From The Schepens Eye Research Institute, Harvard Medical School, Boston, Massachusetts 02114
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ABSTRACT |
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Previous studies have shown that Src is required
for platelet-derived growth factor (PDGF)-dependent cell
cycle progression in fibroblasts. Since fibroblasts usually express
both PDGF receptors (PDGFRs), these findings suggested that Src was
mandatory for signal relay by both the and
PDGFRs. In this
study, we have focused on the role of Src in signal relay by the
PDGFR. In response to stimulation with PDGF-AA, which selectively
engages the
PDGFR, Src family members (Src) associated with the
PDGFR and Src kinase were activated. A mutant receptor, in which
tyrosines 572 and 574 were replaced with phenylalanine (F72/74), failed
to efficiently associate with Src or activate Src. The wild type (WT)
and F72/74 receptors induced the expression of c-myc and
c-fos to comparable levels. Furthermore, an equivalent
extent of PDGF-dependent soft agar growth was observed in
cells expressing the WT or the F72/74
PDGFR. Comparing the ability
of these two receptors to initiate tyrosine phosphorylation of
signaling molecules indicated that both receptors mediated
phosphorylation of the receptor itself, phospholipase C
1, and SHP-2
to similar levels. In contrast, the F72/74 receptor triggered
phosphorylation of Shc to 1 and 20% of the WT levels for the 55- and
46-kDa Shc isoforms, respectively. These findings indicate that after
exposure of cells to PDGF-AA, Src stably associates with the
PDGFR,
and Src activity is increased. Furthermore, Src is required for the
PDGF-dependent phosphorylation of signaling molecules such
as Shc. Finally, activation of Src during the
G0/G1 transition does not appear to be required
for latter cell cycle events such as induction of c-myc or
cell proliferation.
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INTRODUCTION |
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Platelet-derived growth factor
(PDGF)1 is a homo- and
heterodimer consisting of two homologous chains, A and B. All possible isoforms exist and are biologically active. Like the ligand, the receptor is also a dimer that can exist as a homo or hetero combination of the and
subunits of the PDGF receptor (PDGFR). Exposure of
cells to PDGF assembles the monomeric receptor subunits into dimers, in
which each of the subunits binds one of the chains of the dimeric
ligand. The PDGFR subunits differ in their ability to bind to the two
PDGF chains. The
subunit binds only the B chain with high affinity,
whereas the
subunit binds both PDGF chains. Consequently, the PDGF
isoform will determine the subunit composition of the PDGFR. PDGF-AA
assembles only
homodimers, PDGF-AB induces
and
dimers, and PDGF-BB, the universal ligand, leads to formation of all
possible PDGFR subunit dimers (1). In this work we will focus on the
PDGFR, which is activated by all forms of PDGF and can be
selectively activated with PDGF-AA.
Once the PDGFR has been activated, it triggers a number of signaling
cascades. Comparison of the signal relay pathways engaged by the and
PDGFRs demonstrates that although there are many similarities,
there are also a number of differences. For instance, the SH2
domain-containing phosphotyrosine phosphatase, SHP-2, binds to the
kinase insert of the
PDGFR (2), but to the tail of the
PDGFR (3).
Furthermore, although both of the receptors recruit and associate with
phosphatidylinositol 3-kinase, phospholipase C
(PLC
), and SHP-2,
only the
PDGFR stably associates with GTPase-activating protein of
Ras (RasGap) (4, 5). In regard to which of these signaling molecules
are required for sending a biological signal, reports to date have
shown that the
PDGFR initiates multiple signal relay cascades and
that at least some of the receptor-associated proteins are required for
PDGF-dependent DNA synthesis (6, 7). In contrast,
preventing the
PDGFR from individually associating with
phosphatidylinositol 3-kinase, PLC
, or SHP-2 does not severely impair the mitogenic signal of the receptor (2, 8-10). Thus, which if
any of these recruited signaling molecules is required for mitogenic
signal relay from the
PDGFR remains an open question.
