated by examining the
development of rhodopsin, the level of Akt, p70S6K-1 and PKC gamma threonine
phosphorylation after pharmacological inhibition of PDK-1. Treatment of retinal
explants with PDK-1 inhibitor (BX795) for four days increased the number of rod
photoreceptors, which suggests that inhibition of PDK-1 promotes the
differentiation of rod photoreceptors. IC50 of BX795 on stimulating differentiation
of rod photoreceptors was approximately 100nM.
In previous experiment, IGF-1 as a PI3-kinase activator and PMA as the direct
stimulator of PKCs increased the number of rods after four days explant culture.
The effects of IGF-1, and PMA on rod photoreceptor differentiation were
enhanced after PDK-1 inhibition (Figure 16), which suggest that inhibition of
PDK-1 has a synergistic effect with activation of PI3 kinase and PKCs in
regulating rod photoreceptor differentiation. One mechanism by which inhibition
60
of PDK-1 results in increased number of rods is modulating the activity of STAT3
through suppression of its tyrosine phosphorylation. It is supported by the fact
that inhibition of PDK-1 decreased tyrosine 705 phosphorylation of STAT3.
This result suggests that PDK-1 modulates the effect of STAT3 in rod
photoreceptor development.
IGF-1
IR
PIP2
PIP3
PI3K
PDK-1
BX795
Akt
PKC
P70 S6K1
Plasma membrane
PMA
STAT3
Nuclear
membrane
Rod photoreceptor
differentiation
Gene expression
Figure 21. A diagram showing the relationship of PI3-kinase/PKCs/PDK1/STAT3 signaling pathway
61
The interaction mechanisms of PDK-1 with Akt, p70S6K1 and PKCs are different.
Activation of Akt by PDK-1 is PIP3 dependent. PIP3 binds to the PH domain of
Akt and uncovers the Akt active sites, hence allowing phosphorylation from PDK1 (Alessi et al, 1998). It is found that the level of Thr308 phosphorylation of Akt1
decreased gradually under the inhibition of PDK-1 (Figure 18). However, 100nM
of BX795 does not change the phosphorylation of Akt.
p70S6K1 is a member of the AGC kinase family. Activation of p70S6K1 by PDK1 is PIP3 independent. PDK-1 has a PDK1-interacting fragment pocket (PIF) that
is accessible for interactions. p70S6K1 has hydrophobic motif (HM) domains that
are capable of possessing the PIF. The binding of HMs with PDK1 PIF pocket
promotes activation of PDK1, enabling the phosphorylation of substrates at the
activation loop and promoting the binding of the phosphorylated HMs to their own
PIF pockets, which helps stabilize the active conformations of p70S6K1 (Pullen
et al, 1998). Classical PKCs also have HM domains. Their activation is achieved
by Ca2+ and DAG. PKCs interact with PDK-1 through their dephospho-HMs and
become phosphorylated at their activation loop, which is required for activity and
molecular stability (Davey et al, 2000). We found that inhibition of PDK-1
decreased the phosphorylation of p70S6K1, however, activation of PKCs did not
change the level of p70S6K1. On the other hand, phospho-PKCγ at Thr514
decreased in BX795 treated retinas, yet in PMA treated retinas there was no
statistical difference (Figure 16). These data suggested that PKCs is downstream
of PDK-1 and PDK-1 can activate phosphorylation of PKCγ. 100nM of BX795
was able to affect the phosphorylation of p70S6K1 and PKCγ. However, 100nM
62
of BX795 did not affect the phosphorylation of Akt. Therefore, our results suggest
that the effects of PDK-1 during rod development may be related to p70S6K1
phosphorylation but independent of Akt activity.
In summary, p55α or p50α, p55γ subunits are recognized as the functional
subunits controlling PI3-kinase class IA activity during retinal photoreceptor
development, however, the functional role of each subunit is not yet elucidated.
In addition, we also identified PDK-1 as a key intermediate molecule involved in
the
PI3-Kinase/PKCs/STAT3
pathway
that
regulates
rod
photoreceptor
differentiation. Inhibition of PDK-1 promotes photoreceptor differentiation through
the modulating of STAT3 activity.
63
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