(Received for publication, December 29, 1994; and in revised form, June 16, 1995)
From the
Because the protein-tyrosine phosphatase (PTP) Syp associates
with the tyrosine-phosphorylated platelet-derived growth factor
receptor (
PDGFR), the
PDGFR is a likely Syp substrate. We
tested this hypothesis by determining whether recombinant Syp (rSyp)
and a control PTP, recombinant PTP1B (rPTP1B), were able to
dephosphorylate the
PDGFR. The
PDGFR was phosphorylated at
multiple tyrosine residues in an in vitro kinase assay and
then incubated with increasing concentrations of rSyp or rPTP1B. While
the receptor was nearly completely dephosphorylated by high
concentrations of rPTP1B, receptor dephosphorylation by rSyp plateaued
at approximately 50%. Two-dimensional phosphopeptide maps of the
PDGFR demonstrated that rSyp displayed a clear preference for
certain receptor phosphorylation sites; the most efficiently
dephosphorylated sites were phosphotyrosines (Tyr(P))-771 and -751,
followed by Tyr(P)-740, while Tyr(P)-1021 and Tyr(P)-1009 were very
poor substrates. In contrast, rPTP1B displayed no selectivity for the
various
PDGFR tyrosine phosphorylation sites and dephosphorylated
all of them with comparable efficiency. A Syp construct that lacked the
SH2 domains was still able to discriminate between the various receptor
phosphorylation sites, although less effectively than full-length Syp.
These in vitro studies predicted that Syp can
dephosphorylate the receptor in vivo. Indeed, we found that a
PDGFR mutant (F1009) that associates poorly with Syp, had a much
slower in vivo rate of receptor dephosphorylation than the
wild type receptor. In addition, the GTPase-activating protein of Ras
(GAP) and phosphatidylinositol 3-kinase were less stably associated
with the wild type
PDGFR than with the F1009 receptor. These
findings are consistent with the in vitro experiments showing
that Syp prefers to dephosphorylate sites on the
PDGFR, that are
important for binding phosphatidylinositol 3-kinase (Tyr(P)-740 and
Tyr(P)-751) and GAP (Tyr(P)-771). These studies reveal that Syp is a
substrate-selective PTP and that both the catalytic domain and the SH2
domains contribute to Syp's ability to choose substrates.
Furthermore, it appears that Syp plays a role in PDGF-dependent
intracellular signal relay by selectively dephosphorylating the
PDGFR and thereby regulating the binding of a distinct group of
receptor-associated signal relay enzymes.
Tyrosine phosphorylation modulates the activity of many enzymes
involved in regulation of a wide variety of cellular events and is a
reversible process governed by the opposing effects of protein-tyrosine
kinases and protein-tyrosine phosphatases
(PTPs)()(1, 2, 3) . In addition to
a conserved catalytic domain, most PTPs also contain highly variable
noncatalytic sequences, which can regulate the activity and/or
subcellular location of the PTP(1, 3) . The initially
purified PTPs failed to show a high degree of substrate
specificity(4) , and this led to the hypothesis that
subcellular targeting is the primary determinant in selection of a
substrate. More recent evidence indicates that an interaction may exist
between the amino acids surrounding the phosphotyrosine and the amino
acids surrounding the mouth of the active site of the
PTP(5, 6, 7) . The relative strength of the
interaction between a particular PTP and a tyrosine-phosphorylated
protein may permit the PTP to discriminate between substrates. This
possibility is supported by the identification of PTPs that show a high
degree of substrate specificity: two different mammalian PTPs have been
identified that appear specific for MAP
kinases(8, 9, 10, 11, 12) .
Thus the choice of substrates not only depends on the subcellular
location of a PTP, but at least in some instances on the substrate
specificity of the PTP.
Syp (also commonly called SH-PTP2 or PTP-1D) is one member of a small family of SH2 domain-containing PTPs, which also includes Corkscrew (Csw) and HCP (also called SH-PTP1 or PTP-1C)(13, 14) . All three of these PTPs appear to play important roles in signal transduction. Together with the Drosophila homologues of c-Raf and Ras, Csw acts downstream of the Torso receptor tyrosine kinase in a signaling cascade that is essential for normal development of the Drosophila embryo(15, 16) . HCP plays a negative role in signaling of receptors such as the erythropoeitin receptor and the B cell antigen receptor(17, 18) . In contrast, Syp may function more like Csw, in that it appears to play a positive role in signaling. Catalytically inactive Syp constructs block fibroblast growth factor-driven mesoderm induction at a step prior to MAP kinase activation(19) . Microinjection of neutralizing Syp antibodies or Syp SH2 domains severely inhibits growth factor-mediated DNA synthesis in mammalian cells(20, 21) . These studies indicate that the SH2 domain-containing phosphatases play an important role in the regulation of intracellular signal relay cascades.