A number of groups have investigated the role of Src in
PDGF-dependent signal relay. The initial observations were
that Src activity increased in a PDGF-stimulated cell (11, 12).
Subsequent studies demonstrated that all three Src family members that
are expressed in a fibroblast are activated in response to PDGF and associate with the PDGFR (13). Binding of Src family members to the
PDGFR requires the presence of two tyrosine phosphorylation sites
(579 and 581) in the
PDGFR juxtamembrane domain (14). The binding of
Src to the receptor is dependent on the Src SH2 domains, and these
events are thought to contribute to activation of Src in a
PDGF-stimulated cell (14-16).
Recent studies have defined two Src-dependent stages in the cell cycle, mid/late G1, as well as G2/M. PDGF-BB-dependent entry into the S phase of the cell cycle was largely eliminated by microinjection of NIH 3T3 cells with either dominant negative forms of Src and Fyn or a neutralizing antibody that inhibits the activity of Src, Fyn, and Yes (17). Progression to the S phase of the cell cycle could be blocked by microinjecting the reagents at the time of PDGF stimulation or even up to 6 h after stimulating with PDGF (17). Constitutive expression of c-myc, but not fos or jun, rescued the block induced by dominant negative Src (18), leading to the idea that Src contributes to PDGF mitogenic signaling by increasing c-myc expression. The observations that Src is activated during mitosis (19-21) suggested that Src is performing an important function during this stage of the cell cycle as well. Indeed, Roche et al. (22) have demonstrated that the activity of Src family kinases is essential for the G2 to M transition. Thus Src kinase activity is required at mid G1 and at the G2/M transition in order for PDGF-stimulated cells to traverse the cell cycle.
PDGF-dependent signaling is not unique in its requirement for the activity of Src kinases. A number of other signaling pathways, including those initiated by B and T cell receptors, cytokine receptors, integrins, as well as the receptors for epidermal growth factor and colony stimulating factor-1 also involve Src family members (reviewed in Ref. 23). Thus, Src appears to be a key member of signal relay pathways in a variety of signaling systems, and it is important to precisely define what events Src is regulating.
Although Src is activated at both the G0/G1
transition and at the G2/M transition, the relative importance of Src
activation at the early point in the cell cycle has not been thoroughly
investigated. In addition, the role of Src in signaling by the PDGFR
has not been addressed directly. In this study, our goal was to assess the importance of the initial increase in Src activity for signal relay
by the
PDGFR. Our approach was to generate a receptor mutant that
was unable to activate Src and then to compare the ability of the WT
and mutant receptors to mediate signal transduction events and drive
cell proliferation. In our system, the Src family members were not
manipulated and were presumably available to execute necessary
functions at latter stages in the cell cycle. We found that the wild
type
PDGFR activated Src and that a mutant receptor that did not
initiate this event also failed to mediate efficient phosphorylation of
a small subset of signaling molecules that included Shc. In contrast,
activation of Src at the G0/G1 transition did
not appear to be required for induction of c-myc or for cell
proliferation.
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EXPERIMENTAL PROCEDURES |
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Cell Lines--
The mouse embryo 3T3 Patch B (PhB) cell line was
derived from Ph/Ph mouse embryos and was kindly provided by
Dr. Dan Bowen-Pope (24). PhB cells were maintained in Dulbecco's
modified Eagle's medium supplemented with 5% calf serum. The human WT
and F72/74 PDGFR constructs were subcloned into the
pLNCX2 retroviral vector, which is a modification of the
pLNCX vector (25) in that the polylinker of pLNCX2 contains
the following unique restriction sites: HindIII,
NotI, HpaI, SalI, BglII,
ClaI. The
PDGFR constructs in the retroviral vector were
transfected into GP+E cells, and the viral supernatant from these cells
was used to infect GP+E/Am12 cells (generous gift of Dr. Arthur Banks).
