Department of Pharmacology and the Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill School of Medicine, Chapel Hill, North Carolina 27599-7365
Received for publication, November 9, 2000, and in revised form, March 6, 2001
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ABSTRACT |
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The p21-activated kinases (PAKs) are important
mediators of cytoskeletal reorganization, cell motility and
transcriptional events regulated by the Rho family GTPases Rac and
Cdc42. PAK activation by serum components is strongly dependent on cell
adhesion to the extracellular matrix (ECM). PAK binds directly to the
Nck adapter protein, an interaction thought to play an important role in regulation and localization of PAK activity. This report
demonstrates that the interaction of PAK with Nck is regulated
dynamically by cell adhesion. PAK-Nck binding is rapidly lost after
cell detachment and rapidly restored after re-adhesion to the ECM
protein fibronectin, suggesting a rapidly reversible mode of
regulation. Furthermore, the loss of Nck binding correlates with
changes in the phosphorylation state of PAK in nonadherent cells, as
evidenced by electrophoretic mobility shift and phosphorylation within
a sequence known to mediate interaction with Nck. The ability of cell
adhesion to regulate PAK phosphorylation and interaction with Nck may
contribute to the anchorage-dependence of PAK activation as well as
to the localization of activated PAK within a cell.
The p21-activated kinase
(PAK)1 family comprises the
best characterized effectors for the Rho family GTPases Cdc42 and Rac (1). As such, PAK activity has been implicated in Cdc42- and Rac-mediated regulation of gene expression and modulation of the actin
cytoskeleton and cell motility (2, 3). PAK is also involved in the
regulation of signaling cascades involving the MAPK (4-6) and Jun
N-terminal kinases (JNK) (5, 7, 8). PAK activity is stimulated by a
variety of soluble factors (e.g. insulin, thrombin,
PDGF (1, 2, 9)) as well as by cellular adhesion to ECM (10, 11).
Recently, it has been reported that activation of PAK by soluble
factors is highly dependent on cell adhesion (12, 13), and this may
contribute to the anchorage-dependence of MAPK activation (13, 14). One
of these reports offers strong evidence that the lack of PAK activation
in nonadherent cells is due to an uncoupling of PAK from activated Rac
at the plasma membrane (12). Specifically, the deficiency in PAK
activation in nonadherent cells is not due to a dramatic reduction in
Rac activation but correlates with a marked decrease in the recruitment of activated Rac to the cell membrane. This is particularly
interesting given observations that forced localization of PAK to the
plasma membrane strongly activates PAK (15-17). One hypothesis to
explain this activation is that membrane localization of PAK is likely to increase the efficiency of its interaction with other
membrane-associated regulatory elements, such as activated Rac (18) and
sphingolipids (17, 18). Taken together, these data strongly suggest
that membrane localization is a crucial aspect of PAK activation
in vivo.
The recruitment of PAK to the cell membrane may be mediated by its
direct interaction with the adapter protein Nck (19-22). The three SH3
domains mediate interaction with a large number of binding partners
(23), while the SH2 domain binds to phosphotyrosine-containing proteins
such as activated receptor tyrosine kinases (RTKs; e.g. PDGFR (20, 24, 25)) and the Crk-associated substrate
p130CAS (26). Nck-PAK interaction is mediated through the
second SH3 domain of Nck, which binds to a proline-rich region in the N
terminus of PAK (19-21). In fibroblasts, this interaction occurs even
in the absence of extracellular stimuli (19-21). This suggests
that in unstimulated cells, PAK and Nck exist in a constitutive complex that can be rapidly relocated to membrane-associated,
phosphotyrosine-rich sites generated by growth factor stimulation or
adhesion to ECM. The importance of this interaction in regulating PAK
function is underscored by the observations that expression of a
membrane-targeted Nck SH3 domain leads to constitutive PAK activation
(15) and that a PAK mutant unable to bind Nck is severely impaired in
its ability to promote cytoskeletal organization (27).