Recent studies have revealed that Syp can act as an adapter protein.
In response to PDGF stimulation, Syp becomes phosphorylated at tyrosine
542 (YTNI)(22) , which is a good binding site for
the Grb2 SH2 domain(23) . Grb2 binds Syp at this
phosphotyrosine site in vitro, and both Grb2 and the guanidine
nucleotide-releasing factor Sos coimmunoprecipitated with Syp from
lysates of PDGF-stimulated cells(22, 24) . Moreover,
studies with the add-back PDGF
receptor (
PDGFR) mutants
indicate that binding of Syp is one of the mechanisms by which the
PDGFR is able to mediate activation of Ras(25) . Thus, one
of the apparent functions of Syp is to serve as an adapter protein in
the process of PDGF-stimulated Ras activation.
Upon activation, the
PDGFR is phosphorylated at multiple tyrosine residues and thereby
becomes a docking site for SH2-domain-containing signal transduction
proteins. These proteins play important roles in the relay and control
of mitogenic signals sent from the receptor to the nucleus. Some of the
proteins that associate with the activated
PDGFR include
phospholipase C-
1, the GTPase-activating protein of Ras (GAP),
phosphatidylinositol 3-kinase (PI 3-kinase), Nck, Shc, Grb2,
pp60
,
p62
, p59
, an
as yet unidentified 120-kDa protein, and Syp (26, 27, 28) . Some of the
receptor-associated proteins undergo tyrosine phosphorylation in a
PDGF-stimulated cell, and this event affects their ability to send a
biological signal. Given that Syp is brought into close proximity to
these tyrosine-phosphorylated signaling molecules, it is likely that
Syp regulates
PDGFR signal transduction by modulating the
phosphorylation state of the members of the
PDGFR complex.
To
test this hypothesis, we determined the in vitro ability of a
recombinant form of Syp to dephosphorylate the PDGFR. Our studies
showed that Syp was capable of dephosphorylating the receptor and that
there was a rank order for dephosphorylation of the various
PDGFR
phosphorylation sites. Investigation of the basis for Syp's
substrate specificity identified two main factors: 1) positioning of
the Syp catalytic domain on the
PDGFR, as directed by the binding
of Syp's SH2 domains to the receptor; and 2) an intrinsic
preference of the catalytic domain for certain phosphotyrosine
residues. To ascertain whether these in vitro studies
reflected the in vivo situation, we compared the rate of
receptor dephosphorylation of the wild type and the F1009 mutant, which
binds Syp poorly. After stimulation with PDGF, the wild type receptor
was dephosphorylated more rapidly than the F1009 mutant, suggesting
that Syp functions to dephosphorylate the receptor in an intact cell.
In addition, we found that the p85 subunit of PI 3-kinase, and to a
greater extent GAP, are less stably associated with the wild type
receptor than the F1009 mutant, which is consistent with Syp's
ability to dephosphorylate the PI 3-kinase and GAP binding sites. Our
studies show that Syp displays a high degree of substrate specificity
and that one of its substrates appears to be the activated
PDGFR.
To
assay dephosphorylation of the PDGFR and receptor-associated
proteins,
PDGFR immunoprecipitates were prepared as follows. HepG2
cells expressing the wild type
PDGFR (25) were grown to
near confluence in Dulbecco's modified Eagle's medium, 10%
fetal bovine serum, incubated overnight in Dulbecco's modified
Eagle's medium, 0.1% calf serum, and were left resting or
stimulated for 5 min at 37 °C with 50 ng/ml PDGF-BB. The cells were
lysed in EB (58) and the receptor was immunoprecipitated with
the 30A polyclonal anti-
PDGFR antibody, as described
previously(30) . The
PDGFR immune complexes were
radiolabeled in a standard in vitro kinase assay in the
presence of [
P]ATP as described previously (59) and washed 3
in 2
phosphatase buffer.