Virus from mass populations of G418-resistant GP+E/AM12 cells was used
to infect PhB cells, and the infected Ph cells were selected in the
presence of 1 mg/ml G418. Mass populations of drug-resistant PhB cells
were sorted by FACS to obtain populations of WT and F72/74 PhB cells
expressing equal levels of receptor. The sorted populations were used
for all of the experiments.
Site-directed Mutagenesis--
The 1.7-kilobase pair
PstI-BamHI fragment of the human PDGFR was
subcloned into the pBS+ plasmid, and the resulting
construct was called 19E. Site-directed mutagenesis was carried out by
using the Amersham oligonucleotide-directed in vitro
mutagenesis kit. To introduce the phenylalanine changes at Tyr-572 and
Tyr-574, the following oligonucleotide, which introduced an
EcoRI site without changing the amino acid sequence, was
used: 5'-GTCCACAAAAATGAATTCATGTCC3-'. Mutants were initially identified by restriction enzyme digestion using EcoRI and then
verified by sequencing. The PstI-BamHI fragment
of 19E was then subcloned into 18F, which is a 3.5-kilobase pair
NotI-BamHI insert subcloned into pBluescript
SKII+ and includes the entire
PDGFR cDNA (2).
Finally, the NotI-BamHI fragment from 18F was
subcloned into the NotI-BamHI-digested
pLNCX2 retroviral vector.
Antibodies--
The PDGFR antibodies were crude rabbit
polyclonal antibodies raised against a glutathione
S-transferase fusion protein including either the carboxyl
terminus (amino acids 951-1089; 27P) or a portion of the first
immunoglobulin domain (amino acids 52-94; 80.8) of the human
PDGFR.
The
PDGFR antisera recognize the mouse and human
PDGFR and do not
cross-react with the
PDGFR of either species.2 The Src-2 antibody
used for immunoprecipitation of Src family members was purchased from
Santa Cruz and is an affinity-purified rabbit polyclonal antibody
raised against a peptide corresponding to amino acids 509-533 of
c-Src. This antibody recognizes all three of the Src family members
that are expressed in fibroblasts, Src, Yes, and Fyn. The Src antibody
used for Western blotting, 327, is a mouse monoclonal that is thought
to recognize the SH3 domain of c-Src and v-Src (26) (Oncogene Science
Inc.) and was used at a 1:1,000 dilution. The immunoprecipitating
anti-phosphotyrosine antibody used in these studies was PY20, a
monoclonal antibody purchased from Transduction Labs. For
anti-phosphotyrosine Western blot analysis, a combination of PY20
(Transduction Labs) and 4G10 (Upstate Biotechnology, Inc,), each at a
1:1,000 dilution, was used. To immunoprecipitate PLC
, we used a
polyclonal antibody, 36.3, which was raised against a glutathione
S-transferase-PLC
1 fusion protein that included amino
acids 550-850 of rat PLC
1. PLC
Western blot analysis was
performed using a mixture of monoclonal anti-PLC
1 antibodies
(Upstate) at a concentration of 0.25 µg/ml. SHP-2 was
immunoprecipitated subjected to Western blot analysis as described
previously (27). Shc was immunoprecipitated and immunoblotted (using a
dilution of 1:100) with an anti-Shc polyclonal antibody purchased from
Upstate.
Immunoprecipitation and Western Blot Analysis of Src--
PhB
cells expressing the WT or F72/74 PDGFR were grown to confluence,
incubated overnight in DME containing 0.1% CS, and were left resting
or stimulated with 50 ng/ml PDGF-AA for 5 min. The cells were washed
and lysed in EB (10 mM Tris-HCl (pH 7.4), 5 mM
EDTA, 50 mM NaCl, 50 mM NaF, 0.1% bovine serum
albumin, 1% Triton X-100, 1 mM phenylmethylsulfonyl
fluoride, 2 mM Na3VO4, and 20 µg/ml aprotinin), and Src was immunoprecipitated using the Src-2
antibody. Immune complexes were bound to formalin-fixed Staphylococcus aureus membranes, spun through EB + 10%
sucrose, washed twice with 1.0 ml of radioimmune precipitation buffer
(150 mM NaCl, 10 mM NaPO4 (pH 7.0),
2 mM EDTA, 1% sodium deoxycholate, 1% Nonidet-P40, 0.1%
SDS, 20 µg/ml aprotinin, 50 mM NaF, 2 mM Na3VO4, 0.1% 2-mercaptoethanol), twice with
1.0 ml of PAN (10 mM PIPES (pH 7.0), 100 mM
NaCl, 20 µg/ml aprotinin) + 0.5% Nonidet P-40, twice with 1.0 ml of
PAN, and finally resuspended in PAN and stored at
70 °C.
