(Received for publication, December 21, 1994; and in revised form, April 3, 1995)
From the
p120 GTPase-activating protein (GAP) is a negative regulator of
Ras that functions at a key relay point in signal transduction pathways
that control cell proliferation. Among other proteins, p120 GAP
associates with p190, a GAP for the Ras-related protein, Rho. To
characterize the p120 In mammalian cells, Ras acts as a molecular relay in a mitogenic
signal transduction network that ultimately regulates initiation of DNA
replication(1, 2) . Upon growth factor stimulation of
receptor tyrosine kinases or activation of nonreceptor tyrosine
kinases, Ras is activated by binding to GTP(3) . Activated Ras
subsequently binds downstream effectors, including the Raf-1
serine/threonine kinase, which in turn activates a cytoplasmic kinase
cascade, thus conveying mitogenic signals from the cell surface to
their eventual nuclear destination (4, 5, 6, 7, 8, 9) .
In normal cells, inactivation of Ras is mediated by the 120 kDa Ras
GAP, The
p120 GAP can be subdivided into several domains, including a
species-specific N-terminal hydrophobic region, an SH3 domain flanked
by two SH2 domains, pleckstrin homology, and putative calcium-dependent
binding domains, as well as the C-terminal catalytic
domain(14) . In many signaling proteins, SH2 domains have been
shown to mediate protein-protein interactions through their ability to
bind phosphorylated tyrosine
residues(15, 16, 17, 18, 19, 20, 21) .
This role is also performed by the p120 SH2 domains, which are
necessary for interactions between p120 and kinases, including the
platelet-derived growth factor receptor and the Src tyrosine kinase, as
well as cytoplasmic proteins such as the p120 GAP-associated protein,
p62 (22, 23, 24, 25) . While the
importance of the N-terminal SH2 domain in p120 for protein-protein
interactions has been
illustrated(26, 27, 28) , the necessary
presence of the C-terminal SH2 domain has not previously been
demonstrated, raising the question of the significance of two SH2
domains in p120. p120 GAP associates with several proteins that are
potentially important for the regulation or function of p120 and Ras,
including the 190-kDa phosphoprotein, p190. Despite its large size and
multiple domains, only two functions have been ascribed thus far to
p190, that of being a GAP for the Rho/Rac family of GTPases (29) and binding directly to GTP(30) . To characterize in more detail the p120
Figure 1:
Domains
of p120 GAP expressed as GST fusion proteins. Regions of human Ras GAP
containing the hydrophobic N terminus, different combinations of the N-
and C-terminal SH2 and SH3 domains, or the C-terminal regions were
subcloned into pGEX-2T and expressed in E. coli. Amino acid
residues are numbered at the junctions of the various domains. C-1 contains the pleckstrin homology and calcium-dependent binding
domains, while C-2 contains the GAP catalytic domain. Proteins
were purified using glutathione-coated Sepharose beads (as described under ``Materials and Methods''), and immobilized
fusion proteins were used in subsequent binding
assays.
Results show that none of the individual domains of
p120 GAP bound p190 to an appreciable extent (Fig. 2A).
Similarly, each SH2 domain in conjunction with the SH3 domain bound
very little p190. In contrast, the N- and C-terminal SH2 domains
together with the SH3 domain contained in the (N+C)SH fusion
protein bound p190 to a greater extent. This enhanced binding is not
due simply to an effect of the SH3 domain upon the individual SH2
domains, as neither NSH2+SH3 nor CSH2+SH3 fusion proteins
bound p190 at levels significantly above those seen with NSH2 or CSH2
fusion proteins alone. Only when both SH2 domains were present did the
levels of binding increase, suggesting that the binding of p120 to p190
is mediated by both SH2 domains of p120. Comparison with the levels of
p190 in whole cell lysate indicates that approximately 2-3% of
total p190 bound to (N+C)SH fusion protein in this experiment.
Levels of GST fusion proteins used in each precipitation were
determined by Coomassie staining of the same gel (Fig. 2B). Because SH2 domains interact with
phosphorylated tyrosine residues, we examined the effect of increasing
p190 tyrosine phosphorylation upon its interaction with the GST-p120
fusion proteins.
