INTRODUCTION

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The nonreceptor protein tyrosine kinase Src is critical for normal cellular processes such as proliferation and differentiation, and certain mutations in Src cause uncontrolled cell proliferation and transformation (11). Under normal conditions, the enzymatic activity of Src is tightly regulated. Biochemical (13, 20, 45, 64) and structural (75, 92) analyses have shown that the kinase activity of the c-Src protein is intramolecularly regulated by conserved modular domains, the Src homology regions 2 and 3 (SH2 and SH3) (18). Consistent with their regulatory role, mutations within these domains render the kinase active and oncogenic (11). In addition, upon Src activation, these domains mediate protein-protein interactions and are thought to determine substrate selectivity and signaling specificity (18, 28).

Traditionally, studies aimed at elucidating the signaling properties of c-Src have used constitutively active and transforming Src alleles as models. Activated Src alleles exhibit deregulated kinase activity and are known to induce multiple signaling responses due to promiscuous substrate phosphorylation. Thus, it has been difficult to determine which of the many responses is responsible for the signaling properties of Src. In addition, despite the identification of a plethora of putative Src substrates in v-Src-transformed cells, the importance of these substrates in the physiologic and/or tumorigenic effects of c-Src has been difficult to ascertain.

To gain insight into the signaling mechanisms of wild-type c-Src and given that the c-Src SH3 domain has been shown to participate in the intramolecular negative inhibition of the c-Src kinase activity (55, 79), we used physiological ligands for the conserved SH3 domain of c-Src to activate the enzyme. At the same time, we used these ligands as links to downstream events to study the signaling mechanisms and specificity of c-Src. The molecules used for our studies consist of a protein that we previously identified, Sin, and the homologous protein p130^Cas (1, 72). Cas was first identified as a highly phosphorylated protein in v-Src- and v-Crk-transformed cells (72); Sin was independently cloned as the Fyn embryonic substrate Efs (40). These molecules specifically bind to Src family SH3 domains with high affinity through proline-rich motifs (2, 57, 72). Sin and Cas comprise a multiadapter protein family that also includes HEF1/CasL independently cloned as a human enhancer of filamentation in yeast and as a focal adhesion kinase (FAK)-binding protein expressed in lymphocytes (48). All of these proteins exhibit conserved secondary structures, which in turn consist of many conserved modules that mediate protein-protein interactions. Thus, Cas proteins have conserved N-terminal SH3 domains, central regions comprised of repeated tyrosine-containing residues, Src SH3-binding proline-rich motifs (except HEF1/CasL), and conserved C termini that have been implicated in homo- or heterodimerization between family members (61). The presence of these conserved domains and their ability to promote protein-protein interactions suggest that members of the Cas family mediate the formation of multiprotein complexes in a phosphotyrosine-dependent manner. These protein-protein interactions are thought to subsequently activate intracellular signaling pathways with pleiotropic effects on cellular behavior (52, 61).

The most extensively studied member of this family, p130^Cas, becomes highly phosphorylated on multiple tyrosine residues in response to a variety of stimuli. For example, mitogens such as epidermal growth factor, platelet-derived growth factor, and lysophosphatidic acid have been shown to induce tyrosine phosphorylation of Cas (15, 59). In addition, integrin engagement or stimulation of serpentine receptors such as the bombesin and the endothelin receptors stimulate Cas phosphorylation (15, 47, 87, 88). Cas phosphorylation in turn has been implicated in multiple cellular processes such as integrin receptor signaling (36, 50, 58, 88), cell migration and survival (14, 16, 17, 44), regulation of the cell cycle (60, 93), and apoptosis (7). Furthermore, Cas has been implicated in cellular transformation, as demonstrated by its presence as a tyrosine-phosphorylated protein in v-Src- and v-Crk-transformed cells (72), by the fact that p130^Cas/ cells cannot be transformed by Src (37), and by antisense RNA experiments showing that Cas-specific antisense RNA partially reverts v-Src transformation (6).