Anti-phosphotyrosine Western Blot Analysis of Immunoprecipitated
Proteins--
The PDGFR, PLC
, SHP-2, and Shc were
immunoprecipitated from resting or PDGF-stimulated cells, as described
above, using the appropriate antibodies, except the first set of washes
were with EB instead of radioimmune precipitation buffer.
Immunoprecipitates representing 1.5 × 106 cells were
then subjected to an anti-phosphotyrosine Western blot.
In Vitro Kinase Assay--
Src immunoprecipitates from
approximately 1-2 × 104 cells were incubated with
1.6 µg of acid-denatured rabbit muscle enolase (Sigma), 20 mM PIPES (pH 7.0), 10 mM MnCl2, 20 µg/ml aprotinin, and 10 µCi of [-32P]ATP for 10 min at 30 °C in a total reaction volume of 20 µl. The reaction was
stopped by adding 20 µl of 2× sample buffer (10 mM EDTA,
4% SDS, 5.6 M 2-mercaptoethanol, 20% glycerol, 200 mM Tris-HCl (pH 6.8), 1% bromphenol blue). Kinase assays
with the PDGFR were performed under identical conditions, except that
0.5 µgs of glutathione S-transferase-PLC was used as the
exogenous substrate instead of enolase. After the kinase assay, the
samples were incubated for 4 min at 98 °C and spun at 8,000 rpm for
5 min, and the supernatant was resolved on a 10% SDS-polyacrylamide electrophoresis gel.
Soft Agar Assays--
Six-well tissue culture plates were coated
with a layer of DME, 5% CS containing 0.6% Sea-Plaque-agarose (FMC
Bioproducts, Rockland, ME). Subconfluent Ph cells expressing the WT or
F72/74 PDGFR were trypsinized, washed in PBS twice, and resuspended in DME 5% CS at 1 × 105 cells/ml. One ml of cells
was then added to 1 ml of DME 5% CS containing 0.9% Low melting point
agarose and 200 ng/ml PDGF-AA or buffer (10 mM acetic acid,
2 mg/ml bovine serum albumin). The cells were plated onto the coated
tissue culture plates, allowed to solidify, and then placed in a
37 °C incubator. After 10 days, colonies were visualized with an
inverted microscope. Colonies were scored as having 10 or more cells.
Each of the conditions were assayed in triplicate.
Northern Blotting--
RNA was isolated from the PhB cells after
lysis in guanidine isothiocyanate and centrifugation through a CsCl
gradient (28). RNA (10 µg) was separated on a 1.2%
formaldehyde-agarose gel and electro-transferred to Nytran (Schleicher
and Schuell). RNA was cross-linked to the filter with UV light using a
Stratalinker (Stratagene). Hybridizations were performed at 45 °C in
50% formamide, 5× SSC (1× SSC is 150 mM NaCl, 15 mM sodium citrate), 100 mM sodium phosphate,
0.2% polyvinylpyrrolidone, 0.2% Ficoll, 0.2% bovine serum albumin,
0.1% SDS, 250 µg/ml salmon sperm DNA. After hybridization, blots
were washed twice at room temperature with 2× SSC, 0.5% SDS and three
times at 45 °C with 0.1× SSC, 0.5% SDS. 32P-labeled
probes were prepared with random primers using the Prime-It II kit
(Stratagene) and [-32P]dCTP. The following cDNAs
were used as probes: a 548-base pair HindIII-XbaI
fragment of pKS321 containing the human glyceraldehyde-3-phosphate dehydrogenase gene (29), a 1.5-kilobase pair
EcoRI-PstI fragment of murine c-fos/pGEM3 (30), a
400-base pair PstI fragment of murine c-myc that
includes exon 2.