Figure 2:
The
p120
Figure 3:
Phosphorylation of p190 by activated c-Src
tyrosine kinase. Sf9 insect cells were infected with wild-type
baculovirus (Bac), baculoviruses encoding c-Src or p190, or
were coinfected with c-Src and p190 baculoviral recombinants, as
indicated. Cells were lysed 48 h postinfection, and clarified lysates
were loaded in triplicate for separation by SDS-PAGE and analysis by
Western blot using anti-Src (A), anti-p190 (B), or
anti-phosphotyrosine antibodies (C). Molecular mass markers
are indicated in kDa at the right of each
blot.
We assayed for p190 binding after incubating Sf9 cell lysates of
p190 virus singly-infected cells or p190/Src virus-coinfected cells
with purified GST-p120 fusion proteins. Levels of p190 bound to the
p120 fusion proteins were determined by Western blot using anti-p190
antibodies (Fig. 4A), and levels of GST-p120 fusion
proteins used in each incubation are shown by Coomassie staining of the
same gel (Fig. 4B). We quantified the relative binding
of p190 to GST-p120 fusion proteins after normalization with respect to
levels of p190, as well as to levels of GST fusion proteins used in
each incubation (Fig. 4C). Significantly, the SH3
domain of p120 bound neither the phosphorylated nor unphosphorylated
form of p190, leading us to conclude that it does not directly mediate
the interaction between p120 and p190. Similarly, GST-p120 fusion
proteins containing C-terminal p120 GAP regions did not bind
phosphorylated p190 (data not shown), implying that these domains do
not directly mediate the interaction between p120 GAP and
phosphorylated p190. On the other hand, tyrosine phosphorylation of
p190 did enhance its binding to the isolated N- and C-terminal SH2
domains to similar low extents. p190 phosphorylation also enhanced its
binding to the NSH2+SH3 and CSH2+SH3 constructs to
approximately the same extent as the individual SH2 domains,
demonstrating that the SH3 domain does not substantially enhance
binding of p190 to the individual SH2 domains. Importantly, when both
SH2 domains were present on the same polypeptide, binding to
phosphorylated p190 was increased in a synergistic manner compared with
that of the individual SH2 domains. In this experiment, approximately
30-40% of total phosphorylated p190 bound to (N+C)SH fusion
protein, as determined by comparison with the levels of p190 in whole
cell lysates. Tyrosine phosphorylation of p190 increased its binding to
(N+C)SH by at least 10-fold compared with unphosphorylated p190.
These findings show the dependence on tyrosine phosphorylation of p190
and the necessary presence of both N- and C-terminal p120 SH2 domains
for maximal complex formation.
Figure 4:
Two
SH2 Domains in p120 GAP synergistically bind tyrosine phosphorylated
p190. Sf9 cells infected singly with p190 or coinfected with p190 and
c-Src baculovirus recombinants were lysed in RIPA buffer. Clarified
cell lysates were incubated for 1 h at 4 °C with purified GST-p120
fusion proteins that had been immobilized on glutathione-Sepharose
beads. Following incubation, beads were washed, and complexes were
separated by SDS-PAGE. A, levels of unphosphorylated p190
(- Src) or p190 phosphorylated in the presence of Src (+ Src) that bound to the p120 GAP fusion proteins were
assayed by Western blot using anti-p190 antibodies. 10% of
p190-infected and p190/Src-coinfected whole cell lysates (lanes15 and 16) used in each incubation were loaded
for comparison, and nonspecific binding was determined by incubating
lysates with GST alone (lanes13 and 14). B, levels of GST-p120 fusion proteins used in each incubation
are shown by Coomassie stain of the lower portion of the same gel. C, relative binding of p190 (openbars) and
p190 phosphorylated by c-Src (shadedbars) to GST
fusion proteins was quantified. Normalization was with respect to
levels of phosphorylated and unphosphorylated p190 used in each
precipitation, as well as to levels of GST fusion proteins used in each
incubation.