Given the tight regulation of PAK activation by cell adhesion to ECM
and the importance of Nck binding in regulating PAK activity, I
investigated whether adhesion might affect PAK-Nck interaction. Herein,
I show that the ability of PAK to bind Nck is highly dependent on cell
adhesion and correlates with an anchorage-dependent change in PAK phosphorylation. These data have significant implications for
the regulation and localization of PAK activity.
Antibodies--
Polyclonal antibodies against the N- or C
terminus of PAK1 were from Santa Cruz Biotechnology. Antibodies
against Nck included a monoclonal from Transduction Laboratories and a
polyclonal from Santa Cruz Biotechnology. Polyclonal antibody against
the PDGF Cell Culture--
NIH3T3 cells were cultured as described
previously (13). Briefly, cells were serum-starved overnight and then
either harvested or cultured in suspension. For suspension culture,
cells were trypsinized, collected by centrifugation, washed once with
DMEM, 2% BSA, 2 mg/ml trysin inhibitor and once with DMEM, 2% BSA,
and then resuspended and rotated in the same at 37 °C for the
indicated times. Where indicated, cells were replated, after 1 h
in suspension, onto FN-coated tissue culture plates (13) for the
indicated times.
Cell Lysis, Immunoprecipitation, and Western Blotting--
Cells
were washed twice in ice-cold phosphate-buffered saline and then
scraped or resuspended in lysis buffer (50 mM Tris, pH 7.6, 150 mM NaCl, 1 mM each EDTA and EGTA, 1%
Triton X-100 containing protease and phosphatase inhibitors (Sigma)).
Lysates were transferred to microcentrifuge tubes, vortexed
vigorously for 30 s, incubated on ice for 10 min, and centrifuged
at 4 °C for 10 min at ~15,000 × g. Precleared
lysates were either frozen at Phosphatase Treatment--
PAK immunoprecipitates were washed
three times with lysis buffer, twice with 0.05 M Tris, pH
7.0, 0.1 M NaCl, and once with buffer A (0.05 M
Tris-HCl, pH 7.9, 0.1 M NaCl, 0.01 M magnesium chloride). Immunoconjugates were resuspended in 50 µl of buffer A
alone or buffer A containing 0.5 units of calf intenstinal alkaline phosphatase (New England Biolabs) and incubated at 30 °C for 30 min.
Immunoconjugates were then washed twice in buffer A and either resuspended in 1× Laemmli sample buffer and boiled for 5 min or resuspended in 50 µl of 0.02 M MES, pH 6.0, containing
0.75 units of potato acid phosphatase (Calbiochem) and incubated for 15 min at 30 °C. These latter samples were then washed in buffer A and boiled in Laemmli sample buffer as described above.
Phosphopeptide Analysis--
Cells were serum-starved overnight
then labeled for 4 h with 1.0 mCi/ml
32P-labeled orthophosphate in phosphate-free DMEM
containing 2% BSA. For the suspension culture, the labeling medium was
removed, and the cells were rapidly washed, trypsinized, collected, and resuspended in the original labeling medium and then incubated at
37 °C with rotation for 1 h. Cells were harvested, and PAK was
immunoprecipitated as described above. Radiolabeled PAK was purified on
SDS-PAGE gels, and individual bands were excised and subjected to
in-gel trypsinization as described elsewhere (13). Phosphopeptides were
analyzed by two-dimensional separation on thin layer cellulose plates
(13) or by HPLC (AP Biotechnology) on a reverse-phase C18 column
(Vydac) developed with a linear gradient of aqueous acetonitrile. For
some experiments, an excess of a synthetic peptide with the sequence
NTSTMIGAGSK (single-letter code), corresponding to the tryptic peptide
containing Ser21 of PAK1, was added to the mix of
radioactive peptides before HPLC separation. Fractions were then
analyzed by scintillation counting and spectrophotometry at 215 nm. The
peptide was synthesized and purified by the University of North
Carolina Peptide Synthesis Facility.