Multiadapter molecules, such as the Cas protein, depending on the type and number of conserved motifs they contain, can form interactions with unique sets of cytoplasmic intermediates and thus determine signaling specificity. These conserved motifs have tyrosine-containing sequences (Y motifs) which upon phosphorylation by tyrosine kinases recruit cytoplasmic substrates through phosphotyrosine-SH2 domain interactions. The type and number of Y motifs present on the different members of the Cas family differ, suggesting that these proteins may have similar but not identical functions. Consistent with the model that Y motifs can determine signaling specificity, the multiadapters and insulin receptor substrates IRS1 and -2 (56, 94) have been shown to mediate distinct functions of the insulin receptor (5, 80, 89). The different signaling properties of each molecule have been attributed to unique Y motifs that bind to distinct SH2 domain-containing cytoplasmic intermediates.

We have previously shown that coexpression of Src and Sin activates Src signaling, as measured by serum response element (SRE)-mediated transcriptional activation (1). In this study we compared the abilities of the Src SH3-binding proteins Cas and Sin to bind to and stimulate the enzymatic activity of c-Src. We then analyzed the molecular events that mediate Sin-Cas-induced Src signaling, using the phosphorylated forms of these proteins as links to downstream signaling events. In addition, we compared the signaling mechanisms of ligand-activated c-Src to signaling mediated by a constitutively active and oncogenic Src mutant. In these experiments, we used full-length and truncated forms of Sin and Cas proteins, and we found quantitative differences in the ability of the different Sin and Cas proteins to activate c-Src signaling. In addition, we found that ligand-activated and constitutively active Src proteins utilize different signaling mechanisms to induce SRE-dependent transcription.

	MATERIALS AND METHODS

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Cells and antibodies. Human embryonic kidney carcinoma 293 cells were grown as previously described (62). Cells were maintained in Dulbecco modified Eagle medium containing 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 mg/ml). The Sin monoclonal antibody, raised against a fragment of Sin encompassing amino acids 142 to 258, was generated by Transduction Laboratories. Cas polyclonal antibody CasN-17 was purchased from Santa Cruz Biotechnology. The Src mouse monoclonal antibody 327 was provided by Joan Brugge (Harvard Medical School); the antiphosphotyrosine monoclonal antibody pY20 was purchased from Transduction Laboratories. Antiphosphotyrosine SrcY416 was kindly provided by A. Laudano (University of New Hampshire). Other reagents used were anti-phospho-ERK monoclonal antibody (E-4), anti-ERK, anti-C3G (Santa Cruz), anti-Ras, anti-Rap1, anti-Crk (Transduction Laboratories), and anti-hemagglutinin epitope (HA) (BAbCo, Richmond, Calif.).

DNA constructs. DNA manipulations were performed by standard protocols. Full-length Sin and truncated SinC (amino acids 1 to 335) were cloned into the SpeI-NotI sites of the pEBB expression vector. pEBB was derived from the pEF-BOS expression vector driven by the human elongation factor 1- promoter (53). In SinC, a deletion of amino acids 340 to 560 removes one of the proline-rich Src SH3-binding sites, the last three Y motifs in the substrate region, and the C-terminal homologous region. PCR-amplified Cas proteins were cloned into the BamHI-NotI sites of pEBB. Full-length Cas expresses a short form of the protein that is alternatively spliced, missing amino acids 5 to 99 (72). CasUR contains an internal deletion of a Bsu36I fragment encompassing amino acids 496 to 713 that removes the unique region of Cas. CasURC contains, in addition to the Bsu36I fragment deletion, a C-terminal deletion that encompasses amino acids 748 to 968. This construct was generated by PCR amplification using CasUR as a template and synthetic oligonucleotides containing 5' BamHI and 3' NotI restriction sites. CasURCP* was generated as the CasURC construct except that the 3' oligonucleotide that was used for PCR amplification contained two base pair substitutions that produce two point mutations (Ser-Ala and Pro-Leu) in the sequence within the core of the PXXP motif of Cas. These substitutions change the Src SH3-binding site of Cas for that of Sin (PSPP to PALP). All sequences were confirmed by automated sequence analysis. The different SinC YDVP mutants were generated by PCR by substituting tyrosines 148, 188, and 253 with phenylalanine residues, using mutated oligonucleotides. The amplified fragments were cloned into the SpeI-NotI sites of pEBB, and mutations were confirmed by automated sequencing. Plasmids expressing the RasN17 and CrkW170K mutants were previously described (30, 81). The RapN17 mutant was provided by Philip Stork (Oregon Health Sciences University) (86). The SRE-luciferase reporter construct was generated as previously described (1); the AP-1 luciferase reporter was provided by C. A. Hauser (La Jolla Cancer Research Foundation). Plasmids that expressed wild-type Src protein and SrcY527F have been described elsewhere (45), as has the v-Raf construct (68).