Figure 5:
Binding of p120 double point mutant,
RSH2E, to tyrosine phosphorylated p190 is reduced. Lysates of Sf9 cells
infected with either the p120 mutant RSH2E or wild type p120 virus were
mixed with Sf9 lysates of p190 virus singly infected or p190/Src virus
coinfected cells for 10 min on ice. Mixed lysates were
immunoprecipitated with monoclonal anti-GAP antibody 2A5B4, loaded in
duplicate, and analyzed by Western blot using anti-p120 polyclonal
antibody (A) and anti-p190 polyclonal antibody (B). C, the nitrocellulose membrane from panelB was stripped and reprobed with anti-phosphotyrosine monoclonal
antibody.
3Y1 or SR3Y1 cells were lysed and
incubated with either anti-p120 or anti-p190 monoclonal antibodies.
Resulting precipitates were resolved by SDS-PAGE, and immunoblots were
probed with anti-p120, anti-p190 or anti-phosphotyrosine antibodies (Fig. 6, A-C). The p190 in the v-Src transformed
SR3Y1 whole cell lysates is phosphorylated on tyrosine to a higher
extent than that in normal 3Y1 cells (panel C). Consistent
with our findings using Sf9 cell lyates, the level of p120 GAP bound to
p190 in anti-p190 immunoprecipitates was increased in SR3Y1 cells (panelA). Essentially the same result was obtained
when monoclonal anti-p120 antibodies were used to immunoprecipitate the
p120
Figure 6:
Tyrosine phosphorylation of p190
correlates with enhanced binding to full-length p120 GAP in rat
fibroblasts. Confluent 10-cm plates of either normal 3Y1 or
v-Src-transformed SR-3Y1 cells were lysed in RIPA buffer and clarified.
p120 GAP and p190 were precipitated from lysates with anti-p120
monoclonal antibody 6F2H5G1 (6F2) or monoclonal anti-p190 antibody 8C10
(p190-M) as indicated at the top of each lane.
Immunocomplexes were loaded in duplicate and analyzed by Western blot
with anti-p120 polyclonal antibody (A) and anti-p190
monoclonal antibody, 8C10 (B). C, the nitrocellulose
membrane from panel B was stripped and reprobed with
monoclonal anti-phosphotyrosine antibody to determine the levels of
protein tyrosine phosphorylation in the
immunocomplexes.
Figure 7:
Enhanced binding of tyrosine
phosphorylated p190 to full-length p120 GAP detected with different
monoclonal anti-p120 antibodies. Confluent 10 cm plates of either 3Y1
or SR-3Y1 cells were lysed in RIPA buffer and clarified. p120 GAP and
p190 were precipitated from lysates with anti-p120 monoclonal antibody
3F3E6 (3F3) or anti-p190 polyclonal antibody (p190) as indicated at the top of each lane. Resulting complexes were analyzed
by Western blot with anti-p190 polyclonal antibodies (A) or
anti-phosphotyrosine monoclonal antibodies (B). C, to
extend results shown in panel A, 3Y1 and SR-3Y1 lysates were
also precipitated with 2A5B4 (2A5) or 10E6F3 (10E)
anti-p120 monoclonal antibodies. Immunopreciptates, along with 2% of
the whole cell lysates (WCL) used in each precipitation were
analyzed by immunoblotting with polyclonal anti-p190 antibodies for
levels of p190 co-precipitating with p120
GAP.