Recruitment of PAK to Activated RTK Complexes Requires Cell
Adhesion--
Nck can recruit PAK to activated RTKs (e.g.
PDGFR) upon growth factor stimulation, which may play a role in
regulating PAK activity (20). Given the strong anchorage dependence of
PAK activation by growth factors (12, 13), the effect of cell adhesion
on recruitment of PAK to activated PDGFR was investigated (Fig.
1). As previously reported (29),
PDGF-stimulated tyrosine phosphorylation of the PDGFR occurred
efficiently in both adherent and nonadherent NIH3T3 cells. Growth
factor stimulation of adherent cells induced the formation of a complex
containing tyrosine-phosphorylated PDGFR, Nck, and PAK. In nonadherent
cells, Nck was still recruited to the activated PDGFR, whereas PAK was
completely absent from this complex; this suggested that although the
SH2-dependent recruitment of Nck to activated receptor is
anchorage-independent, the SH3-mediated interaction of Nck with PAK
might be regulated by cell adhesion.
The Interaction between PAK and Nck Is
Anchorage-dependent--
To directly investigate the effects of
cell adhesion on PAK-Nck interaction, anti-Nck Western blots were
performed on PAK immunoprecipitates from stably adherent cells and
cells that had been incubated in suspension (Fig.
2). Even in the absence of serum and
growth factors, PAK immunoprecipitates from stably adherent cells
contained readily detectable levels of Nck, consistent with previous
reports (19, 20). However, little or no Nck was detected in PAK
immunoprecipitates from nonadherent cells (Fig. 2). The lack of PAK-Nck
association was not caused by a decrease in the amount of Nck protein
in nonadherent cells, as shown by Nck Western blots of adherent and
nonadherent cell extracts (data not shown and Fig. 4). Interestingly,
the interaction between Nck and PAK was also ablated by treatment of
adherent cells with cytochalasin D (Fig. 2). It is important to point
out that although the concentration of cytochalasin D used in these
experiments allowed cells to remain loosely attached, it completely
disrupted the actin cytoskeleton and promoted cell rounding and
complete tyrosine dephosphorylation of FAK (data not shown).
This indicates that in NIH3T3 cells the growth factor-independent
interaction between Nck and PAK requires a fully functional cellular
interaction with the ECM.
PAK-Nck Interaction Is Dynamically Regulated by Cell
Adhesion--
To further investigate this
anchorage-dependent interaction, the kinetics of PAK-Nck
dissociation after cellular detachment were determined (Fig.
3). The amount of Nck co-precipitating
with PAK dropped dramatically (~8-fold by densitometry) within 10 min after detachment and was essentially undetectable at 30 and 60 min.
Interestingly, the loss of PAK-Nck interaction after detachment was
rapidly reversed by replating cells onto the ECM protein fibronectin (FN). The amount of co-precipitating Nck in cells replated on FN for 15 min was ~90% of the level seen in stably adherent cells, whereas the
level at 30 min after replating was indistinguishable from that of
adherent cells (Fig. 3).
Nck Binding Correlates with PAK Phosphorylation--
The rapid
kinetics of PAK-Nck dissociation and re-association suggest that the
complex might be regulated by a rapidly reversible post-translational
modification, such as phosphorylation. Both PAK and Nck are known
phosphoproteins (1, 23). However, the role of phosphorylation in
regulating Nck function has yet to be determined, and while many of the
details of PAK phosphorylation in vitro have been
elucidated, the complexity of its regulation in vivo is not
completely understood, and its regulation by cell adhesion has never
been investigated.
Importantly, a recent report demonstrates that the association of Nck
and PAK can be regulated by phosphorylation (22). In this report,
strong biochemical data indicated that phosphorylation of
Ser21, a putative autophosphorylation site at the
C-terminal end of the Nck-binding region of PAK1, dramatically reduced
the interaction between PAK and Nck (22). Reduced Nck binding was also
observed in cells in which PAK was robustly activated and
hyperphosphorylated either by mutation or expression of a
constitutively active mutant of Cdc42 (22), suggesting a negative
feedback loop in which PAK activation down-regulates interaction with Nck.