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RESULTS |
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Characterization of the F72/74 PDGFR--
To determine whether
Src family members (hereafter collectively referred to as Src) are
involved in
PDGFR signaling relay, we tested if Src is activated
after engagement of the
PDGFR. Our preliminary experiments (data not
shown; see Figs. 2 and 3 below) indicated that
PDGFR is indeed able
to activate Src; we next wanted to generate an
PDGFR mutant that
failed to stimulate the kinase activity of Src. For the
PDGFR,
phosphorylation of tyrosines 579 and 581 in the juxtamembrane domain
enables Src to associate with the receptor and also appears to activate
the kinase activity of Src (14). Since these two tyrosines, as well as
the flanking sequences are identical in the
and
PDGFRs, we
reasoned that mutating them in the
PDGFR would generate a mutant
PDGFR that would be unable to activate or bind Src. To test this
hypothesis, we mutated tyrosine 572 and 574 to phenylalanine residues,
and the resulting receptor was expressed in Ph cells, a fibroblast cell
line that expresses the
PDGFR, but not the
PDGFR.
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Activation of Src--
Stimulation of quiescent fibroblasts with
PDGF-BB has been shown to increase the kinase activity of Src, Fyn, and
Yes by 2-5-fold (13). Since PDGF-BB activates both and
PDGFRs,
which are expressed in most fibroblasts, these studies did not
distinguish the contribution of the two receptors to the Src activation
event. Mori et al. (14) clearly demonstrated that the
PDGFR can activate Src; thus we were interested in learning whether
the
PDGFR also activates Src and if so, whether association of Src
with the receptor was required for this event. Src was
immunoprecipitated from WT and F72/74
PDGFR expressing PhB cells
that were either left resting or stimulated with 35 ng/ml PDGF-AA for
5, 15, 30, 60, or 120 min. The kinase activity of the
immunoprecipitated Src was measured in an in vitro kinase
assay, using enolase as an exogenous substrate (Fig.
3A). PDGF-AA stimulation of WT
cells for 5 min resulted in a 1.8-fold increase in the in
vitro phosphorylation of enolase, which was 1.9-fold after 15 min
and peaked at 30 min with a 2.2-fold increase. After 60 min, enolase
phosphorylation began to decrease with only a 1.6-fold and 1.2-fold
increase observed at 60 and 120 min, respectively. In Src
immunoprecipitates from F72/74 cells, enolase phosphorylation did not
increase after 5 min and was only slightly increased (1.2-fold) after
15 min of stimulation. After 30 min, enolase phosphorylation decreased
below the level seen before PDGF stimulation. Western blotting the Src
immunoprecipitates with an anti-Src antibody indicated that comparable
amounts of Src were present in all of the samples (Fig. 3B).
These experiments demonstrate that engagement of the
PDGFR leads to
activation of Src. Furthermore, the F72/74 receptor, which associates
with Src poorly, is unable to efficiently activate Src, suggesting that
binding of Src to the
PDGFR is required for maximal
PDGF-dependent activation of Src kinase activity.
