©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Ras GTPase-activating Protein (GAP) Is an SH3 Domain-binding Protein and Substrate for the Src-related Tyrosine Kinase, Hck (*)

Scott D. Briggs (1), Sophia S. Bryant (2), Richard Jove (2), Sam D. Sanderson (1), Thomas E. Smithgall (1)(§)

From the (1)Eppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Nebraska 68198 and the (2)Cellular and Molecular Biology Program, University of Michigan Medical School, Ann Arbor, Michigan, 48109

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The Ras GTPase-activating protein (GAP) is a target for protein tyrosine kinases of both the receptor and cytoplasmic classes and may serve to integrate tyrosine kinase and Ras signaling pathways. In this report, we provide evidence that GAP is an SH3 domain-binding protein and substrate for the Src-related tyrosine kinase Hck, which has been implicated in the regulation of myeloid cell growth, differentiation, and function. Wild-type (WT) or kinase-inactive (K269E) mutant Hck proteins were co-expressed with bovine GAP using the baculovirus/Sf-9 cell system. GAP was readily phosphorylated on tyrosine by WT but not K269E Hck. GAP was present in WT Hck immunoprecipitates from the co-infected cells, indicative of HckGAP complex formation. Unexpectedly, GAP also associated with the kinase-inactive mutant of Hck, suggesting that tyrosine autophosphorylation of Hck is not required for complex formation. The WT and K269E forms of Hck also associated with GAP mutants lacking either the C-terminal catalytic domain (CAT) or the Src homology region (SH), indicating that these GAP domains are dispensable for complex formation. Recombinant GST fusion proteins containing the Hck, Src, Fyn, or Lck SH3 domains associated with full-length GAP, CAT, and SH, all of which share an N-terminal proline-rich region resembling an SH3-binding motif (PPLPPPPPQLP). Deletion of the highly conserved YXY sequence from the Hck SH3 domain abolished binding. GAP-SH3 interaction was also inhibited by the proline-rich peptide GFPPLPPPPPQLPTLG, which corresponds to N-terminal amino acids 129-144 of bovine GAP. An N-terminal deletion mutant of GAP lacking this proline-rich region did not bind to the Hck SH3 domain. These data implicate the Hck SH3 domain in GAP interaction, and suggest a general function for the SH3 domains of Src family kinases in recognition of GAP via its proline-rich N-terminal domain.


INTRODUCTION

Hck is a member of the Src family of cytoplasmic protein tyrosine kinases and is expressed primarily in hematopoietic cells of the myeloid and B-lymphoid lineages(1, 2, 3) . Previous studies have linked Hck activation to granulocyte-macrophage colony-stimulating factor signal transduction (4) as well as macrophage activation by bacterial lipopolysaccharide(5, 6, 7) . In addition, Hck expression is significantly enhanced during the terminal differentiation of myeloid leukemia cells in vitro(4, 8) . These findings suggest a diverse role for Hck in signal transduction processes required for the growth, differentiation, and function of hematopoietic cells. A more recent study has linked Hck activation to leukemia inhibitory factor signaling in embryonic stem cells, suggesting that Hck may play a role in early development as well(9) . However, the cellular targets for the Hck kinase are currently undefined.

Hck shares several structural features with Src. These include a C-terminal tail with a negative regulatory tyrosine residue (Tyr-501), a tyrosine kinase domain, non-catalytic Src homology 2 and 3 (SH2 and SH3) domains, and a unique N-terminal domain (reviewed in Ref. 10). SH2 domains have been postulated to regulate the kinase activity of cytoplasmic protein tyrosine kinases by binding phosphorylated tyrosine residues in the catalytic domain and tail region(10) . SH3 domains also help to regulate tyrosine kinase activity and may cooperate with SH2 domains in this regard(11, 12, 13) .

SH2 and SH3 domains are also found in other proteins associated with tyrosine kinase signal transduction, including phospholipase C-, the 85-kDa subunit of phosphatidylinositol 3`-kinase (p85 subunit), and the Ras GTPase-activating protein (GAP;()reviewed in Refs. 14-16). Other SH2- and SH3-containing proteins lack an associated catalytic function and serve as molecular adaptors to join proteins with the appropriate Src homology recognition motifs(15, 16) . SH2 domains bind to tyrosine-phosphorylated sequences with high affinity and specificity and help to mediate protein-protein interactions between autophosphorylated tyrosine kinases and downstream effector proteins. SH3 domains are also involved in protein-protein interaction. Unlike SH2 domains, SH3 domains bind to proline-rich sequences in target proteins in a phosphorylation-independent manner (17). Recent evidence suggests a central role for SH3 domains in mediating subcellular localization and substrate recognition by Src-related kinases (see ``Discussion'').