Previous work has correlated PAK phosphorylation with an
electrophoretic mobility shift on SDS-PAGE gels; specifically,
increasing phosphorylation of PAK correlates with slower migration (22, 30). This correlation was utilized to investigate whether the loss of
Nck binding in nonadherent cells coincided with a change in the
phosphorylation state of PAK. Previous experiments in the current
report were performed using standard mini-gels, and no shift in PAK
mobility was readily apparent. Therefore, to better visualize potential
PAK mobility shifts, subsequent experiments utilized larger, higher
cross-linking gels (see "Experimental Procedures"). Using this
method, PAK isolated from adherent, serum-starved cells runs as a
discrete doublet, whereas in nonadherent cells, only the faster
migrating form is present (Fig.
4A). A slower migrating form
of PAK reappears within an hour of replating cells on FN (Fig.
4A). As expected, this mobility shift is due to a difference
in phosphorylation, as the slower migrating species present in PAK
immunoprecipitates from adherent cells are almost completely converted
to the faster migrating form by phosphatase treatment in
vitro (Fig. 4B). Significantly, this differential phosphorylation correlates closely with the loss of Nck binding in
nonadherent cells (Fig. 4A) and suggests that Nck binds
exclusively to the more highly phosphorylated, slower migrating form of
PAK. This finding was confirmed in reciprocal
co-immunoprecipitation experiments in which the PAK present in Nck
immunoprecipitates co-migrated exclusively with the slower migrating
form of PAK that is present in adherent cells but completely absent
from nonadherent cells (Fig. 4C).
The loss of PAK-Nck interaction in nonadherent cells and its
correlation with PAK dephosphorylation indicate that Nck binding is
compromised under conditions in which PAK activity is very low, which
is somewhat surprising in light of the recent report suggesting high
levels of PAK activity are responsible for disrupting PAK·Nck
complexes (22). However, that report is in stark contrast to an earlier
report in which a constitutively active double-point mutant of PAK
showed enhanced binding to Nck (27). The recovery of PAK·Nck
complexes from cells stimulated with growth factors and other agonists
also supports the notion that Nck remains associated with activated PAK
under physiological conditions (15, 16, 19-21, 31, 32). Therefore,
there are some apparent contradictions regarding the role of PAK
activity or autophosphorylation in regulating Nck binding in
vivo. However, there is still strong in vitro evidence indicating that PAK-Nck interaction can indeed be regulated by phosphorylation near the proline-rich Nck-binding region (22). How
might these observations be reconciled with the current data?
One possible explanation is that Ser21 appears to be a
relatively poor autophosphorylation site (30) and may be modified in this way only when PAK is activated to supraphysiological levels (e.g. by an excess of constitutively active Cdc42 or Rac).
It is therefore possible that phosphorylation of this site is indeed responsible for regulating Nck binding but that in vivo this
modification is catalyzed by a kinase other than PAK. Results thus far
suggest that such a kinase may be activated upon cellular detachment. To directly investigate the possibility that cellular detachment might
affect phosphorylation of this region, phosphopeptide mapping was
performed on radiolabeled PAK immunoprecipitated from
32P-labeled adherent cells and cells in suspension.
Separation of tryptic phosphopeptides by two-dimensional mapping or by
HPLC revealed several adhesion-dependent changes in PAK
phosphorylation (Fig. 4D and data not shown). To identify
specific changes in phosphorylation in and around
Ser21, an excess of purified, synthetic peptide
corresponding to amino acids 19-29 of PAK1 (the tryptic fragment
containing Ser21) was added to the phosphopeptide mixtures
to serve as a marker during HPLC. Although PAK from adherent cells
contained several phosphopeptides not present in the nonadherent sample
(Fig. 4D, fractions 20, 29, and 30), PAK from nonadherent
cells contained a phosphopeptide that co-eluted with the synthetic
peptide (Fig. 4D, fraction 26). These data suggest that
cellular detachment induces phosphorylation of PAK within a region
known to regulate interaction with Nck.