Washed immune complexes, representing approximately 4.25
10
cells or 6.5
10
µg of
PDGFR, were resuspended in 20 µl of 2
phosphatase
buffer and then added to microfuge tubes containing 20 µl of the
appropriate amount of rPTP or rPTP storage buffer. Equivalent amounts
of rSyp and rPTP1B (standardized according to enzymatic activity toward
pNPP) were used, and the reactions were allowed to proceed for 30 min
at 30 °C in a total volume of 40 µl. Typically 0.1-5.0
µg (1.4-70 pmol) of rSyp or 0.05-0.3 µg
(0.08-4 pmol) of rPTP1B was used. Reactions were terminated by
adding 40 µl of 2
sample buffer (10 mM EDTA, 4%
SDS, 5.6 M
-mercaptoethanol, 20% glycerol, 200 mM Tris-HCl, pH 6.8, 2% bromphenol blue). The samples were boiled and
resolved on a 7.5% SDS-PAGE gel. The gel was KOH-treated (60) and then subjected to autoradiography. The extent of
protein dephosphorylation was determined by densitometric analysis of
the autoradiogram.
To
investigate whether Syp-mediated receptor dephosphorylation affects
receptor binding to signal transduction proteins, we compared the
ability of the WT and F1009 PDGFRs to bind to GAP and the p85
subunit of PI 3-kinase in vivo at 5 and 25 min post-PDGF
stimulation. HepG2 cells expressing either the WT or F1009 receptor
were left resting, were stimulated with 50 ng/ml PDGF-BB for 5 min, or
were stimulated for 5 min, and then the medium was replaced with
prewarmed Dulbecco's modified Eagle's medium containing
0.1% calf serum and allowed to incubate for another 20 min at 37
°C. Finally, the cells were lysed as described above, the
PDGFR was immunoprecipitated, and immunoprecipitates were resolved
on a 7.5% SDS-PAGE gel, transferred to Immobilon, and subjected to
anti-PDGFR (30A 1:1000), anti-p85 (1:500), or anti-GAP (69.3 1:5000)
Western blot analysis. Immunoreactive proteins were detected and
analyzed as described above.
Figure 1:
Expression and purification of
His-tagged PTPs. Bacteria harboring Syp or PTP1B expression plasmids
were grown to log phase and induced with 0.4 mM
isopropyl-1-thio--D-galactopyranoside; the bacteria were
lysed and the PTPs were purified by nickel affinity chromatography. LaneM, protein molecular mass markers; the mass of
each marker (in kDa) is indicated in the leftmargin. Lanes1 and 2, crude and purified rSyp,
respectively; lanes3 and 4, crude and
purified rPTP1B, respectively.
To characterize the
recombinant enzymes, we determined their K and k
using the low molecular weight substrate pNPP.
The linear range of PTP activity was determined for both enzymes as a
function of time and enzyme concentration (data not shown). Staying
within the linear range for time and enzyme concentration, the activity
of each enzyme was measured as the concentration of pNPP was varied (Fig. 2). The phosphatase activity of Syp (k
= 4.09 s
) was 18.2-fold lower than that
of PTP1B (k
= 74.34
s
). The K
values for Syp and
PTP1B toward pNPP were 2.8 and 3.8 mM, respectively (Fig. 2). This standardization was necessary in order to
meaningfully compare the ability of the two PTPs to dephosphorylate
candidate physiological substrates described below.
Figure 2:
Phosphatase activity of rSyp and rPTP1B
using pNPP as a substrate. A, rSyp; B, rPTP1B. Assays
were performed as described under ``Experimental
Procedures.'' Values for K and K
were derived from Lineweaver-Burke plots of
the data.
In contrast, the degree of dephosphorylation of the
PDGFR differed for the two PTPs. Receptor dephosphorylation increased
linearly as the amount of rPTP1B was increased, and at the highest dose
approximately 70% of the receptor's phosphate was removed (Fig. 3B). Adding 5 times more rPTP1B resulted in the
loss of more than 90% of the label from the receptor (data not shown).
Incubation of receptor immunoprecipitates with increasing amounts of
rSyp increased the degree of receptor dephosphorylation up to a maximum
of approximately 50% (Fig. 3A, lanes5 and 6). This appeared to be the maximal extent of
dephosphorylation, since the receptor was not further dephosphorylated
when 5 times more rSyp was added (Fig. 3A, lanes7 and 8). Note that although a different number
of moles of rPTP1B and rSyp were used in this experiment, the number of
units of PTP activity for the two enzymes was identical. These
experiments demonstrated that rPTP1B efficiently dephosphorylated the
PDGFR, and at high concentrations removed greater than 90% of the
label. In contrast, rSyp-mediated
PDGFR dephosphorylation reached
a plateau, and further dephosphorylation was not observed even with
high concentrations of rSyp.
