©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Two SH2 Domains of p120 Ras GTPase-activating Protein Bind Synergistically to Tyrosine Phosphorylated p190 Rho GTPase-activating Protein (*)

(Received for publication, December 21, 1994; and in revised form, April 3, 1995)

Sophia S. Bryant (1)(§) Scott Briggs (4) Thomas E. Smithgall (4) George A. Martin (5) Frank McCormick (5) Jin-Hong Chang (6) Sarah J. Parsons (6) Richard Jove (1) (2) (3)(¶)

From the  (1)Cellular and Molecular Biology Program, the (2)Department of Microbiology and Immunology, and the (3)Comprehensive Cancer Center, University of Michigan Medical School, Ann Arbor, Michigan 48109, the (4)Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Nebraska 68198, (5)Onyx Pharmaceuticals, Richmond, California 94806, and the (6)Department of Microbiology, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 p120p190 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 p120p190 complexes formed in vivo. These findings suggest that SH2-phosphotyrosine interaction is one mechanism by which the cell regulates p120p190 association and thus may be a means for coordinating the Ras- and Rho-mediated signaling pathways.


INTRODUCTION

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,()p120, which was originally identified by its ability to stimulate the intrinsic GTPase activity of Ras over 100-fold(10, 11, 12, 13) .

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) .()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.

To characterize in more detail the p120p190 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 p120p190 complex formation in the cell and in this way may coordinate the Ras- and Rho-mediated signaling pathways during cell proliferation.


MATERIALS AND METHODS

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-p120p190 Complexes

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 -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).


RESULTS

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.


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 p120p190 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.


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.



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 p120p190 GAP interaction is mediated by p120 SH2 domains and depends on p190 tyrosine phosphorylation for maximal complex formation.


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.



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 p120p190 complex all recognized the SH3 domain of p120 GAP (data not shown).

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 p120p190 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 p120p190 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 p120p190 interaction in intact cells is enhanced by p190 tyrosine phosphorylation.


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.




DISCUSSION

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 p120p190 association, may coordinate changes in cell morphology with stimulation of DNA replication.

To characterize further the p120p190 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 p120p190 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.

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 p120p190 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 p120p190 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) .

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 p120p190 complex.

To extend our findings from the baculovirus/Sf9 cell system, we examined p120p190 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 p120p190 complexes paralleled the increase in tyrosine phosphorylation of p190, consistent with the importance of tyrosine phosphorylation for complex formation. Previous studies of p120p190 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).

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 p120p190 complex is predominantly cytosolic, complex formation may prevent access of both GAP proteins to their membrane-associated substrates. Thus elevated p120p190 complex formation, promoted by tyrosine phosphorylation and possibly by other regulatory events, might simultaneously activate both Ras- and Rho-mediated signaling pathways.


FOOTNOTES

*
This was supported in part by Grants CA58667 (to T. E. S.), CA39438 (to S. J. P.), and CA55652 (to R. J.) from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Fellow of the University of Michigan Cancer Biology Training Program.

To whom correspondence should be addressed. Dept. of Microbiology and Immunology, 6606 Medical Science II, University of Michigan Medical School, Ann Arbor, MI 48109-0620.

The abbreviations used are: GAP, GTPase-activating protein; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis.

R. Roof, J.-H. Chang, and S. J. Parsons, manuscript submitted.

S. Bryant and R. Jove, unpublished data.

G. Martin and F. McCormick, unpublished data.


ACKNOWLEDGEMENTS

We thank members of the lab for stimulating discussions and K. Pumiglia for comments on the manuscript.