Several lines of evidence strongly implicate Ras as an essential downstream component of Src and other protein tyrosine kinase growth-regulatory signaling pathways. Cells stimulated with mitogenic growth factors or transformed by v-src and other tyrosine kinase oncogenes contain elevated levels of Ras in its active GTP-bound form(18, 19, 20) . Conversely, microinjection of antibodies to Ras blocks cellular transformation by tyrosine kinase oncogenes as well as the mitogenic response to epidermal growth factor and platelet-derived growth factor(21, 22) . Similarly, dominant-negative Ras mutants block tyrosine kinase-mediated signal transduction(23) .

GAP may serve as part of the biochemical link between protein tyrosine kinases and Ras signal transduction(24, 25) . Several members of the Src family have been shown to associate with and phosphorylate GAP, including v-Src, c-Src, Lck, Yes, Fyn, and Lyn(26, 27, 28, 29, 30, 31, 32, 33, 34) . Structurally, GAP consists of a C-terminal catalytic domain that interacts with Ras and an N-terminal regulatory region containing SH2 and SH3 domains (reviewed in Ref. 35; see Fig. 1). GAP enhances the intrinsic GTPase activity of Ras, promoting its conversion from the active GTP-bound form to the inactive GDP-bound form(24, 25) . Although this catalytic function of GAP suggests that it is primarily a negative regulator of Ras, other studies suggest an effector function for GAP as well. For example, expression of deletion mutants of GAP that lack the C-terminal catalytic domain can induce gene expression from a Ras-dependent reporter gene construct (36) and cytoskeletal reorganization(37) . Furthermore, antibodies to the GAP SH3 domain block Ras-dependent germinal-vesicle breakdown in Xenopus oocytes(38) .


Figure 1: Structures of Hck and bovine GAP mutants used in this study. Numbering above the diagrams indicates the amino acid boundaries of each of the various domains and the locations of the deletions. SH, Src homology; PPPP, putative proline-rich SH3 target region; CAT, GAP catalytic domain.



In the present study, we provide evidence that GAP is a substrate for the Src family kinase, Hck. GAP associates with Hck in a phosphotyrosine-independent manner involving the Hck SH3 domain and a proline-rich sequence found in the N-terminal domain of GAP. Identification of an SH3-binding motif within GAP suggests a previously unrecognized mode of protein-protein interaction for this critical Ras regulator/effector.


EXPERIMENTAL PROCEDURES

Generation of Recombinant Hck and GAP Baculoviruses

A full-length human Hck cDNA was obtained from the American Type Culture Collection (ATCC, Rockville, MD). This clone was originally isolated by Ziegler et al.(1) , and contains artifactual 5` sequences that lack proper initiation signal sequences for translation. These sequences were deleted and a functional initiation codon was restored using the polymerase chain reaction (PCR). The conserved kinase domain residue Lys-269 of Hck was converted to Glu using standard PCR-based techniques. The resulting wild-type and K269E mutant cDNAs were inserted into the baculovirus transfer vector pVL1393 (Pharmingen, San Diego, CA) and used to generate recombinant baculoviruses using Baculogold DNA and the manufacturer's protocol (Pharmingen).

The N GAP mutant was prepared by digesting a pBlueBac transfer vector (InVitrogen) containing the p120 bovine GAP cDNA with MscI and BglII. A NotI linker was added to the MscI site of the resulting 1-kilobase fragment, which was then subcloned into NotI/BglII-digested pBlueBac-p120 GAP transfer vector. The recombinant N GAP baculovirus was prepared by co-transfection of Sf9 cells with the transfer vector and wild-type baculoviral DNA, followed by plaque purification using standard protocols.()Preparation of baculoviruses for the expression of full-length, SH, and CAT GAP is described in detail elsewhere(29) .

Antibodies

Monoclonal antibodies to GAP were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Epitope mapping experiments (not shown) indicate that this antibody recognizes part of the GAP SH3 domain, and therefore does not recognize the GAP SH mutant. To allow detection of the GAP SH mutant on immunoblots, rabbit antiserum was raised against a recombinant GST fusion protein containing the first 181 amino acids of human GAP, immediately N-terminal to the first SH2 domain (Panigen, Blanchardsville, WI; see Fig. 1). Antiserum to Hck was purchased from Santa Cruz Biotechnology. This antiserum recognizes the unique N-terminal region of Hck. Monoclonal antibody PY20 to phosphotyrosine was purchased from Transduction Laboratories (Lexington, KY).