Figure 3:
Dephosphorylation of the PDGFR in
vitro. The
PDGFR was immunoprecipitated from unstimulated
cells(-) or cells stimulated with 50 ng/ml PDGF-BB (+),
radiolabeled in a standard in vitro kinase assay, washed in
phosphatase buffer, and then incubated with increasing amounts of rSyp (A) or rPTP1B (B) at 30 °C for 20 min. In panel B the phosphatase reactions also contained the indicated amount of
fusion protein encoding both of Syp's SH2 domains. Samples were
resolved by SDS-PAGE, the gel was exposed to film, and the resulting
autoradiogram is shown. The numbersabove the lanes indicate the number of pmol of PTP that was added to the
reaction. The amount of PTP activity used in each set of lanes in panelsA and B was the same, as
normalized to pNPP activity. For example, the 4 pmol of rPTP1B used in lanes7 and 8 of panelB has the same number of units of PTP activity as the 70 pmol of
rSyp used in the same lanes in panelA.
Similar results were obtained in four independent
experiments.
There are at least three explanations for why Syp was not able to efficiently dephosphorylate the receptor as did rPTP1B. First, rSyp may be a less stable enzyme than rPTP1B, so that over the time course of the phosphatase assay rSyp activity declines. We tested this possibility and found that both enzymes remain fully active during the entire duration of the assay (data not shown). Second, the SH2 domains of Syp may bind phosphorylated receptor tyrosine residues and thereby protect these sites from the PTPase activity of Syp. To test if Syp SH2 domains are able to protect the PDGFR from PTPs in our assay we added the SH2 domains of Syp to the reaction containing PTP1B. The amount of Syp SH2 fusion protein added to the phosphatase reaction corresponded to the molar concentration of rSyp that was used. Addition of Syp's SH2 domains did not affect the ability of PTP1B to dephosphorylate the receptor (Fig. 3B and data not shown). This indicated that the inability of Syp to efficiently dephosphorylate the PDGFR is not merely due to protection of receptor phosphotyrosine residues by Syp's SH2 domains. The third explanation for the difference observed between rSyp and rPTP1B is that rPTP1B is a very general phosphatase and acts indiscriminately, while rSyp is a selective phosphatase capable of dephosphorylating only a subset of the receptor's phosphotyrosine residues.
Figure 4:
Phosphopeptide maps of the PDGFR
dephosphorylated by rSyp or rPTP1B. In vitro labeled
PDGFR immunoprecipitates were subjected to a phosphatase assay
using rSyp, rPTP1B, or buffer (as in Fig. 3), and then the
receptor was analyzed by two-dimensional tryptic/thermolytic mapping.
Equal amounts of sample (based on protein) were spotted onto the thin
layer plates. PanelsA and D show maps of
the
PDGFR after incubation with buffer alone. PanelsB and E show maps of the
PDGFR after
incubation with a low dose of rPTP1B (0.08 pmol) or rSyp (1.4 pmol),
respectively. PanelsC and F show maps of
the
PDGFR after incubation with a high dose of rPTP1B (0.8 pmol)
or rSyp (14 pmol), respectively. Dephosphorylation with PTP1B was done
in the presence of Syp SH2 domains, 1.4 pmol (panelB) or 14 pmol (panelC). In each of the
sets (panelsB and E or C and F), the same amount of PTP activity (normalized by pNPP
dephosphorylation) was added. Spot1 corresponds to
tyrosine 751; spot3 corresponds to tyrosine 1009; spot6 corresponds to tyrosine 740; spot8b corresponds to tyrosine 1021; spot9 corresponds to tyrosine 771, and spots2, 7, and 8a are as yet unidentified
phosphopeptides(29) . Similar results were obtained in four
independent experiments.
While all
phosphotyrosine sites on the PDGFR are equally good targets for
rPTP1B, maps of the
PDGFR dephosphorylated by rSyp showed that
rSyp had a distinct preference for certain sites (Fig. 4, D-F). The low dose of rSyp primarily dephosphorylated spots 1, 6, 7, 9, and to a
lesser extent 8a (compare E and D). A
10-fold increase in the amount of rSyp resulted in increased
dephosphorylation of spot 1 as well as spots 6, 7, and 9, while other spots, 2, 3, 8a, and 8b, retained most if
not all of their original phosphotyrosine (Fig. 4, compare D and F). Note that these Syp-resistant spots were
effectively dephosphorylated by rPTP1B (compare 4, A and C, with 4, D and F). The selective nature of
rSyp's activity on the
PDGFR is perhaps best appreciated by
comparing the progressive dephosphorylation of spot 1 relative to spots
2 and 3 when the receptor is exposed to rPTP1B versus rSyp.