REFERENCES

  1. Kung, H. F., Smith, M. R., Bekesi, E., Manne, V., and Stacey, D. W.(1986) Exp. Cell Res. 162, 363-371 [Medline] [Order article via Infotrieve]
  2. Stacey, D. W., DeGudicibus, S. R., and Smith, M. R.(1987)Exp. Cell Res. 171, 232-242 [Medline] [Order article via Infotrieve]
  3. Lowy, D., and Willumsen, B.(1993)Annu. Rev. Biochem. 62, 851-891 [CrossRef][Medline] [Order article via Infotrieve]
  4. Heidecker, G., Kolch, W., Morrison, D. K., and Rapp, U. R.(1992)Adv. Cancer Res. 58, 53-73 [Medline] [Order article via Infotrieve]
  5. Roberts, T. M. (1992)Nature360,534-535 [CrossRef][Medline] [Order article via Infotrieve]
  6. Crews, C. M., and Erikson, R. L.(1993)Cell 74, 215-217 [Medline] [Order article via Infotrieve]
  7. Moodie, S. A., Willumsen, B. M., Weber, M. J., and Wolfman, A.(1993)Science 260, 1658-1661 [Medline] [Order article via Infotrieve]
  8. Van Aelst, L., Barr, M., Marcus, S., Polverino, A., and Wigler, M.(1993)Proc. Natl. Acad. Sci. U. S. A. 90, 6213-6217 [Abstract]
  9. Vojtek, A. B., Hollenberg, S. M., and Cooper, J. A.(1993)Cell 74, 205-214 [Medline] [Order article via Infotrieve]
  10. Trahey, M., and McCormick, F.(1987)Science 238, 542-545 [Medline] [Order article via Infotrieve]
  11. Trahey, M., Wong, G., Halenbeck, R., Rubinfeld, B., Martin, G. A., Ladner, M., Long, C. M., Crosier, W. J., Watt, K., Koths, K., and McCormick, F.(1988) Science 242, 1697-1700 [Medline] [Order article via Infotrieve]
  12. Vogel, U. S., Dixon, R. A., Schaber, M. D., Diehl, R. E., Marshall, M. S., Scolnick, E. M., Sigal, I. S., and Gibbs, J. B.(1988)Nature 335, 90-93 [CrossRef][Medline] [Order article via Infotrieve]
  13. Boguski, M. S., and McCormick, F.(1993)Nature 366, 643-654 [CrossRef][Medline] [Order article via Infotrieve]
  14. Marshall, M., Hill, W., Ng, A., Vogel, U., Schaber, M., Scolnick, E., Dixon, R., Sigal, I., and Gibbs, J.(1989)EMBO J. 8, 1105-1110 [Abstract]
  15. Pawson, T., and Gish, G. D.(1992)Cell 71, 359-362 [Medline] [Order article via Infotrieve]
  16. Mayer, B. J., Hamaguchi, M., and Hanafusa, H.(1988)Nature 332, 272-275 [CrossRef][Medline] [Order article via Infotrieve]
  17. Musacchio, A., Noble, M., Pauptit, R., Wierenga, R., and Saraste, M.(1992) Nature 359, 851-855 [CrossRef][Medline] [Order article via Infotrieve]
  18. Waksman, G., Kominos, D., Robertson, S. C., Pant, N., Baltimore, D., Birge, R. B., Cowburn, D., Hanafusa, H., Mayer, B. J., Overduin, M., Resh, M., Rios, C., Silverman, L., and Kuriyan, J.(1992)Nature 358, 646-653 [CrossRef][Medline] [Order article via Infotrieve]
  19. Birge, R. B., and Hanafusa, H.(1993)Science 262, 1522-1524 [Medline] [Order article via Infotrieve]
  20. Ren, R., Mayer, B. J., Cicchetti, P., and Baltimore, D.(1993)Science 259, 1157-1161 [Medline] [Order article via Infotrieve]
  21. Marengere, L. E., Songyang, Z., Gish, G. D., Schaller, M. D., Parsons, J. T., Stern, M. J., Cantley, L. C., and Pawson, T.(1994)Nature 369, 502-505 [CrossRef][Medline] [Order article via Infotrieve]
  22. Kaplan, D. R., Morrison, D. K., Wong, G., McCormick, F., and Williams, L. T.(1990) Cell 61, 125-133 [Medline] [Order article via Infotrieve]
  23. Kazlauskas, A., Ellis, C., Pawson, T., and Cooper, J.(1990)Science 247, 1578-1581 [Medline] [Order article via Infotrieve]