Regulation of PAK phosphorylation in vivo is complex and not
fully understood. The shift in electrophoretic mobility observed in
Fig. 4A suggests a net dephosphorylation of PAK in
nonadherent cells. However, Fig. 4D demonstrates that under
these conditions PAK retains some phosphates and even acquires new
modifications that are induced specifically, and perhaps uniquely, by
the state of nonadherence. Considerable effort has established that PAK is a substrate for autophosphorylation (1, 30) as well as phosphorylation by other kinases (e.g. PDK1 (34), PKA (13)). Clearly, further analysis is required to address fully the regulation of PAK phosphorylation by cell adhesion. Among the most important issues to be addressed are the complete profile of PAK phosphorylation in adherent versus nonadherent cells and the kinases or
phosphatases involved in this regulation.
The importance of Nck-mediated PAK localization to the membrane is
likely to be 2-fold. First, relocation to the membrane places PAK in
close proximity with membrane-associated activating factors, such as
activated Rac (12) and sphingolipids (17), increasing the efficiency of
their interaction and thereby the efficiency of PAK activation (18).
Second, membrane localization of PAK places it in an appropriate
setting to exert its effects on lamellipodia extension and cell
motility, events that involve modulation of the actin cytoskeleton near
the cell membrane (36). Another possibility is that by recruiting PAK
to sites of active growth factor and/or cell adhesion signaling, the
Nck-PAK interaction may be important in translating extracellular
gradients into intracellular asymmetry and thus in establishing cell
polarity (3). Given the ability of PAK to regulate the actin
cytoskeleton, cell motility, cytoplasmic signaling cascades, and gene
expression, the regulation of PAK-Nck interaction by cell adhesion
provides an intriguing new facet of regulation to this important
signaling complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
receptor was a generous gift from K. DeMali and A. Kazlauskas and was used as described (28). Phosphotyrosine was detected using monoclonal antibody 4G10 from Upstate Biotechnology.
80 °C or further clarified by
incubation with protein G-Sepharose beads (PharMingen) for 1 h at
4 °C and then used for immunoprecipitation. The lysates were
incubated with antibodies for 1-2 h at 4 °C and then with protein
G-coupled Sepharose beads (PharMingen) for 1 h. In most cases,
complexes were washed three times with lysis buffer. For polyclonal Nck
immunoprecipitations, complexes were washed once with lysis buffer and
three times with phosphate-buffered saline containing 1% Tween 20. Washed complexes were boiled in 1× Laemmli sample buffer for 5 min.
Samples were run on standard Bio-Rad mini-gels (10% for Nck Western
blots, 7.5% for PAK and PDGFR Western blots) or on higher resolution
gels contained 8% acrylamide and 0.5% bisacrylamide and were cast in
15 × 15-cm cassettes (Hoefer Scientific). Separated proteins were
transferred to nitrocellulose or polyvinylidene difluoride membranes,
which were then blocked and blotted using standard techniques.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (77K):
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Fig. 1.
Recruitment of PAK to activated PDGF receptor
is anchorage-dependent. PDGFR was immunoprecipitated
from adherent cells (Adh) or cells cultured in suspension
for 1 h (Susp) before or after stimulation with 20 ng/ml PDGF-BB for 5 min as indicated. Immunocomplexes were
separated by SDS-PAGE and analyzed by Western blotting with antibodies
against the PDGFR (to confirm equal loading), Nck, and PAK. For
analysis of PDGFR tyrosine phosphorylation, anti-PDGFR blots were
stripped and reprobed with anti-phospotyrosine (P-Tyr)
antibody.
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Fig. 2.
Cell adhesion regulates PAK-Nck
interaction. PAK was immunoprecipitated from serum-starved, stably
adherent cells (Adh), cells cultured in suspension
(Susp), or adherent cells treated with 2.5 µM
cytochalasin D for 30 min (CD). Immunocomplexes were
separated by SDS-PAGE and analyzed by Western blotting with antibodies
against PAK and Nck. 10 µg of whole cell extract
(wce) from stably adherent cells was run alongside the
immunoprecipitates to more easily distinguish bands of interest
(indicated by arrowheads) from background Ig bands.