Co-expression of Hck and GAP Proteins in Sf9 Insect Cells

For co-expression studies, Sf9 cells were grown to 50% confluence in T-25 flasks and infected at a multiplicity of infection of 5-10 with recombinant Hck and GAP baculoviruses. Forty-eight hours postinfection, cells were lysed by sonication in 1.0 ml of lysis buffer (50 mM Tris-HCl, pH 7.4, 50 mM NaCl, 1 mM EDTA, 1 mM MgCl, and 0.1% Triton X-100) supplemented with 25 µg/ml aprotinin, 50 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 20 mM NaF, 1 mM NaVO, and 50 µM NaMoO. To test for GAP tyrosine phosphorylation, the GAP monoclonal antibody and protein G-Sepharose (10 µl of a 50% slurry; Pharmacia Biotech Inc.) were added to the clarified lysates and incubated at 4 °C for 1 h. Immune complexes were pelleted by centrifugation and washed with three 1.0-ml aliquots of RIPA buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1 mM EDTA, and 1% sodium deoxycholate). Immunoprecipitated proteins were resolved by SDS-PAGE, transferred to polyvinylidine difluoride membranes, and probed with antibodies to phosphotyrosine.

To test for the presence of HckGAP complexes, immunoprecipitates were prepared from lysates of co-infected Sf9 cells with the anti-Hck antiserum. Immunoprecipitated Hck proteins were analyzed for associated GAP or the GAP CAT mutant by immunoblotting with the anti-GAP monoclonal antibody or for the presence of the GAP SH mutant by immunoblotting with the anti-GAP N-terminal antiserum described above.

Expression of Hck-GST Fusions in Escherichia coli and in Vitro Association Reactions

Using PCR, the coding sequences of the Hck unique N-terminal domain (amino acids 1-62), SH3 domain (amino acids 52-122), SH2 domain (amino acids 123-220), and various combinations thereof (see Fig. 6) were amplified and cloned into the bacterial expression vector, pGEX-2T (Pharmacia). Similarly, the coding sequence of the SH3 domain of v-Src (amino acids 76-147) was amplified by PCR and cloned into pGEX-2T. The resulting plasmids were used to express these domains as GST fusion proteins in E. coli(39) . The fusion proteins were purified using glutathione-agarose beads as described elsewhere(40, 41) . GST fusion proteins containing the SH3 domains of Lck and Fyn were purchased from Santa Cruz Biotechnology. The coding region for the highly conserved YXY motif found in the SH3 domain of many Src kinase family members (42) was deleted from the Hck SH3 domain coding sequence using PCR. The resulting Hck SH3 YDY mutant was also expressed as a GST fusion protein for binding studies.


Figure 6: The Hck SH3 domain binds GAP, SH, and CAT in vitro. The noncatalytic domains of Hck (N-terminal, SH3, and SH2 domains) were expressed either alone or in the combinations shown as GST fusion proteins in E. coli and immobilized on glutathione-agarose beads. Immobilized fusion proteins or GST as a negative control were mixed with lysates from Sf9 cells expressing full-length GAP or the GAP deletion mutants CAT, SH, or N. Following incubation and washing, bound GAP proteins were visualized by immunoblotting. The SH3 binding activity of full-length GAP (A) and the GAP deletion mutants SH (B), CAT (C), and N (D) are shown. To verify that N was expressed in part D, an aliquot of the N cell lysate was immunoprecipitated with the GAP monoclonal antibody and immunoblotted in parallel with the other samples (lane marked CON).



For GST fusion protein binding experiments, aliquots of clarified lysates from Sf9 cells expressing full-length and mutant forms of GAP were incubated with Hck-GST fusion proteins or GST (10 µg) immobilized on glutathione-agarose beads in a final volume of 1.0 ml of lysis buffer. Following incubation at 4 °C for 1 h, protein complexes were pelleted by centrifugation and washed three times with RIPA buffer. Associated proteins were eluted by heating in SDS-PAGE sample buffer, and the presence of GAP or GAP mutants was determined by immunoblotting with either the GAP monoclonal antibody (to visualize full-length, CAT and N proteins) or the GAP N-terminal antiserum (for SH GAP mutant).