Transfections. 293 cells were transfected as previously described (1, 62). Two micrograms of pMHHB(c-Src) or pc-SrcY527F expression vector or pEVX empty vector was used in all transfections. The transfection mixtures also included 1 µg of vector (pEBB) used to express the different Sin or Cas proteins or 1 µg of Sin- and Cas-expressing pEBB and different concentrations of the inhibitors as shown in the figures. SRE and AP-1 luciferase reporters and the MFG-lacZ plasmid expressing -galactosidase (1 µg of each) were also included in the transfection mixtures. When necessary, the total amount of DNA in each transfection mixture was kept constant by the addition of empty vector in the DNA-calcium phosphate coprecipitate. Luciferase activity was determined using a Promega kit according to the manufacturer's protocol; -galactosidase activity to normalize for transfection efficiency between the different samples was determined according to standard protocols (73).

Immunoprecipitations. Immunoprecipitations were performed as previously described (1). Briefly, cells were lysed in 1 ml of ice-cold NP-40 lysis buffer (1% NP-40, 20 mM Tris-HCl [pH 8.0], 150 mM NaCl, 10% glycerol, 10 mM NaF, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride, 10 µg of aprotinin/ml, 10 µg of leupeptin/ml) and incubated on ice for 10 min. The cell debris and nuclei were removed by centrifugation in an Eppendorf centrifuge for 10 min at 4°C. The cell lysates were then incubated with the specified antibodies at concentrations suggested by the manufacturers for 2 h at 4°C. The immune complexes were collected after the addition of 20 µl of protein G-plus-protein A-agarose (Oncogene Science) and incubation at 4°C for 30 min. The pellets of agarose beads were washed three times with 1 ml of lysis buffer and then subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting.

Western blot analysis. Total cell extracts or immunoprecipitates normalized for protein content were boiled in Laemmli sample buffer, separated by SDS-PAGE on a 10% gel, and transferred to nitrocellulose membranes. Filters were blotted with the appropriate monoclonal antisera according to manufacturer's protocol in TBST-milk at 4°C overnight (16 h). Rabbit polyclonal antibodies were used at 1:500 dilution. Monoclonal antibodies were each used at 1 µg/ml of TBST-milk. The filters were washed in TBST and consequently incubated with anti-mouse or anti-rabbit immunoglobulin G-conjugated horseradish peroxidase at a 1:4,000 dilution in TBST at room temperature for 1 h. Filters were then washed and developed by enhanced chemiluminescence (ECL) (Amersham) as described by the manufacturer.