While the catalytic activity of p120 GAP toward Ras has been
studied
extensively(10, 11, 12, 14, 43, 44) ,
the characterization of its interactions with associated proteins, such
as p62 and p190, has been less well studied. In v-Src-transformed
cells, only a small fraction of total cellular p120 GAP is found in the
particulate membrane fraction along with Ras and p62. The majority of
p120 GAP is complexed with p190 in the cytosolic fraction of the
cell(34, 35, 38) . Interest in p190 was
heightened after the discovery that it too had GAP-like activity, but
it was toward members of the Rho subfamily of small GTP-binding
proteins(29, 45) . The Rho GTPases (RhoA, RhoB, RhoC,
Rac1, Rac2, CDC42Hs, and TC10) are Ras-related proteins that contribute
to changes in cell morphology, including membrane ruffling and actin
stress fiber formation(13) . Because p190 exhibits Rho GAP
activity, an intriguing model has emerged in which the cell, through
p120 To characterize further the
p120 We show that tyrosine phosphorylation of p190 enhances its
binding to the individual p120 SH2 domains to an equivalent limited
extent and that the two SH2 domains together synergistically bind
approximately 10-fold more p190. Although others have shown examples of
p120 SH2/SH3 domains synergistically mediating interactions with the
platelet-derived growth factor receptor in fibroblasts, only a small
increase in binding of the entire SH2/SH3/SH2 region to the receptor
relative to the individual N-terminal SH2 domains was
observed(26) . In addition, only a low level of the isolated
C-terminal SH2 domain bound to the receptor in comparison with the
isolated N-terminal SH2 domain, raising a question as to the
significance of the presence of the C-terminal SH2 domain(26) .
In the cases of p120 GAP-associated proteins p62 and the epidermal
growth factor receptor, it has been reported that these interactions
are mediated primarily by the N-terminal SH2 domain and only weakly by
the C-terminal SH2 domain, although potential synergistic effects of
both SH2 domains were not determined(28) . In our experiments,
we show that the presence of the C-terminal SH2 domain is necessary for
maximal p120 While
the necessity of both p120 SH2 domains for maximal binding is evident
from our results, the role that the SH3 domain plays in this
interaction is less clear. From these experiments, we can conclude that
the SH3 domain does not bind directly to p190 nor does it contribute to
the ability of individual SH2 domains to bind p190. We cannot, however,
rule out the possibility that the SH3 domain might have a structural
role, such as providing a spatial requirement between both SH2 domains,
that is only possible when all three domains are on the same
polypeptide. Consistent with the conclusion that the SH3 domain does
not directly bind p190 is our finding that when full-length p120 GAP
was immunoprecipitated from rat fibroblasts with monoclonal anti-p120
antibodies directed against the SH3 domain, these antibodies did not
disrupt the p120 To extend our findings from the
baculovirus/Sf9 cell system, we examined p120 A
model consistent with our results is one in which a kinase like Src
phosphorylates p190 on two tyrosine residues, thereby stabilizing its
association with p120 GAP via the SH2 domains. There is some evidence
suggesting that p120 in a complex with p190 has reduced catalytic
activity toward Ras(35) , raising the possibility that complex
formation contributes to Ras activation. Similarly, p190 in the complex
may have reduced GAP activity toward Rho family proteins.
Alternatively, because the p120
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
p190 interaction further, we used
bacterially expressed glutathione S-transferase fusion
polypeptides to map the regions of p120 necessary for its interactions
with p190. Our results show that both the N-terminal and the C-terminal
SH2 domains of p120 are individually capable of binding p190 expressed
in a baculovirus/insect cell system. Moreover, the two SH2 domains
together on one polypeptide bind synergistically to p190, and this
interaction is dependent on tyrosine phosphorylation of p190. In
addition, mutation of the highly conserved Arg residues in the critical
FLVR sequences of both SH2 domains of full-length p120 reduces binding
to tyrosine-phosphorylated p190. The dependence on p190 phosphorylation
for complex formation with p120 SH2 domains observed in vitro is consistent with analysis of the native p120
p190 complexes
formed in vivo. These findings suggest that
SH2-phosphotyrosine interaction is one mechanism by which the cell
regulates p120
p190 association and thus may be a means for
coordinating the Ras- and Rho-mediated signaling pathways.
(
)p120, which was originally identified by
its ability to stimulate the intrinsic GTPase activity of Ras over
100-fold(10, 11, 12, 13) .