GAP Proline-rich Peptide Synthesis and Inhibition of SH3 Binding

The proline-rich peptide GFPPLPPPPPQLPTLG, corresponding to bovine GAP N-terminal domain residues 129-144, was synthesized by standard solid phase methods on an Applied Biosystems Model 430A peptide synthesizer. An unrelated peptide with the sequence KPQIAALKEETEEEV was synthesized for use as a negative control. Syntheses were performed on a 0.25 mmol scale on [p-(hydroxymethyl)phenoxy]methylpolystyrene resins (0.88 meq/g substitution). N-Amino groups were protected with the base-labile N-(9-fluorenyl)methyloxycarbonyl (Fmoc) group. Side chain functional groups were protected as follows: Gln (Trt or trityl); Glu (O-tert-butyl ester); Lys (Boc or tert-butyloxycarbonyl); and Thr (tert-butyl). Synthesis was initiated by the in situ coupling of the C-terminal residue to the [p-(hydroxymethyl)phenoxy]methylpolystyrene resin in the presence of excess N,N`-dicyclohexylcarbodiimide and 1-hydroxybenzotriazole with 4-(dimethylamino)pyridine as coupling catalyst. Peptide chain elongation was accomplished by repetitive Fmoc deprotection in 50% piperidine in N-methylpyrrolidinone followed by residue coupling in the presence of 2-(1H-benzotriazol-1-yl)-1,1,1,3,3,-tetramethyluronium hexafluorophosphate. Peptides were purified by high performance liquid chromatography according to previously published methods(43) . The acetate salt form of the peptides were generated in each case and purity (>96%) was assessed as the integrated single peak area on analytical high performance liquid chromatography. Peptides were characterized by amino acid compositional analysis. For inhibition studies, the peptides were added to in vitro association reactions at various concentrations (see legend to Fig. 8) with the immobilized Hck GST-SH3 fusion protein and Sf9 cell lysates containing full-length GAP or the deletion mutants as described above.


Figure 8: Inhibition of GAP-SH3 interaction in vitro with a proline-rich peptide corresponding to GAP N-terminal residues 129-144. In vitro SH3 domain binding assays were conducted with the immobilized Hck SH3 domain and full-length GAP and the GAP deletion mutants in the presence of 1, 3, 10, 30, 100, and 300 µM (left to right) of the synthetic peptide GFPPLPPPPPQLPTLG. This sequence corresponds to residues 129-144 of the bovine GAP N-terminal domain and resembles a proline-rich SH3 domain-binding sequence. The amount of GAP bound in each reaction was assessed by immunoblotting as described under ``Experimental Procedures'' and in the legend to Fig. 6. Shown are the effects of the peptide on the binding activity of full-length GAP (A), and the GAP deletion mutants SH (B) and CAT (C). Control lanes include GST without the SH3 sequence (GST), and binding activity in the absence of the peptide (SH3).




RESULTS

Phosphorylation of GAP by Hck in a Baculovirus Co-expression System

Previous studies have established that GAP is a target for both normal and transforming cytoplasmic protein tyrosine kinases of the Src family(26, 27, 28, 29, 30, 31, 32, 33, 34) . To determine whether or not GAP is a substrate for Hck as well, we used a baculovirus/Sf9 cell system for the co-expression of these proteins. Sf9 insect cells were infected with recombinant baculoviruses containing the full-length bovine GAP cDNA either alone or in combination with baculoviruses containing either wild-type Hck (WT) or a kinase-inactive Hck mutant (K269E; Fig. 1). GAP was immunoprecipitated from clarified cell lysates, resolved by SDS-PAGE, transferred to polyvinylidine difluoride membranes, and probed with antibodies to either phosphotyrosine or GAP. As shown in Fig. 2, GAP was readily phosphorylated on tyrosine residues in cells in which it was co-expressed with WT Hck but not in cells where it is expressed either alone or with the kinase-inactive form of Hck. Control blots show that the WT and K269E Hck proteins were expressed at approximately equal levels.


Figure 2: Phosphorylation of GAP by Hck in a baculovirus/Sf9 cell coexpression system. Sf9 cells were infected with a bovine GAP baculovirus alone (GAP) or with the GAP and Hck wild-type (WT) or Hck kinase-inactive (K269E) baculoviruses in combination. GAP was immunoprecipitated from the infected cell lysates with anti-GAP monoclonal antibodies and analyzed by immunoblotting with either the antiphosphotyrosine antibody, PY20 (left), or the GAP monoclonal antibody (center). To verify the expression of the Hck K269E mutant, the cell lysates were analyzed directly by immunoblotting with the anti-Hck antiserum (right).