Ras and Rap1 binding assays. Transfected 293 cells were lysed with cold lysis buffer (50 mM Tris [pH 7.5], 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 0.1% 2-mercaptoethanol, 0.5 mM sodium orthovanadate, 50 mM NaF, 5 mM sodium pyrophosphate, 10 mM sodium glycerophosphate, 0.1 mM phenylmethylsulfonyl fluoride), and aliquots of the cell extracts containing 50 µg of total protein were mixed with 40 µl crude bacterial extracts expressing glutathione S-transferase (GST)-RalGDS-RBD (Rap1-binding domain) or Raf1-RBD (Ras-binding domain) that had been precoupled to glutathione beads in the presence of 125 mM NaCl. The aliquots were incubated 1 h at 4°C with rotation. The protein complexes were washed, separated by SDS-PAGE on a 15% polyacrylamide gel, transferred on nitrocellulose membranes, and blotted with Ras- and Rap1-specific antibodies.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Differential activation of Src signaling by Sin and Cas. To gain insight into the specificity of c-Src signaling, we compared the abilities of wild-type and mutated forms of Sin and Cas proteins to bind to and activate Src. Although the Sin and Cas proteins do not share extensive primary sequence homology, they are closely related in their overall secondary structure (Fig. 1). For example, they both contain proline-rich consensus motifs that exhibit high binding affinity to the Src SH3 domain (1, 72) and are important for Src binding. The proline-rich motif of Cas has been shown to mediate association of this protein with Src and is important for Src-dependent phosphorylation of Cas (57). A point mutation within the proline-rich motif of a truncated fragment of Sin or deletion of the Sin proline-rich consensus leads to inhibition of Src signaling (reference 1 and unpublished observations). The N-terminal SH3 domains and the C termini of Cas and Sin are conserved (90 and 57%, respectively), and their central regions contain multiple tyrosine motifs that mediate substrate binding through phosphotyrosine-SH2 domain interactions. In addition, Cas contains a unique region of unknown function between its substrate-binding region and its C terminus. Given the homology of these proteins, we compared the abilities of Sin and Cas to bind to and activate c-Src, as well as their abilities to become phosphorylated and promote Src-dependent intracellular signaling.

View larger version (25K): [in this window] [in a new window]   FIG. 1.   Schematic representation of full-length and truncated Sin and Cas proteins and comparison of their Y motifs. The constructs were generated as described in Materials and Methods. SR, substrate-binding regions of Sin and Cas that contain the motifs shown on the right; P, proline-rich sequences that bind to the Src SH3, RPLPALP, and RPLPPPP for Sin and RPLPSPP for Cas. Y motifs that are deleted in truncated Sin and Cas proteins are boxed. CasURCP* is the same as CasURC except that the PSPP motif has been changed to PALP.

View larger version (50K): [in this window] [in a new window]   FIG. 2.   Sin and Cas associate with and are phosphorylated by c-Src. (A) 293 cells were cotransfected with a full-length or truncated Sin or Cas expression vector (1 µg of each) and a c-Src expression plasmid, the pEVX empty vector used to express Src, or the c-SrcK295R kinase mutant (2 µg of each). Cell lysates were subjected to immunoprecipitation (IP) using anti-Src antiserum, and Western blots of the immunoprecipitates were blotted with pY20, an antibody against phosphotyrosine. In lane 1, cells were transfected with pEBB vector backbone used to express Sin and Cas proteins; lanes 2 to 9 contain the different Sin and Cas proteins as shown at the bottom. (B) 293 cells were transfected with Src and Sin or Cas expression constructs as in panel A. Untransfected controls (U) were included to compare the levels of endogenous versus overexpressed Src. Cell lysates were immunoprecipitated with Src antibody; immune complexes were separated by SDS-PAGE, transferred to nitrocellulose, and blotted with antibody pY416, which recognizes the active form of Src (top panel). The blots were stripped and reprobed with anti-Src antibody to determine the relative levels of Src in the different lanes (lower panel). (C) The top panel represents total lysates from cells transfected with 1 µg each of the Sin and Cas proteins on a Western blot probed with Sin-, Cas-, or HA-specific antibodies. The bottom panel contains the same extracts blotted with the CasN-17 antibody. Protein bands on the different blots were visualized using horseradish peroxidase-conjugated secondary antibody and ECL reagents. All blots were exposed on film for the same amount of time (10 s).

View larger version (31K): [in this window] [in a new window]   FIG. 3.   The Sin and Cas proteins mediate differential activation of Src signaling. 293 cells were cotransfected as in Fig. 2 with a Sin or Cas expression construct (as shown, lanes 2 to 9) and wild-type c-Src (A and B, top) or the Src kinase mutant (A and B, bottom); 1 µg of luciferase reporter construct containing the SRE (left) or AP-1 (right) promoter was included in the transfection mix, along with -galactosidase expression plasmid (1 µg). Luciferase activity was measured as described in Materials and Methods. The results represent the average of at least five experiments and are expressed as fold activation relative to the values obtained with the vector backbone (pEBB) that was used to express Sin and Cas proteins and was given a value of 1. The results shown represent the mean ± standard deviation.