  24. Brott, B., Decker, S., Shafer, J., Gibbs, J., and Jove, R.(1991)Proc. Natl. Acad. Sci. U. S. A. 88, 755-759 [Abstract]
  25. Park, S., Liu, X., Pawson, T., and Jove, R.(1992)J. Biol. Chem. 267, 17194-17200 [Abstract/Free Full Text]
  26. Anderson, D., Koch, C., Grey, L., Ellis, C., Moran, M., and Pawson, T.(1990) Science 250, 979-982 [Medline] [Order article via Infotrieve]
  27. Moran, M., Koch, A., Anderson, D., Ellis, C., England, L., Martin, G., and Pawson, T. (1990)Proc. Natl. Acad. Sci. U. S. A. 87, 8622-8626 [Abstract]
  28. Marengere, L., and Pawson, T.(1992)J. Biol. Chem. 267, 22779-22786 [Abstract/Free Full Text]
  29. Settleman, J., Albright, C. F., Foster, L. C., and Weinberg, R. A.(1992) Nature 359, 153-154 [CrossRef][Medline] [Order article via Infotrieve]
  30. Foster, R., Hu, K.-Q., Shaywitz, D., and Settleman, J.(1994)Mol. Cell. Biol. 14, 7173-7181 [Abstract]
  31. Fantl, W. J., Escobedo, J. A., Martin, G. A., Turck, C. W., del Rosario, M., McCormick, F., and Williams, L. T.(1992)Cell 69, 413-423 [Medline] [Order article via Infotrieve]
  32. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., and Hall, A.(1992) Cell 70, 401-410 [Medline] [Order article via Infotrieve]
  33. Ridley, A. J., and Hall, A.(1992)Cell 70, 389-399 [Medline] [Order article via Infotrieve]
  34. Ellis, C., Moran, M., McCormick, F., and Pawson, T.(1990)Nature 343, 377-381 [CrossRef][Medline] [Order article via Infotrieve]
  35. Moran, M., Polakis, P., McCormick, F., Pawson, T., and Ellis, C.(1991)Mol. Cell. Biol. 11, 1804-1812 [Medline] [Order article via Infotrieve]
  36. O'Reilly, D., Miller, L., and Luckow, V. (1992) Baculovirus Expression Vectors: A Laboratory Manual, W. H. Freeman and Company, New York, NY
  37. Kawai, S.(1980) J. Virol. 34, 772-776 [Medline] [Order article via Infotrieve]
  38. Park, S., and Jove, R. (1993)J. Biol. Chem.268,25728-25734 [Abstract/Free Full Text]
  39. Park, S., Marshall, M., Gibbs, J., and Jove, R.(1992)J. Biol. Chem. 267, 11612-11618 [Abstract/Free Full Text]
  40. Hjermstad, S., Briggs, S., and Smithgall, T.(1993)Biochemistry 32, 10519-10525 [Medline] [Order article via Infotrieve]
  41. Chang, J.-H., Sutherland, W. M., and Parsons, S. J.(1995)Methods Enzymol. 254, 430-445 [Medline] [Order article via Infotrieve]
  42. Chang, J.-H., Wilson, L., Moyers, J., Zhang, K., and Parsons, S. J.(1993) Oncogene 8, 959-967 [Medline] [Order article via Infotrieve]
  43. DeClue, J., Zhang, K., Redford, P., Vass, W., and Lowy, D.(1991)Mol. Cell. Biol. 11, 2819-2825 [Medline] [Order article via Infotrieve]
  44. Gideon, P., John, J., Frech, M., Lautwein, A., Clark, R., Scheffler, J., and Wittinghofer, A. (1992)Mol. Cell. Biol. 12, 2050-2056 [Abstract]
  45. Settleman, J., Narasimhan, V., Foster, L., and Weinberg, R.(1992) Cell 69, 539-549 [Medline] [Order article via Infotrieve]
  46. Koch, C., Moran, M., Anderson, D., Liu, X., Mbmalu, G., and Pawson, T.(1992) Mol. Cell. Biol. 12, 1366-1374 [Abstract]
  47. McGlade, J., Brunkhorst, B., Anderson, D., Mbamalu, G., Settleman, J., Dedhar, S., Rozakis-Adcock, M., Chen, L., and Pawson, T.(1993) EMBO J.12,3073-3081 [Abstract]

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