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Induction of Immediate Early Genes--
Previous studies have
linked PDGF-BB-dependent Src activation in NIH 3T3 cells
with induction of c-myc but not fos or
jun, (18). We were curious whether the PDGFR was able to
trigger an increase in the myc mRNA level and if this
event required activation of Src. To this end, RNA was harvested from
resting cells and cells treated with 35 ng/ml PDGF-AA for 20, 60, or
120 min. In both WT and F72/74 cells, PDGF-AA increased transcription
of myc (Fig. 4). The
fos mRNA level also rose in both WT and F72/74 cells after PDGF-AA treatment (Fig. 4). In both cell types, the amount of
fos mRNA decreased slightly after 20 min and then
increased at the 60 and 120 min time points. Equal loading of RNA was
confirmed by probing with a glyceraldehyde-3-phosphate dehydrogenase
probe. These studies indicated that the
PDGFR activated
c-myc and c-fos, and furthermore, that robust
activation of Src was not required for these events. It is likely that
the
PDGFR is engaging other signaling pathways that lead to
c-myc activation. For instance, the protein kinase C
activator 12-O-tetradecanoylphorbol-13-acetate has been
shown to activate c-myc in fibroblasts (31, 32), and the
PDGFR is able to recruit and activate PLC
(8).
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Cell Proliferation--
We also evaluated the importance of the
increase in Src activity in PDGF-stimulated cells for
PDGF-dependent cell proliferation. Ph cells expressing an
empty vector or the WT or F72/74 PDGFR were plated in soft agar
supplemented with PDGF or buffer. After 10 days, the cultures were
photographed, and the number of colonies was counted (Fig.
5). Although this assay is a commonly
used indication of cell transformation, since colony formation requires
cell proliferation, we have used it here to evaluate
PDGF-dependent cell proliferation. All three cell types
were able to form a limited number of small colonies in the absence of
PDGF, and this background level of colony formation was comparable in
all of the cell lines. Adding PDGF-AA, which selectively activates the
PDGFR, had only a marginal effect on the empty vector expressing
cells, whereas PDGF-BB, which activates the endogenous
PDGFR,
triggered a 9.5-fold increase in colony formation. Activation of the
PDGFR with PDGF-AA stimulated a 27.5-fold increase in colony number
for the WT receptor-expressing cells and a 25.5-fold increase in the
F72/74 receptor-expressing cells. In addition, the colonies that grew
in the presence of PDGF-AA were of similar size and morphology for the
WT and F72/74 cells. Finally, PDGF-BB was also able to drive colony
formation in the cells expressing the WT and F72/74 receptors, and the
colony number and morphology was comparable. A likely reason that
PDGF-BB (the universal ligand) stimulated more colonies in the WT and F72/74 cells as compared with the vector-expressing cells is that the
WT and F72/74 cells have two forms of PDGFRs, whereas the vector-expressing cells have only a single receptor, the endogenous
PDGFR. These data demonstrate that the F72/74 receptor is able to
drive growth of cells in soft agar to a comparable extent as the WT
PDGFR and thus suggest that Src activation during the G0/G1 transition is not required for
PDGF-AA-dependent cell growth.
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Tyrosine Phosphorylation of Proteins--
The observation that
activation of Src during the first 2 h of PDGF-AA stimulation is
not mandatory for driving the mitogenic signal of the PDGFR raises
the question of the nature of the role of Src in
PDGFR signaling.
Hence, we tested the hypothesis that Src is required for
PDGF-dependent tyrosine phosphorylation of proteins. To
this end, Ph cells expressing the empty vector, WT, or F72/74
constructs were grown to 85-90% confluence, arrested by serum
starvation, and then left resting or stimulated with 50 ng/ml PDGF AA
for 5 min. The cells were washed and lysed, and several different
proteins were immunoprecipitated and subjected to anti-phosphotyrosine
Western blot analysis (Fig. 6). To check the levels of immunoprecipitated protein in the samples, the
anti-phosphotyrosine blots were stripped and then reprobed with the
immunoprecipitating antibody (Fig. 6, lower panel of each
pair). Stimulation of the empty vector-expressing cells did not
increase the level of phosphotyrosine in any of the proteins. Upon
stimulation of the WT receptor-expressing cells with PDGF AA, PLC
,
Shc, and SHP-2 were robustly tyrosine-phosphorylated. Stimulation of
cells expressing the F72/74 receptor increased tyrosine phosphorylation
of PLC
and SHP-2, and the extent of phosphorylation was comparable
to that seen in the WT receptor-expressing cells (Fig. 6, A
and B). In contrast, Shc phosphorylation was dramatically
reduced in the F72/74 receptor-expressing cells (Fig. 6C).