(
)This subfamily of Ras-related proteins is involved
in membrane ruffling and formation of actin stress fibers in response
to growth factor stimulation(32, 33) . In
v-Src-transformed or epidermal growth factor-stimulated cells, the
majority of cytoplasmic p120 is complexed with
p190(34, 35) . The biological significance of this
interaction between the p120 and p190 GAPs is not yet clear. One
attractive possibility is that through this complex, cells may couple
the Ras-mediated signaling pathway to that of other GTPases, such as
Rho or Rac, thereby coordinating DNA replication with changes in cell
morphology.
p190
interaction, we have used a series of bacterially expressed GST fusion
proteins containing isolated regions of p120 to precisely map the
domains that interact with p190 expressed in a baculovirus/insect cell
system. Our results show that the two SH2 domains of p120 bind in a
synergistic manner to tyrosine phosphorylated p190. Upon mutating
critical SH2 residues, which are involved in mediating interactions
with phosphotyrosine, binding of full-length p120 to tyrosine
phosphorylated p190 is reduced. Consistent with these in vitro binding studies, p120 association with p190 in rat fibroblasts
transformed by v-Src correlates with tyrosine phosphorylation of p190.
These results suggest that SH2-phosphotyrosine interactions contribute
to regulation of p120
p190 complex formation in the cell and in
this way may coordinate the Ras- and Rho-mediated signaling pathways
during cell proliferation.
Cell Culture
Spodoptera frugiperda (Sf9) insect cells (American Type Culture Collection) were
cultured as described previously(36) . For protein production,
Sf9 cells were singly infected or coinfected with recombinant
baculovirus stocks using a multiplicity of infection of 10 for each
virus(25) . Untransformed (3Y1) and v-Src-transformed (SR3Y1)
rat fibroblasts (37) were maintained in Dulbecco's
modified Eagle's medium as described previously(38) .Baculovirus Recombinants
Construction of the
following recombinant baculoviral vectors has been described
elsewhere(39) : bSrc coding for full-length chicken c-Src, bGAP
encoding full-length bovine p120 GAP, GAPdSH encoding p120 GAP with a
deletion of amino acids 166-518, and GAPdCAT encoding p120 GAP
with a deletion of amino acids 751-983. The baculovirus RSH2E
mutant encodes full-length bovine p120 GAP containing two Arg
Glu point mutations at amino acids 203 and 373.
(
)The complete human p190 cDNA was also expressed from
a baculoviral recombinant.
(
)
GST Fusion Proteins
Regions of human
p120 GAP containing the variable N-terminal region, various
combinations of the SH2 and SH3 domains, or the C-terminal domains were
subcloned into pGEX-2T and expressed in Escherichia
coli(40) . Cells were pelleted and resuspended in buffer
containing phosphate-buffered saline, 1% Triton X-100, 1 mM EDTA, 0.1% -mercaptoethanol, 0.2 mM phenylmethanesulfonyl fluoride, and 5 mM benzamidine.
Lysozyme was added to a final concentration of 0.5 mg/ml, and then
cells were lysed by sonication and clarified. The soluble fraction was
incubated with glutathione-coated Sepharose beads (Pharmacia Biotech
Inc.) for 30 min at 4 °C and washed 3 times in washing buffer.
Beads were stored in 50 mM HEPES, pH 8.0, 150 mM
NaCl, 10% glycerol, 0.1 mM dithiothreitol, and 5 mM benzamidine at 4 °C. Immobilized fusion proteins were used in
subsequent binding assays.
Reconstitution of GST-p120
Lysates of Sf9 insect cells infected with various
baculovirus recombinants were incubated with different purified
GST-p120 domain fusion proteins immobilized on glutathione-Sepharose
beads for 1 h at 4 °C. The resulting complexes were collected by
centrifugation, washed 3 times with RIPA buffer (150 mM NaCl,
50 mM Tris-HCl, pH 7.5, 1% Nonidet P-40, 0.25% sodium
deoxycholate, 1 mM sodium orthovanadate, 1 mM phenylmethanesulfonyl fluoride, 1 µM leupeptin, 1
µM antipain, 0.1 µM aprotinin, 10 µg/ml
p190
Complexes
-macroglobulin, 2 mM EGTA) containing 1
mM sodium orthovanadate and lacking protease inhibitors,
boiled in SDS-gel sample buffer, and resolved by SDS-PAGE. Levels of
baculoviral-expressed recombinant p190 bound to the GST-p120 fusion
proteins were assayed by Western blot analysis using polyclonal
anti-p190 antibodies followed by horseradish peroxidase-conjugated goat
anti-rabbit IgG antibodies for detection by enhanced chemiluminescence
(ECL) (Amersham Corp.). Normalization of GST-p120 fusion proteins used
in each incubation with p190 was confirmed by Coomassie stain of the
lower portion of the same gel used for Western blot analysis of p190.