The ratio of spots 1 to 2 or 1 to 3 remained relatively constant when
the receptor was dephosphorylated by increasing concentrations of
rPTP1B but became more dissimilar as increasing concentrations of rSyp
were used (Fig. 4). These experiments indicate that the reason
Syp is able to only partially dephosphorylate the receptor (Fig. 3A) is because only a subset of the
PDGFR's phosphorylation sites are good Syp substrates.
While all sites on the PDGFR were available for
dephosphorylation (rPTP1B dephosphorylates them), the phosphotyrosine
sites 1009 (spot 3) and 1021 (spot 8b and a portion of 8a) found
within the carboxyl-terminal tail of the
PDGFR, appeared to be
very resistant to dephosphorylation by rSyp. In contrast, the sites
most affected by the activity of rSyp, Tyr(P)-771, and Tyr(P)-751
(spots 9 and 1, respectively) and to a lesser extent Tyr(P)-740 (spot
6), are found within the kinase insert domain of the
PDGFR. Thus
it appears that this particular region of the
PDGFR is the primary
target of rSyp phosphatase activity. We wanted to determine the
mechanism behind rSyp's preference for dephosphorylating sites in
the kinase insert domain of the PDGFR over those found in the
carboxyl-terminal tail of the receptor. We previously determined that
it is not merely due to the presence of the SH2 domains, since they do
not protect the receptor from dephosphorylation by PTP1B (Fig. 3). Since Syp binds to the tail of the
PDGFR(32, 33) , it may have poor access to the
immediately proximal tail phosphorylation sites. Thus positioning of
rSyp on the
PDGFR may mediate the ability of rSyp to
dephosphorylate different sites on the receptor. Alternatively, the
catalytic domain of Syp itself may be able to discriminate between the
various phosphorylation sites.
To investigate the relative
contribution of Syp's SH2 and catalytic domains to Syp's
ability to act in a substrate-specific manner, we repeated our
dephosphorylation assay using a recombinant protein encoding only the
catalytic domain of Syp (rCAT). Kinetic analysis of rCAT demonstrated
that deletion of Syp's SH2 domains results in a dramatic increase
in catalytic activity toward pNPP (k = 5,
700 s
) but no change in K
(K
= 3.1 mM). When the
tyrosine-phosphorylated PDGFR was incubated with a concentration of
rCAT containing approximately 4
more PTPase activity than the
high concentrations of rSyp or rPTP1B used in Fig. 3, spots 9
(site 771) and 1 (site 751) were selectively dephosphorylated (Fig. 5, compare A and B). In contrast spots 6
(site 740), 7 (a peptide mixture), and 8b (site 1021) were relatively
untouched ( Fig. 5compare A to B). However,
unlike full-length Syp, dephosphorylation of spots 2 (unidentified) and
3 (site 1009) is also observable at this low concentration of rCAT (Fig. 5, A and B). In the presence of 5 or 10
times more rCAT, both spots 3 and 8b (sites 1009 and 1021,
respectively) were efficiently dephosphorylated (Fig. 5, compare C to D and E), indicating that the
specificity for distinct autophosphorylation sites is greatly
diminished when high concentrations of enzyme that lacks the SH2
domains is used. Therefore, rSyp's selection of substrates is
determined by at least two variables: 1) the intrinsic specificity of
the catalytic domain for certain phosphotyrosine residues, and 2) the
SH2 domains, which bind to the tail of the
PDGFR, and thereby may
position Syp's catalytic domain over the kinase insert domain of
the
PDGFR.
Figure 5:
Phosphopeptide maps of the PDGFR
dephosphorylated by rCAT. In vitro labeled
PDGFR
immunoprecipitates were subjected to a phosphatase assay using rCAT or
buffer, and the receptor was analyzed by two-dimensional
tryptic/thermolytic mapping as in Fig. 4. PanelsA and C show maps of the
PDGFR after incubation with
buffer alone. PanelsB, D, and E show maps of the
PDGFR after incubation with 0.06, 0.03, or
0.6 pmol of rCAT respectively. This amount of PTP activity corresponds
to 4
, 8
, and 40
the highest amount of PTP
activity used with rSyp and rPTP1B. PanelE was
exposed to film 3
longer than panelsA-D. Similar results were obtained in three
different experiments.
Figure 6:
Comparison of the in vivo dephosphorylation rate of the wild type and F1009 PDGFRs.