GAPHck Complex Formation Does Not Require Hck Autophosphorylation

Previous studies have shown that GAP is not only phosphorylated by tyrosine kinases of the Src family, but often forms complexes with Src kinases as well(26, 27, 28, 29, 30, 31, 32, 33, 34) . To investigate the possibility of HckGAP complex formation and its dependence on tyrosine phosphorylation, WT and kinase-inactive Hck were co-expressed with GAP in Sf9 insect cells. Hck proteins were immunoprecipitated from clarified cell lysates and tested for the presence of associated GAP by immunoblotting with the anti-GAP monoclonal antibody. As shown in Fig. 3A, GAP was present in the Hck immunoprecipitates from cells co-infected with WT Hck and GAP. Surprisingly, GAP was also present in Hck immunoprecipitates from cells co-infected with the kinase-inactive Hck mutant and GAP, suggesting that Hck autophosphorylation is not required for complex formation. No GAP was observed in anti-Hck immunoprecipitates from cells expressing GAP alone. Immunoblots of the clarified cell lysates show that Hck and GAP were expressed at approximately equal levels in each of the cultures (Fig. 3, B and C).


Figure 3: Association of wild-type and kinase-inactive forms of Hck with GAP. Anti-Hck immunoprecipitates were prepared from lysates of Sf9 cells expressing wild-type Hck (WT), kinase-inactive Hck (KE), bovine GAP (GAP), or a combination of GAP and Hck WT or KE. Hck immunoprecipitates from Sf9 cells infected with wild-type baculovirus (AcMNPV) were included as an additional negative control. A, the presence of GAP in the Hck immunoprecipitates was analyzed by immunoblotting with the anti-GAP monoclonal antibody. B, equivalent expression of WT and KE Hck proteins was verified by immunoblotting aliquots of the Sf9 cell lysates with the anti-Hck anti-serum. C, expression of GAP was verified by immunoblotting the lysates with the anti-GAP serum.



Deletion of the Src Homology Region and Tyr-457 of GAP Does Not Abolish Complex Formation with Hck or Tyr Phosphorylation of GAP

The observation that the kinase-inactive mutant of Hck is able to associate with GAP suggests that mechanisms other than phosphotyrosine-SH2 interaction are responsible for complex formation. To test this hypothesis further, a mutant of GAP lacking both SH2 domains, the SH3 domain, and Tyr-457 (the major Tyr phosphorylation site for v-Src; Ref. 27) was co-expressed with wild-type and kinase-inactive Hck in the baculovirus system. The structure of this mutant, known as SH, is shown in Fig. 1. Immunoprecipitates were prepared with the Hck antiserum and probed on immunoblots with either the N-terminal GAP antiserum or with the antiphosphotyrosine antibody, PY20. As shown in Fig. 4A, GAP SH co-precipitated with both forms of Hck, indicating that the GAP Src homology region is not required for association to occur. In addition, this GAP mutant was readily phosphorylated on tyrosine by WT Hck (Fig. 4B), despite the fact that the major site of phosphorylation by v-Src has been deleted (Tyr-457). Control blots verified that GAP SH was expressed at equal levels in the three cultures infected with the SH baculovirus (data not shown).


Figure 4: GAP-Hck association does not require the Src homology region of GAP. Anti-Hck immunoprecipitates were prepared from lysates of Sf9 cells expressing wild-type Hck (WT), kinase-inactive Hck (KE), a deletion mutant of GAP lacking the Src homology region and Tyr-457 (SH), or a combination of GAP SH and Hck WT or KE. Hck immunoprecipitates from Sf9 cells infected with wild-type baculovirus (AcMNPV) were included as an additional negative control. A, the presence of SH in the Hck immunoprecipitates was assessed by immunoblotting with the GAP anti-serum. B, the tyrosine phosphorylation state of SH was determined by immunoblotting the Hck immunoprecipitates with the antiphosphotyrosine antibody, PY20. C, expression of WT and KE Hck proteins was verified by immunoblotting the lysates with the Hck antibody.



The GAP Catalytic Domain Is not Required for Interaction with Hck

To assess whether the catalytic domain of GAP is involved in HckGAP complex formation, co-expression experiments were conducted with a GAP mutant lacking a large C-terminal fragment within the catalytic domain that interacts with Ras (CAT mutant; see Fig. 1). Immunoprecipitates were prepared with the anti-Hck antiserum and blotted with the GAP monoclonal antibody or with antiphosphotyrosine. As shown in Fig. 5A, the CAT mutant co-precipitated with both the wild-type and kinase-inactive Hck proteins but did not bind directly to the Hck antibody, indicative of complex formation. Antiphosphotyrosine immunoblotting of the precipitated proteins indicates that the CAT mutant is phosphorylated on tyrosine (Fig. 5B). This mutant retains the major site of tyrosine phosphorylation by v-Src (Tyr-457; 27) which may be utilized by Hck as well. Control blots shown in Fig. 5C indicate that WT and KE Hck proteins are expressed at approximately equal levels in the co-infected cultures. Control blots also verified that GAP CAT was expressed at equal levels in the three cultures infected with the CAT baculovirus (data not shown). This experiment suggests that the C-terminal catalytic function of GAP is dispensable for interaction with Hck.