Levels of p190 bound to GST fusion proteins were quantified from films
exposed in the linear range using the AMBIS optical imaging system.
Cell Lysis and Immunoprecipitations
48 h
postinfection, Sf9 cells were lysed in RIPA buffer for 15 min at 4
°C. Lysates were clarified by centrifugation for 15 min and used in
subsequent binding assays. Confluent 3Y1 or SR3Y1 fibroblasts (10-cm
plates) were lysed in 1 ml of RIPA lysis buffer as described above.
Proteins in cell lysates were immunoprecipitated with antibodies for 1
h at 4 °C, incubated with protein A-Sepharose beads (Pharmacia) for
30 min at 4 °C and then collected by centrifugation.
Immunoprecipitates were washed 3 times with RIPA buffer, and resolved
by SDS-PAGE. Levels of p120 GAP, p190, and their tyrosine
phosphorylation states were determined by Western blot analysis using
monoclonal anti-phosphotyrosine antibodies, and monoclonal or
polyclonal anti-p120 or anti-p190 antibodies followed by horseradish
peroxidase-conjugated anti-mouse or anti-rabbit antibodies for
detection by ECL.Antibodies
Anti-p120 rabbit polyclonal sera was
raised against the N terminus of human p120 GAP (amino acid residues
1-181). Preparation of anti-p120 and anti-p190 monoclonal
antibodies as well as anti-p190 polyclonal sera have been
described(41) . Anti-phosphotyrosine monoclonal antibody 4G10
was obtained from UBI (Lake Placid, NY).
p120 GAP SH2 Domains Mediate Its Interaction with
p190
Previous studies indicated that the majority of p120 GAP
associates with a cellular phosphoprotein, p190, in v-Src-transformed
cells(35) . To determine which domains of p120 GAP mediate this
interaction, we used a panel of GST fusion proteins spanning the entire
length of human p120 (Fig. 1). Within this panel, p120 SH2 and
SH3 domains are present singly or in combination with each other,
allowing us to define individual as well as any possible synergistic
contributions of these domains to formation of the p120p190
complex. After expressing these constructs in E. coli, cells
were lysed, and fusion proteins were purified using
glutathione-Sepharose beads. These immobilized GST fusion proteins were
then incubated with Sf9 insect cell lysates containing
baculoviral-expressed p190, and the levels of p190 that co-precipitated
with the p120 fusion proteins were analyzed by SDS-PAGE and
immunoblotting.
p190 interaction is mediated by p120 SH2 domains. Clarified
lysate of Sf9 insect cells infected with a baculovirus recombinant
encoding p190 was incubated with purified GST-p120 domain fusion
proteins immobilized on glutathione-Sepharose beads. The resulting
complexes were collected by centrifugation, washed, and resolved by
SDS-PAGE. A, levels of p190 bound to the GST fusion proteins
were assayed by Western blot analysis using anti-p190 polyclonal
antibodies. 1% of the Sf9 whole cell lysate (WCL) containing
p190 used in each incubation was loaded for comparison. As a negative
control, GST bound to glutathione-beads alone (GST) was
incubated with p190-containing lysates. B, levels of GST
fusion proteins used in each incubation with p190 are shown by
Coomassie stain of the lower portion of the same gel. Numbers at right indicate molecular mass markers in
kDa.