HepG2 cells expressing the WT or F1009
PDGFR were left
unstimulated(-) or were exposed to 30 ng/ml PDGF (+) for the
indicated length of time (in minutes). The cells were lysed, the
receptor was immunoprecipitated and resolved by SDS-PAGE, and the
region of the gel containing proteins of 120-240 kDa was
subjected to antiphosphotyrosine (topportion of panelA) or antireceptor (bottomportion of panelA) Western blot analysis. The extent of
receptor tyrosine phosphorylation was determined by densitometry,
normalized for the amount of receptor, and it is represented
graphically as a percentage of receptor phosphotyrosine at the 4-min
time point. Similar results were obtained in three different
experiments. The difference in phosphorylation of the WT and F1009
receptors at the 40-min time point is statistically significant (p < 0.02).
To assess whether binding of Syp
results in dephosphorylation of the receptor at key tyrosine residues in vivo, we performed the following experiments. First,
phosphopeptide maps of the WT and F1009 PDGFR labeled in vivo were compared at 5 and 25 min post-PDGF-stimulation, however no
consistent differences were observed (data not shown). Consequently, we
turned to a less direct method to examine receptor phosphorylation,
namely by the ability to bind the various receptor-associated proteins.
We speculated that Syp-mediated receptor dephosphorylation may affect
the ability of some of the proteins to bind to the receptor. In our in vitro studies, Syp dephosphorylates Tyr(P)-771, and
Tyr(P)-751, sites involved with the binding of GAP and PI 3-kinase,
respectively, so we focused on these two receptor-associated proteins.
HepG2 cells expressing both WT or F1009 PDGFRs were left unstimulated
or exposed to PDGF for 5 or 25 min, the cells were lysed, the receptor
was immunoprecipitated and the immunoprecipitates were analyzed for the
presence of p85 and GAP. As expected, at 5 min post-PDGF stimulation,
when the phosphotyrosine content of both the WT and F1009 mutant is
high (Fig. 6A), both receptors are able to associate
with comparable amounts of p85 (Fig. 7). The level of GAP that
associated with the F1009 receptor was somewhat greater than the amount
that coprecipitated with the WT receptor, and is consistent with our
previous observations (35) . At 25 min post-PDGF stimulation,
when the phosphotyrosine content of the WT PDGFR is lower than that of
the F1009 mutant, the amount of p85 bound to the WT receptor is
noticeably diminished. In contrast, the amount of p85 bound to the
F1009 receptor at 25 min remained similar to that observed at the 5-min
time point (Fig. 7). This trend was even more striking when we
compared GAP binding at the two time points. The amount of GAP that
associated with the wild type receptor at 25 min was reproducibly
reduced to nearly undetectable levels, whereas GAP binding to the F1009
receptor was only modestly reduced at the 25 min time point (Fig. 7). In contrast to p85 and GAP, binding of phospholipase
C-
1 to the WT and F1009 receptors was diminished to a similar
extent at the 25-min time point (data not shown), and is consistent
with the observation that the Syp does not efficiently dephosphorylate
the tyrosine residue required for phospholipase C-
1 binding
(Y1021, spot 8b and a fraction of spot 8a). These results support the
idea that binding of Syp to the activated PDGFR enhances the
dephosphorylation of the receptor and regulates the duration of binding
of a select group of SH2 domain-containing signal transduction
proteins.
Figure 7:
Comparison of p85 and GAP binding to the
WT and F1009 PDGFRs. HepG2 cells expressing the WT or F1009
PDGFR were treated in one of three ways: left
unstimulated(-), exposed to 50 ng/ml PDGF for 5 min(5) ,
or exposed to 50 ng/ml PDGF for 5 min, and then the medium was replaced
with prewarmed media lacking PDGF and incubated at 37 °C for 20
additional minutes. The cells were lysed, the receptor was
immunoprecipitated, resolved by SDS-PAGE and transferred to Immobilon,
and the pertinent regions of the blot were subjected to anti-PDGFR,
anti-p85, or anti-GAP Western blot analysis. Similar results were
obtained in three different experiments.
In this study we found that the PDGFR is an in vitro substrate of rSyp and that rSyp dephosphorylated only a subset of
the phosphorylation sites on the
PDGFR. In contrast, rPTP1B was
able to dephosphorylate all of the
PDGFR sites indiscriminately.