Figure 5: GAP-Hck association does not require the catalytic domain of GAP. Anti-Hck immunoprecipitates were prepared from Sf9 cell lysates expressing wild-type Hck (WT), kinase-inactive Hck (KE), a deletion mutant of GAP lacking part of the catalytic domain (CAT), or a combination of GAP CAT and Hck WT or KE. Hck immunoprecipitates from Sf9 cells infected with wild-type baculovirus (AcMNPV) were included as an additional negative control. A, the presence of CAT in the Hck immunoprecipitates was assessed by immunoblotting with the GAP monoclonal antibody. B, the tyrosine phosphorylation state of CAT was determined by immunoblotting the Hck immunoprecipitates with the antiphosphotyrosine antibody, PY20. C, expression of WT and KE Hck proteins was verified by immunoblotting the lysates with the Hck antibody.



Recombinant GST Fusion Proteins Containing the Hck SH3 Domain Bind to GAP, SH, and CAT in Vitro

To map the specific domain of Hck that binds to GAP in a phosphotyrosine-independent manner, the Hck unique N-terminal, SH3, and SH2 domains were expressed singly or in various combinations as glutathione S-transferase (GST) fusion proteins in E. coli. The Hck fusion proteins or GST alone were immobilized on glutathione-agarose beads and incubated with lysates from Sf9 cells expressing full-length GAP, SH, or CAT. Following incubation and washing with buffer containing detergents and salt, bound proteins were resolved by SDS-PAGE and immunoblotted with anti-GAP antibodies. As shown in Fig. 6, A-C, full-length GAP as well as the GAP SH and CAT mutants were able to bind to fusion proteins containing the Hck SH3 domain, but not to the SH2 or unique N-terminal domains. This result implicates the Hck SH3 domain in association with GAP.

SH3 domains contain hydrophobic binding pockets for the proline-rich motif found in target proteins(17, 44) . One of these pockets contains the highly conserved sequence Tyr-X-Tyr (YXY), which is essential for SH3-target interaction(42) . To establish a role for this motif in GAP recognition by the Hck SH3 domain, we deleted the amino acids YDY in two different Hck SH3 GST fusion proteins. As shown in Fig. 7, deletion of the YXY motif from these SH3 proteins almost completely abolished binding to GAP, SH, and CAT.


Figure 7: Deletion of the conserved YXY motif from the Hck SH3 domain abolishes binding to GAP, SH, and CAT in vitro. The YDY motif was deleted from the Hck SH3 domain using PCR, and the resulting mutant SH3 domain was expressed in E. coli as a GST fusion protein either alone (SH3 YDY) or in combination with the adjacent N-terminal domain (N-SH3 YDY). These fusion proteins were compared to their wild-type counterparts in terms of GAP binding activity as described in the legend to Fig. 6. The binding of full-length GAP (A) and the GAP deletion mutants SH (B) and CAT (C) to the mutant and wild-type versions of the fusion proteins are shown. Immobilized GST was included as a negative control.



A possible target sequence for the Hck SH3 domain is defined by GAP N-terminal amino acids 129-144, which encompass the proline-rich sequence GFPPLPPPPPQLPTLG. This region is shared by all of the GAP proteins observed to bind the Hck SH3 domain in vitro (Figs. 6 and 7). To determine whether this region is a possible SH3 target, we synthesized a peptide corresponding to this sequence and tested its ability to interfere with GAP-SH3 association using the in vitro binding assay. As shown in Fig. 8, the GAP proline-rich peptide competed for full-length GAP and GAP deletion mutant binding to SH3 in a concentration-dependent manner with an IC value of approximately 50 µM. An unrelated hydrophilic peptide with the sequence KPQIAALKEETEEEV had no effect on GAP-SH3 binding under these conditions (data not shown). These results are consistent with the hypothesis that the N-terminal hydrophobic domain of GAP encompasses an SH3 domain-binding region.

To establish that the N-terminal region of GAP is involved in SH3 binding, we assessed the ability of an N-terminal deletion mutant to associate with the Hck SH3 domain. This mutant (N) lacks amino acids 25-166, which encompass the proline-rich region described above (see Fig. 1). As shown in Fig. 6D, deletion of this proline-rich region abolished the binding of GAP to the Hck SH3 domain, consistent with the results of the peptide competition experiment.

Identification of an SH3 domain-binding sequence in GAP suggests that GAP may be a target for other SH3 domains as well. To address this possibility, in vitro binding assays were conducted with the SH3 domains from v-Src, Lck, and Fyn in comparison to Hck. As shown in Fig. 9, the SH3 domains from all of the Src kinase family members were able to associate with GAP and the GAP deletion mutants.