Sin and Cas-induced Src signaling is independent or downstream of Ras. Given the quantitative differences between Sin and Cas, we next examined whether these proteins utilize the same or different pathways to propagate Src signaling. It has been previously shown that the small GTP-binding protein Ras mediates signaling by constitutive active and oncogenic Src alleles (26, 51, 67). Thus, we further evaluated the pathway(s) that is activated as a result of Sin and Cas binding to Src and examined whether Ras also plays a role in Sin-Cas-induced Src signaling.

View larger version (42K): [in this window] [in a new window]   FIG. 4.   Induction of ERK1,2 phosphorylation and SRE-dependent transcriptional activation by Cas and Sin are Ras independent. (A) 293 cells were transfected with c-Src and pEBB vector or SinC or CasURC as shown. Also cells were transfected with SrcY527F alone, or empty vector used to express this Src mutant (pEVX), in the presence of the Ras inhibitor (2 µg) or the Zipneo empty vector (2 µg) as shown. Total cell extracts were normalized for protein content, resolved by SDS-PAGE, and blotted with anti-phospho-ERK antibody (E-4) (top panel). The lower panel represents a Western blot probed with anti-ERK antibody that recognizes both phosphorylated and unphosphorylated forms of ERK. (B) 293 cells were transfected as above but in the presence of the Src kinase mutant or SrcY527F as a positive control and in the presence of RasN17 inhibitor or empty vector. Western blots of cell extracts were processed as in panel A. (C) Cells were transfected with c-Src and SinC or CasURC or with SrcY527F alone, as shown, with increasing concentrations of the RasN17 inhibitor in the presence of the SRE-luciferase reporter. Percent stimulation is relative to the activation of luciferase in cells transfected with c-Src and SinC or CasURC, or SrcY527F in the presence of the Zipneo empty vector (2 µg) used to express the RasN17 inhibitor, each of which was given a value of 100 (dark gray bars). Actual fold activation in the absence of the RasN17 inhibitor (dark gray bars) was 32 ± 3.2 for SinC, 14 ± 2.4 for CasURC, and 79 ± 16 for SrcY527F. The percent stimulation obtained with c-Src and SinC or with CasURC or SrcY527F in the presence of the dominant negative inhibitors is the mean ± standard deviation. The data represent the average of at least five experiments.

Sin- and Cas-induced Src signaling is mediated by Crk. In vitro studies have previously shown that a phosphorylated tyrosine within a YDVP consensus sequence (Y motif) exhibits preferential binding to the c-Crk SH2 domain (78). c-Crk is a Grb2-like small adapter molecule with no known enzymatic activity, consisting mainly of modular regions such as SH2 and SH3 domains (31). Three of the Sin Y motifs and the majority of the Cas Y motifs are of the YDVP consensus, suggesting a c-Crk SH2 binding specificity (Fig. 1A). Consistent with the in vitro data, Sin and Cas interact with endogenous c-Crk in a c-Src- and phosphotyrosine-dependent manner (1, 72).

View larger version (38K): [in this window] [in a new window]   FIG. 5.   The adapter Crk mediates Sin- and Cas-induced Src signaling. (A) 293 cells were cotransfected with c-Src (2 µg) and SinC or CasURC (1 µg of each) or with a v-Raf expression plasmid (2 µg) in the presence of empty pEBB vector or increasing concentrations of the CrkK170 inhibitor. Percent stimulation is relative to the activation of luciferase in cells transfected with c-Src and SinC or CasURC, or v-Raf in the presence of the pEBB empty vector (2 µg) used to express the CrkK170 inhibitor, each of which was given a value of 100 (dark gray bars). Actual fold activation in the absence of the CrkK170 inhibitor (dark gray bars) was 39 ± 11 for SinC, 12 ± 2.8 for CasURC, and 126 ± 15 for v-Raf. The percent stimulation obtained with c-Src and SinC or with CasURC or v-Raf in the presence of the dominant negative inhibitors is the mean ± standard deviation. The data represent the average of at least seven experiments. (B) 293 cells were cotransfected with c-Src and wild-type (WT) or mutant SinC (1 µg, as shown) in the presence of the SRE-luciferase reporter construct. Data from several experiments (n = 5) were averaged, and values for samples that expressed the mutant Sin proteins were normalized to the induction of luciferase activity observed with wild-type SinC, which was given a value of 100 (actual increase with wild-type SinC was 42 ± 4.7-fold). The percent stimulation is the mean ± standard deviation. (C) Lysates of 293 cells expressing c-Src and wild-type or tyrosine mutant SinC were immunoprecipitated (IP) with anti-Src antibodies (middle and bottom panels). Immunoprecipitates were blotted and probed with antiphosphotyrosine antibody pY20 (middle) or anti-Sin antibody (bottom). Total cell extracts of the same samples were resolved by SDS-PAGE, and the membranes were blotted with anti-Sin antibody (top panel). Consensus sequences containing Y148, Y188, and Y253 are shown at the bottom of panel B. Amino acids important for kinase specificity are shown in bold.