The 52-kDa Shc isoform was most strongly phosphorylated in cells
expressing the WT receptor and in the F72/74 receptor-expressing cells;
this species was phosphorylated to only 1% of the WT level. In cells
expressing the F72/74 receptor, the 46-kDa Shc isoform was
phosphorylated to 20% that of the level seen in cells expressing the
WT receptor. We also compared the extent of tyrosine phosphorylation of
the receptor itself after PDGF stimulation of cells expressing the WT
or F72/74 receptors. The receptors were immunoprecipitated from resting
or PDGF-AA-stimulated cells, and the samples were subjected to
anti-phosphotyrosine Western blot analysis. We observed that PDGF-AA
initiated comparable phosphorylation of both receptors (Fig. 6D). These
data demonstrate that tyrosine phosphorylation of most proteins in a
PDGF-AA-stimulated cell do not require Src, and also, that some of the
proteins, such as Shc, are not phosphorylated efficiently when Src is
not activated. These findings suggest that the
PDGFR is not able to
phosphorylate all of the proteins that are often thought of as
substrates of receptor tyrosine kinases and that other kinases such as
Src contribute to the overall level of substrate phosphorylation in an
acutely stimulated cell.
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DISCUSSION |
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The present study reveals that in response to stimulation with
PDGF-AA, the PDGFR associates with and activates Src and that these
events are dependent on tyrosines 572 and 574. Furthermore, it sheds
light on two aspects of the signal relay pathway downstream of the
PDGFR: 1) the mechanism by which proteins are
tyrosine-phosphorylated in a PDGF-stimulated cell, and 2) the
importance of the initial increase of Src activity for subsequent cell
cycle events.
Tyrosine Phosphorylation of Proteins in PDGF-stimulated
Cells--
We found that when the PDGFR is activated without the
concurrent activation of Src, then Shc is not efficiently
tyrosine-phosphorylated. There are at least several explanations for
this observation. Src could be responsible for phosphorylating the
receptor at the site required for binding of Shc, and when Shc does not
associate with the receptor it is not efficiently
tyrosine-phosphorylated. We have found that Shc associates with the
PDGFR poorly in fibroblasts (2), so testing this idea would be
technically challenging. Another explanation is that Src alters the
substrate specificity of the
PDGFR. However, the F72/74 receptor is
able to phosphorylate various exogenous substrates to a comparable
extent as the WT receptor (Fig. 1 and data not shown). Thus our
existing data do not support this possibility. Our favored explanation
is that once activated, Src contributes to phosphorylation of a select group of proteins in a PDGF-AA-stimulated cell. Shc was somewhat unique
in its dependence on Src activity to be efficiently
tyrosine-phosphorylated, as the receptor itself, PLC
, and SHP-2 were
all comparably phosphorylated in PDGF-stimulated cells expressing the
WT or F72/74 receptors (Fig. 6). To see if there were additional
proteins that were poorly phosphorylated in the F72/74
receptor-expressing cells, we performed antiphosphotyrosine Western
blots on antiphosphotyrosine immunoprecipitates from resting and
PDGF-AA-stimulated cells. There were numerous proteins detected in this
assay, and we did not see differences in the extent of phosphorylation
in any of them from the WT versus F72/74 receptor-expressing
cells.3 Although this assay
does not detect all tyrosine-phosphorylated proteins in a cell (for
instance, we did not see Shc by this approach), it does suggest that
Src is required for phosphorylation of only a small subset of the
proteins that are tyrosine-phosphorylated in a PDGF-AA-activated cell.
Thus, although the
PDGFR, or perhaps some other kinase,
phosphorylates many of the proteins that associate with the
PDGFR, a
subset of the signaling molecules requires Src for efficient tyrosine
phosphorylation.