Figure 9: Association of GAP with the SH3 domains of Src, Lck, and Fyn. To assess whether GAP binding is a unique property of the SH3 domain of Hck, in vitro binding assays were also conducted with immobilized SH3 domains from v-Src, Lck, and Fyn. Immobilized GST-SH3 fusion proteins or GST alone were mixed with lysates from Sf9 cells expressing full-length GAP (A), or the GAP deletion mutants SH (B) and CAT (C). Following incubation and washing, bound GAP proteins were visualized by immunoblotting.




DISCUSSION

Using a baculovirus/Sf9 cell co-expression system, we have established that the Ras GTPase-activating protein (GAP) is a substrate for the hematopoietic protein-tyrosine kinase, Hck. Hck is a member of the Src family of cytoplasmic tyrosine kinases, several of which have been shown to phosphorylate GAP(26, 27, 28, 29, 30, 31, 32, 33, 34) . Tyrosine phosphorylation of bovine GAP by v-Src occurs primarily at Tyr residue 457 (equivalent to Tyr-460 in the human sequence) both in vitro and in vivo(27) . While Tyr-457 is likely to be utilized by Hck as well, data presented here show that other GAP sites are phosphorylated by Hck. The SH mutant of GAP lacking Tyr-457 was readily phosphorylated by Hck in Sf9 cells. Phosphorylation was demonstrated on antiphosphotyrosine immunoblots of SH in Hck immunoprecipitates from co-infected cells (Fig. 4) and in extracts from co-infected cells lysed directly in SDS sample buffer (data not shown). In addition, co-expression of Hck with the wild-type, SH, and CAT forms of GAP resulted in nearly equal levels of tyrosine phosphorylation of these proteins (as judged by nearly identical levels of antiphosphotyrosine immunoreactivity when all three were run together on the same immunoblot; data not shown). These results are in contrast to previous studies with v-Src, which phosphorylated the SH GAP mutant poorly under essentially identical experimental conditions(27) . The inability of v-Src to phosphorylate GAP SH may be a unique property of this transforming kinase, since c-Src was able to phosphorylate SH to the same extent as Hck in the baculovirus system (data not shown).

Although we have not mapped the additional site(s) of GAP phosphorylation by Hck and c-Src, some evidence suggests that they may be localized to the C-terminal catalytic region. Deletion mutants of human GAP containing N-terminal residues 1-181 or C-terminal residues 705-1047 were not phosphorylated when co-expressed with Hck in the baculovirus system (data not shown). Thus, tyrosine phosphorylation of the bovine GAP SH mutant may occur on one or more of five C-terminal tyrosines between amino acids 518 and 702 (see Fig. 1). Alternatively, the isolated catalytic domain of GAP may not be phosphorylated because it lacks the N-terminal proline-rich region and Src homology domains essential for interaction with the tyrosine kinase. Further experiments are required to distinguish between these possibilities. Association of GAP with Ras, p62, p190, or other proteins has been postulated to affect the conformation of GAP (45) and may uncover sites for tyrosine phosphorylation such as those observed following deletion of the Src homology region.

Data presented here suggest that the SH3 domains of Src family kinases may be previously unrecognized determinants of GAP interaction. Recombinant fusion proteins containing the v-Src, Hck, Lck, and Fyn SH3 domains were able to bind to full-length and mutant forms of GAP. Mutation of a conserved SH3 sequence essential for target recognition (YXY motif; Ref. 42) abolished binding of the Hck SH3 domain to GAP in vitro. Inspection of the N-terminal region shared by the full-length, SH, and CAT forms of GAP reveals a proline-rich sequence which may serve as a putative SH3-binding domain (PPLPPPPPQLPP). A synthetic peptide containing this sequence was able to compete for Hck SH3-GAP interaction in vitro, while an N-terminal deletion mutant of GAP lacking this region did not bind to the Hck SH3 domain. This proline-rich sequence is similar to a region in the protein 3BP-1, a protein identified in an SH3 screen to bind to the Abl and Src SH3 domains (PPPLPPLV; 46, 47). The presence of a possible proline-rich SH3-binding motif within the N-terminal region of GAP suggests that GAP may interact with SH3-containing proteins in addition to Src family tyrosine kinases. Such interactions could affect GAP activity, subcellular localization, or interactions with other proteins.