The N- and C-terminal p120 SH2 Domains Together in One
Polypeptide Synergistically Bind Tyrosine Phosphorylated p190
To
explore the effects of phosphorylation on p120p190 interactions,
p190 was phosphorylated on tyrosine using a baculoviral c-Src
recombinant that exhibits an elevated kinase activity compared with
endogenous rodent or chicken c-Src(25) . Previous studies
suggested that p190 is a substrate of c-Src tyrosine kinase in intact
rodent fibroblasts(42) . Sf9 insect cells were singly infected
with wild-type baculovirus or recombinant baculovirus encoding either
activated c-Src or p190 or were coinfected with both c-Src and p190
viruses. Cell lysates were loaded in triplicate for separation by
SDS-PAGE and assayed by Western blot analysis using anti-Src,
anti-p190, or anti-phosphotyrosine antibodies (Fig. 3, A-C). Results show very little tyrosine phosphorylation
of p190 in infected cells by endogenous insect cell kinases; however,
the level of tyrosine phosphorylation of p190 is markedly increased
upon coinfection with the activated c-Src baculovirus, despite the
lower overall levels of p190 in the p190/Src virus-coinfected cells.
Mutation of Highly Conserved Arg Residues in Both SH2
Domains Reduces Binding of Full-length p120 to Tyrosine Phosphorylated
p190
To examine the contribution of p120 SH2 domains to complex
formation in the context of the full-length protein, we constructed a
p120 GAP baculoviral recombinant (RSH2E) in which the highly conserved
Arg residue of the FLVR sequence in both SH2 domains was mutated to Glu
using polymerase chain reaction mutagenesis. This Arg residue is one
critical component of the SH2 phosphotyrosine binding pocket and forms
an ion pair with the phosphate group(18) . Others have shown
using GST-p120 fusion proteins that when this residue is mutated,
binding of the individual SH2 domains to p120 GAP-associated protein
p62 and the EGF receptor is abolished(28) . We expressed the
p120 double point mutant as well as wild-type p120 GAP in Sf9 cells,
and mixed these p120-containing lysates with either p190 or p190/Src
virus-infected cell lysates. The p120p190 GAP complexes were
immunoprecipitated with an anti-p120 antibody and detected by Western
blot analysis using anti-p120, anti-p190, and anti-phosphotyrosine
antibodies. Equivalent amounts of wild-type and mutant p120 GAP were
immunoprecipitated by the anti-p120 antibody (Fig. 5A).
While both p120 GAPs bound little unphosphorylated p190, the p120
double point mutant bound significantly less phosphorylated p190
compared with wild-type p120 GAP (Fig. 5, B and C). These results are consistent with our GST-p120 GAP
findings suggesting that the p120
p190 GAP interaction is mediated
by p120 SH2 domains and depends on p190 tyrosine phosphorylation for
maximal complex formation.
Tyrosine Phosphorylation of p190 Correlates with Enhanced
p120 GAP Binding in Vivo
Because p120 protein preferentially
binds phosphorylated p190 in vitro, we compared the levels of
native p120p190 complex formation in normal 3Y1 rat fibroblasts
and v-Src-transformed 3Y1 cells (SR3Y1). To study the interaction
between endogenous p120 GAP and p190 in rat fibroblasts, we
characterized several anti-p120 monoclonal antibodies(41) . We
found that those antibodies that specifically immunoprecipitated p120
and maintained the p120
p190 complex all recognized the SH3 domain
of p120 GAP (data not shown).
p190 complex (panelB). To ensure that the
observed phosphorylation-dependent increase in the level of p190
binding to p120 was not an artifact of the particular antibodies being
used, the same immunoprecipitation experiment shown in Fig. 6was performed using three additional monoclonal anti-p120
antibodies. The polyclonal anti-p190 antibody did not immunoprecipitate
the p120
p190 complex well, but it did show the difference in the
phosphorylation state of p190 in v-Src-transformed versus normal 3Y1 cells (Fig. 7B). These additional
monoclonal anti-p120 antibodies confirmed our earlier results; more
p190 bound to the full-length p120 GAP when p190 was phosphorylated in
SR3Y1 cells compared with when it was not, despite higher levels of
p190 in 3Y1 lysates than in SR3Y1 lysates (Fig. 7, A-C). Together, these results suggest that the
p120
p190 interaction in intact cells is enhanced by p190 tyrosine
phosphorylation.
p190 association, may coordinate changes in cell morphology
with stimulation of DNA replication.
p190 association, we have used a panel of GST-p120 fusion
proteins and the baculovirus/Sf9 insect cell system to precisely
determine what regions in p120 GAP are necessary for complex formation
with p190 and to define some of the parameters that affect this
interaction. Our results show that, while none of the individual p120
domains bound significant levels of p190, expression of the entire
SH2/SH3/SH2 region in the (N+C)SH construct mediated p190 binding.