Role of Src in Cell Cycle Progression--
Hooshmand-Rad et
al. (38) have identified tyrosine 572 and 574 as phosphorylation
sites, and in agreement with our results, find that mutating these two
tyrosine residues largely eliminates PDGF-AA-mediated Src activation
but does not prevent DNA synthesis when the receptors are expressed in
pig aortic endothelial cells. Yet published work from several other
groups indicates that Src is required for PDGF-dependent
cell cycle progression. Microinjection of reagents that neutralize Src
function blocked PDGF-BB-induced entry of NIH 3T3 cells into S phase
(17). In addition, kinase-inactive forms of Src were able to inhibit
PDGF-BB-dependent DNA synthesis in cells derived from mice
that have the Src gene knocked out (39). One explanation for the lack
of consensus regarding the apparent importance of Src in PDGF-mediated
signaling is that the PDGFR may not require Src for signaling, as is
the
PDGFR. However, the above-cited studies used fibroblasts, which
usually express both the
and
PDGFRs. Furthermore, PDGF-BB was
used, and this form of PDGF activates both of the PDGFRs. Consequently, the
PDGFR should have been able to signal in these cells, provided that it was expressed. We have recently tested the NIH 3T3 (NIH 3T3
cl7) cells used in the above-mentioned microinjection studies and found
that these NIH 3T3 cells do not express detectable levels of the
PDGFR but do express high levels of the
PDGFR as compared with
the Ph cells used herein. Thus PDGF-BB primarily initiates the
PDGFR
in the NIH 3T3 cl7 cells, and the microinjection experiments do not
address the issue of the contribution of Src to
PDGFR signal relay.
Yet this does not provide a satisfying resolution of the discrepancy
between the various groups, because the signaling by numerous distinct
receptor tyrosine kinases was greatly suppressed when reagents that
eliminate Src activity were microinjected into cells (40). As a result,
it seems likely that the signaling by the
PDGFR would be blocked as
well.
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ACKNOWLEDGEMENTS |
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We thank Charlie Hart (Zymogenetics) for generously supplying PDGF-AA, Dan Bowen-Pope (University of Washington) for providing us with the Ph cell line, and Arthur Banks (Columbia University) for the GP+E/Am12 cell line. We greatly appreciate the critical input of Kris DeMali, Steven M. Jones, and Nader Rahimi. In addition, J. A. G. and S. R. thank Kris DeMali for support and encouragement during the evolution of this project. In addition, we thank Lena Claesson-Welsh and Roya Hooshmand-Rad for sharing unpublished results.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Funded by a postdoctoral fellowship from the Colorado-Wyoming
affiliate of the American Heart Association. Present address: The Wayne
Hughes Institute, 701 Western Ave., Glendale, CA 91201.
§ Funded by a postdoctoral fellowship from the Fritz-ThyssenStiftung, Germany.
¶ Present address: EISAI London Research, University College London, London WC1E6BT, UK.
An Established Investigator of the American Heart Association.
To whom correspondence should be addressed: The Schepens Eye Research
Institute, Harvard Medical School, 20 Staniford St., Boston, MA 02114. Tel.: 617-912-2517; Fax: 617-912-0111; E-mail: kazlauskas{at}vision.eri.harvard.edu.
1 The abbreviations used are: PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PLC, phospholipase C; PhB, Patch B; WT, wild type; DME, Dulbecco's modified Eagle's medium; CS, calf serum; PIPES, 1,4-piperazinediethanesulfonic acid.
2 J. A. Gelderloos, S. Rosenkranz, C. Bazenet, and A. Kazlauskas, unpublished observations.
3 S. Rosenkranz and A. Kazlauskas, unpublished observations.
4 DeMali, K., and Kazlauskas, A. (1998) Mol. Cell. Biol., in press.
5 T. Schlesinger and A. Kazlauskas, manuscript in preparation.
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