Syp's substrate specificity arises from both the catalytic domain
and the SH2 domains. Furthermore, a mutant
PDGFR that does not
efficiently associate with Syp loses its phosphotyrosine content more
slowly than the wild type receptor and displays a prolonged association
with SH2 domain-containing signal transduction proteins, suggesting
that in an intact cell, Syp functions to dephosphorylate the
PDGFR
and thereby regulate the proteins that associate with the
PDGFR.
Our studies described herein
indicate that Syp displays a discernible degree of substrate
specificity by dephosphorylating distinct tyrosine residues found
within the PDGFR. The
PDGFR acted as an in vitro substrate of both rPTP1B and rSyp; however, the susceptibility of
the receptor's phosphotyrosine residues to dephosphorylation by
the two PTPs was dramatically different. While PTP1B dephosphorylated
all of the receptor's phosphotyrosines with similar efficiency,
rSyp was able to dephosphorylate only a subset of the available
phosphorylation sites (Fig. 3Fig. 4Fig. 5).
Tyr(P)-751 and Tyr(P)-771, and to a somewhat lesser extent Tyr(P)-740,
were the primary targets on the
PDGFR for dephosphorylation by
rSyp, and these three sites account for 45% of total phosphotyrosine in
the
PDGFR(29) . Interestingly, this is the extent of
receptor dephosphorylation at 30 min post-PDGF stimulation in vivo (Fig. 6).
Other groups have also found that Syp displays a readily detectable degree of substrate specificity(39, 40, 41) . Surprisingly, our findings that rSyp prefers the kinase inset phosphorylation sites over sites in the carboxyl-terminal tail, are opposite to those reported by Sugimoto et al.(39) . It is possible that the differences arise from using intact proteins that contain multiple phosphorylation sites instead of peptides as a substrate. In addition, the apparent preference for the peptide-containing site 1009 by Syp, over the other phosphorylated peptides in the panel of phosphopeptide substrates, could be due to the activation of Syp by the Tyr(P)-1009 peptide(32) .
The preferential dephosphorylation of phosphorylation sites residing in the kinase insert domain of the PDGFR by Syp seems to be directed by a combination of two main factors. The first is an intrinsic preference by the catalytic domain of Syp. Alignment of the sequences surrounding the PDGFR autophosphorylation sites provides some insight for the specificity displayed by the Syp catalytic domain (Fig. 8). Both sites 751 and 771 are found within the context of a possible consensus sequence for phosphotyrosine recognition by the Syp active site: ESXXY(P)XXXXD. No other autophosphorylation site on the PDGFR corresponds to this sequence. This putative consensus sequence appears to be specific for dephosphorylation by Syp as shown by the inability of the low concentration of rCAT to dephosphorylate Tyr(P)-740, a site that does not correspond to the putative consensus sequence, whereas a concentration of PTP1B containing 20 times less phosphatase activity dephosphorylated this site to completion (compare Fig. 4, A and B, to Fig. 5, A and B). It is possible that the charged amino acids at positions -4 and +5, along with the hydrophilic serine at position -3 facilitate an interaction between the active site of the Syp catalytic domain and the phosphotyrosine residue targeted for dephosphorylation. Ongoing experiments are designed to investigate this possibility.
Figure 8:
Alignment of the amino acid sequences
surrounding PDGFR tyrosine phosphorylation sites. Boldfaceletters represent the phosphorylated tyrosine residue. Underlinedletters represent amino acids that may
contribute to a putative Syp active site recognition
sequence.
The second factor that contributes to Syp's ability to
discriminate substrates is the SH2 domains. They could act by binding
to the receptor's COOH-terminal phosphorylation sites and thus
shielding them from dephosphorylation. However, Syp's SH2 domains
do not protect the receptor from dephosphorylation by PTP1B ( Fig. 3and Fig. 4). Instead binding of Syp to the
PDGFR's COOH terminus may position Syp's catalytic
domain over the receptor's kinase insert region and thereby
greatly increase the effective concentration of Syp's catalytic
domain in the vicinity of the kinase insert phosphorylation sites. If
the SH2 domains do contribute to Syp's substrate specificity,
than a PTP lacking the SH2 domains should have a diminished ability to
choose its substrate. Indeed, removal of Syp's SH2 domains
reduced Syp's specificity when sufficiently high concentrations
of rCAT were used (Fig. 5). Interestingly, spots 3 and 8b,
corresponding to sites 1009 and 1021 of the
PDGFR COOH terminus
respectively, appear to be dephosphorylated as well as or more
efficiently than spot 6 (Tyr(P)-740), when Syp's SH2 domains have
been removed but not when full length Syp is used as the source of
phosphatase. Therefore, Syp's preference for kinase insert domain
phosphorylation sites (particularly Tyr(P)-751 and Tyr(P)-771) appears
to be mediated by both an enhanced interaction of phosphotyrosine
residues found within a putative Syp recognition consensus sequence
with the active site of Syp's catalytic domain, and the
positioning of the Syp catalytic domain over the
PDGFR kinase
insert domain as directed by the binding of Syp's SH2 domains to
the COOH-terminal tail of the
PDGFR.