Although data presented here suggest a function for the SH3 domains of Src family kinases in complex formation with GAP, previous analyses of GAP-Src interaction indicate important roles for other Src domains as well. Using chick embryo fibroblasts as a model system, association of c-Src with GAP was shown to involve tyrosine phosphorylation of the Src negative regulatory tail (Tyr-527) and an intact Src SH2 domain(28) . Involvement of Src Tyr-527 suggests that the C-terminal Src kinase (CSK) may affect GAP-Src interaction, possibly by creating a binding site for the GAP SH2 domains. However, autophosphorylation of c-Src was not required, since a kinase-inactive mutant was observed to associate with GAP as readily as the wild-type kinase. This result is consistent with our finding that a kinase-inactive mutant of Hck can associate with GAP to the same extent as the wild-type kinase (Fig. 3). In another study, individual deletions of the N- or C-terminal halves of the c-Src SH2 domain or the entire SH3 domain did not affect tyrosine phosphorylation of GAP by c-Src in transfected chick embryo fibroblasts (48). This result is consistent with the idea that more than one domain of c-Src is involved in the recognition of GAP in vivo. Whether or not the Hck C-terminal tail or SH2 domain contribute to interaction with GAP is currently under investigation. Differences in the mechanism of interaction of individual members of the Src kinase family with GAP are also possible, which may produce unique effects on GAP function.

The finding that GAP associates with the SH3 domains of Hck, Src, Lck, and Fyn adds to a growing list of substrates that interact with Src kinase family members via this mechanism. Several recent studies have shown that the 85-kDa subunit of phosphatidylinositol 3`-kinase binds to the SH3 domains of Src, Fyn, and Lck(49, 50, 51) . Dynamin, a GTP-binding microtubule-associated protein binds to the SH3 domains of Src, Fyn, and Fgr as well as to the SH3 domains of GRB-2 and p85(52) . In the case of phosphatidylinositol 3`-kinase and dynamin, association with the SH3 domain may modulate the biochemical function of these proteins. In addition, recombinant SH3 domain fusion proteins from Src, Fyn, and Lyn have been shown to interact with discrete populations of proteins from fibroblasts(42, 53) . When similar SH3 domain binding experiments were conducted with whole cell lysates from Src-transformed cells, many of the SH3-associated proteins were found to be phosphorylated on tyrosine. Several of these SH3-binding proteins were later identified and include SHC, a signaling protein that links tyrosine kinases to Ras via GRB-2, the GAP-associated protein p62, heterogeneous nuclear ribonucleoprotein K, and paxillin, a cytoskeleton-associated protein(42, 53) . These findings suggest that the SH3 domains of Src and Src family members are important for substrate recognition in vivo.

Although GAP is generally regarded as a negative regulator of Ras, some evidence suggests that GAP is a downstream effector of Ras as well (see Introduction). Thus, interaction of Hck or other Src kinases with GAP may influence Ras activity and GAP effector function in several ways. Phosphorylation of GAP on novel sites within the C-terminal catalytic region may alter GAP activity toward Ras in vivo. Alternatively, tyrosine phosphorylation of GAP could influence interaction with putative effector proteins such as p62(54, 55) . Another possibility is that binding of the proline-rich N-terminal region of GAP to the SH3 domain of Hck or other SH3-containing proteins may influence its catalytic or signaling functions. Both granulocyte macrophage-colony stimulating factor and leukemia inhibitory factor have recently been linked to cellular Hck activation(4, 9) , and granulocyte macrophage-colony stimulating factor is known to activate Ras through a tyrosine-kinase-dependent mechanism(56) . Phosphorylation of GAP may occur as a result of Hck activation in response to these and other physiological stimuli, suggesting a possible role for GAP in these signaling pathways.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants CA58667 (to T. E. S.) and CA55652 (to R. J.), a grant from the Council for Tobacco Research, U.S.A. (to T. E. S.), and by American Cancer Society Institutional Research Grant SIG-16 and National Cancer Institute Cancer Center Support Grant P30 CA36727 to the Eppley Institute for Research in Cancer. 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.

§
Recipient of an American Cancer Society Junior Faculty Research Award. To whom correspondence should be addressed: Eppley Institute for Research in Cancer, University of Nebraska Medical Center, 600 S. 42nd Street, Omaha, NE 68198-6805. Tel.: 402-559-8270; Fax: 402-559-4651; Internet: tsmithga@unmc.edu.

The abbreviations used are: GAP, GTPase-activating protein; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; Fmoc, N-(9-fluorenyl)methyloxycarbonyl; GST, glutathione S-transferase.

S. Bryant and R. Jove, unpublished results.


ACKNOWLEDGEMENTS

We acknowledge the expert technical assistance of Jan Williamson with the peptide synthesis and Dr. Fulvio Perini for conducting amino acid composition analysis, and Victoria Boryca for generating the kinase-inactive Hck mutant.


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