That the p120
p190 complex is mediated by the p120 SH2/SH3/SH2
region is consistent with other studies, which have localized this
interaction to the N-terminal half of p120 GAP protein (27, 46, 47) . Because SH2 domains bind to
phosphorylated tyrosine residues(18) , we explored the effect
of p190 tyrosine phosphorylation upon its association with p120 GAP
domains.
p190 complex formation, providing evidence for the
importance of p120 having two SH2 domains. Even with both SH2 domains
present, however, tyrosine phosphorylation of p190 is needed for
maximum complex formation. Together, these results suggest that the two
SH2 domains in p120 bind synergistically to phosphorylated tyrosine
residues in p190. Two additional lines of evidence support the
important contribution of SH2-phosphotyrosine interactions to
p120
p190 complex formation. First, point mutations of the
critical Arg residues in the FLVR sequences of both SH2 domains in
full-length p120 reduced binding to p190 (Fig. 5). Second,
earlier studies showed that a tyrosine phosphorylated peptide
corresponding to the region of PDGF receptor that binds p120 SH2
domains competes with p190 for binding to p120 (31) .
p190 complex.
p190 native complex
formation in vivo using rodent fibroblasts. Consistent with
our insect cell data, more p190 co-precipitated with endogenous
full-length p120 GAP from lysates of v-Src-transformed rat fibroblasts
than from their normal counterparts in anti-p120 immunoprecipitates.
Similarly, more p120 GAP complexed with p190 in anti-p190
immunoprecipitates prepared from v-Src-transformed SR3Y1 cells as
compared with normal 3Y1 cells. This increase in co-precipitation of
p120
p190 complexes paralleled the increase in tyrosine
phosphorylation of p190, consistent with the importance of tyrosine
phosphorylation for complex formation. Previous studies of
p120
p190 complex formation yielded apparently conflicting
results. Early experiments indicated that the majority of p120 GAP
associates with p190 after growth factor stimulation and in
v-Src-transformed Rat-1 fibroblasts(35) . Other studies have
suggested that similar levels of p190 associate with p120 GAP
regardless of the level of p190 tyrosine phosphorylation in C3H10T1/2
fibroblasts(42) . Still others have found that stable
expression in Rat-2 fibroblasts of an N-terminal polypeptide derived
from p120 GAP leads to its association with p190 in both growth
factor-stimulated and serum-deprived cells(47) . One possible
explanation for these contrasting findings is that the association
between p120 and p190 could be regulated at multiple levels in addition
to the SH2-phosphotyrosine interactions described here. Indeed, we
detected a low level of p190 binding to (N+C)SH fusion protein in
the absence of p190 tyrosine phosphorylation by c-Src (Fig. 2).
Additional regulatory events might be dependent upon the mammalian cell
type, and may not be evident using isolated domains of p120 expressed
in bacterial cells. For example, we cannot exclude that there may be
other structural features of p120 GAP that contribute to p190
association only in the context of the full-length, native p120
protein. Consistent with this possibility, we observed that point
mutations in the FLVR sequences of both SH2 domains did not completely
abolish binding of full-length p120 GAP to p190 (Fig. 5).
p190 complex is predominantly
cytosolic, complex formation may prevent access of both GAP proteins to
their membrane-associated substrates. Thus elevated p120
p190
complex formation, promoted by tyrosine phosphorylation and possibly by
other regulatory events, might simultaneously activate both Ras- and
Rho-mediated signaling pathways.
We thank members of the lab for stimulating
discussions and K. Pumiglia for comments on the manuscript.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.