The predominant
dephosphorylation of PDGFR at Tyr(P)-751 and Tyr(P)-771 by Syp is
intriguing, as it suggests a potential mechanism for regulating
PDGFR-mediated mitogenic signaling. Tyrosine phosphorylation at
751 plays an important role in the binding and the activation of PI
3-kinase(26) , and one group has found that Tyr(P)-751 is
involved with Nck binding. In HepG2 cells we have found that Tyr-751 is
not required for efficient binding of Nck to the
PDGFR. (
)Nck functions as an oncogenic protein as demonstrated by
experiments in which overexpression of Nck led to cell transformation
and tumor formation in nude mice(45, 46) . Therefore,
Nck binding to the
PDGFR may engage signaling pathway(s) that
promote(s) cellular proliferation, and in certain cell types Syp may be
able to control the initiation of these events.
PDGFR-mediated
PI 3-kinase activation is also potentially regulated by
dephosphorylation of tyrosine 751 by Syp. Evidence from several labs
suggests that the increase in PI 3-kinase activity following growth
factor stimulation is crucial for mitogenic signaling, and requires
binding of PI 3-kinase to the tyrosine-phosphorylated
PDGFR (47, 48, 49, 50) . Two adjacent
sites on the
PDGFR, site 740 and site 751, are capable of binding
to the SH2 domains of the p85 subunit of PI
3-kinase(30, 51) , and in some studies site 740 has
been shown to be the high affinity binding site for PI
3-kinase(29) . rSyp was able to dephosphorylate both Tyr-751
and Tyr-740 ( Fig. 4and Fig. 5), and p85 is released more
quickly from the WT PDGFR than the F1009 mutant receptor. However,
compared with the drastic decrease in the phosphate content of tyrosine
751, the loss of phosphate from tyrosine 740 was marginal. Potential
mechanisms for regulation of
PDGFR-mediated PI 3-kinase activation
by Syp, therefore, probably would not simply involve the elimination of
the PI 3-kinase binding site on the receptor. Perhaps site 740 plays
the major role in the association of PI 3-kinase with the
PDGFR,
while site 751 functions to enhance PI 3-kinase activity. In this case,
removal of the PI 3-kinase activation site by dephosphorylation of
tyrosine 751 would nullify the positive mitogenic signal transmitted by
PI 3-kinase without actually decreasing its association with the
receptor. The viability of this hypothesis is currently being tested.
Like Csw, Syp may act as a positive mediator of signaling. Indeed,
recent studies indicate that Syp plays a positive role in growth
factor-stimulated DNA synthesis(20, 21) . Syp may also
assist the flow of mitogenic signals from the PDGFR.
Phosphorylation of tyrosine 771 enables stable association of GAP with
the
PDGFR, and recent data indicate that this event prevents
PDGF-dependent activation of phospholipase C-
1 and PDGF-dependent
DNA synthesis (52) . GAP has also been shown to enhance the
cleavage of GTP bound to activated Ras to GDP, thus turning off Ras
mediated signaling. Thus GAP binding could function to negatively
modulate
PDGFR-mediated mitogenic signaling. Therefore,
Syp's apparent ability to dephosphorylate the GAP binding site is
a possible mechanism by which Syp can enhance the
PDGFR's
mitogenic signal. This is consistent with a recent report that suggests
that Syp's phosphatase activity is necessary upstream of
PDGF-stimulated MAP kinase activation(53) . A second route by
which Syp enhances
PDGFR signaling relates to Syp's ability
to act as an adapter protein that couples the receptor to proteins such
as Grb2 (24) and thus to Ras activation(54) . Given
that Syp can regulate
PDGFR signaling in both positive and
negative ways, it is possible that Syp has a dual function in the
context of
PDGFR signaling. Immediately following
PDGFR
activation, Syp acts as a positive signal transducer, coupling the
receptor to necessary downstream signaling proteins. Once the necessary
events have occurred for efficient transduction of a mitogenic signal,
Syp acts to turn off this signal by dephosphorylating key sites on the
PDGFR and thus preventing uncontrolled cell proliferation.