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Fig. 3.
PAK-Nck interaction is regulated reversibly
and dynamically by cell adhesion. PAK was immunoprecipitated from
serum-starved, stably adherent cells (Adh) and cells
cultured in suspension (Susp) or replated onto FN-coated
plates (FN) for the indicated periods of time (in minutes).
Immunocomplexes were separated by SDS-PAGE and analyzed by Western
blotting with antibodies against PAK and Nck. 10 µg of whole cell
extract (wce) from stably adherent cells was run alongside
the immunoprecipitates to distinguish more easily the bands of interest
(indicated by arrowheads) from background Ig bands.
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[in a new window]
Fig. 4.
Loss of PAK-Nck interaction correlates with
dephosphorylation of PAK. A, PAK was immunoprecipitated
from serum-starved, stably adherent cells (Adh), cells
cultured in suspension (Susp), or cells replated onto
FN-coated plates (FN) for 1 h. Immunocomplexes were
separated in large, high cross-linker SDS-PAGE gels and analyzed by
Western blotting with antibodies against PAK and Nck. Whole cell
extracts from nonadherent or replated cells were run alongside the
immunoprecipitates to distinguish bands of interest (indicated by
arrowheads). B, PAK was immunoprecipitated from
adherent cells or cells in suspension and incubated in phosphatase
buffer alone or buffer containing potato acid phosphatase
(PAP) and/or calf intenstinal phosphatase (CIP)
as indicated. Proteins were resolved on high cross-linker SDS-PAGE gels
and analyzed by Western blotting with antibodies against PAK.
C, Nck was quantitatively immunoprecipitated from stably
adherent cells, and the supernatant extract (Super) from
this IP was immediately reimmunoprecipitated with antibodies against
PAK. These immunocomplexes were run on high cross-linker gels alongside
PAK IPs from stably adherent cells (Adh) or cells in
suspension (Susp) and analyzed by anti-PAK Western blotting.
The position of a 64,000-Da molecular mass marker
(64) is indicated. D, 32P-labeled
tryptic peptides of PAK isolated from adherent cells (white
bars) or cells in suspension for 1 h (shaded bars)
were doped with an excess of an unlabeled, synthetic peptide
comprising amino acids 19-29 of PAK1 (see "Experimental
Procedures") and separated by HPLC. Portions of the indicated
fractions were analyzed by liquid scintillation counting
(bars) to determine the phosphate content (in counts/minute
(CPM)) or by spectrophotometry at 215 nm (dashed line;
A215) to assess the presence of the synthetic
peptide.
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ACKNOWLEDGEMENTS |
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The guidance and generosity of R. L. Juliano are gratefully acknowledged. Lee Graves (University of North Carolina at Chapel Hill) generously provided advice and equipment for phosphopeptide analyses.
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FOOTNOTES |
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* This work was done in the laboratory of R. L. Juliano, who is supported by National Institutes of Health Grants GM26165 and HL45100.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.
Supported by a postdoctoral fellowship (PF-99-009-01-CSM) from the
American Cancer Society. To whom correspondence should be addressed:
Tel.: 919-966-4343; Fax: 919-955-5640; E-mail: Alan_Howe@med.unc.edu.
Published, JBC Papers in Press, March 13, 2001, DOI 10.1074/jbc.C000797200
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ABBREVIATIONS |
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The abbreviations used are: PAK, p21-activated kinase; BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; ECM, extracellular matrix; FN, fibronectin; GTPase, guanosine triphosphatase; HPLC, high-performance liquid chromatography; IP, immunoprecipitation; MAPK, mitogen-activated protein kinase; MES, 2-(N-morpholino)ethanesulfonic acid; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PAGE, polyacrylamide gel electrophoresis; SH, Src homology.
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