The Rap1 GTPase is involved in Cas- and Sin-induced Src signaling. It has been recently shown that the SH3 domain of c-Crk interacts with proline-rich sequences found on C3G (34, 46, 82), a protein that promotes nucleotide exchange on the small GTP-binding protein Rap1 (38). In our system, we also found a Src-dependent association of endogenous Crk and C3G with phosphorylated SinC and CasURC (Fig. 6A). These interactions were dependent on the kinase activity of Src since the association of Sin and Cas with Crk and C3G was abolished in the presence of the Src kinase mutant (data not shown).

View larger version (39K): [in this window] [in a new window]   FIG. 6.   Sin- and Cas-induced Src signaling is mediated by the Rap1 GTPase. (A) 293 cell lysates expressing c-Src and SinC or CasURC were immunoprecipitated with Crk-specific (left) or C3G-specific (right) antibodies. Immunoprecipitates (IP) were blotted and probed with antibody pY20. Membranes were then stripped and blotted with anti-Crk (bottom left) or anti-C3G (bottom right) antibodies. (B) 293 cells were transfected as in Fig. 5A along with increasing concentrations of the RapN17 inhibitor as shown. Percent stimulation is relative to the activation of luciferase in cells transfected with c-Src and SinC or CasURC, or v-Raf in the presence of the pcDNA empty vector (4 µg) used to express the Rap1N17 inhibitor, each of which was given a value of 100 (dark gray bars). Actual fold activation in the absence of the RapN17 inhibitor (black bars) was 26 ± 6.5 for SinC, 10 ± 3.3 for CasURC, and 86 ± 5.2 for v-Raf. The percent stimulation obtained with c-Src and SinC or with CasURC or v-Raf in the presence of the dominant negative inhibitors is the mean ± standard deviation. The data represent the average of at least six experiments. (C) 293 cell extracts expressing c-Src and vector backbone, SinC, or CasURC were incubated with GST-RalGDS-RBD or GST-Raf-RBD; the protein complexes were harvested, separated by SDS-PAGE, and transferred onto nitrocellulose membranes. The membranes were blotted with Rap1-specific (top) or Ras-specific (bottom) antibodies. Protein bands were visualized using ECL. (D) Total extracts from untransfected or cells expressing pcDNA vector, constitutively active RapV12, or wild-type Rap (RapWT) were blotted with anti-phospho-ERK (top) or anti-ERK (bottom). Protein bands were visualized using ECL.

Signaling through the constitutively active SrcY527F mutant is mediated by both Ras and Rap1. The lack of an inhibitory effect with the Ras inhibitor on Sin and Cas induced activation of c-Src is surprising in that SRE-mediated transcription in response to tyrosine kinase activation is predominantly the result of Ras-mediated activation of the ERK1,2 MAP kinases (23, 49, 71, 91). It has also been shown that transcriptional activation induced by constitutively active and oncogenic forms of Src is Ras dependent (45, 63, 67). Consistent with these observations, we found that the Ras inhibitor blocked SrcY527F-induced SRE activation (Fig. 4C). We next examined whether the SrcY527F constitutively active mutant could activate the Crk-Rap1 signaling pathway, using dominant negative inhibitors and assaying for Rap activation. We found that both the CrkK170 and RapN17 mutants inhibited SrcY527F-induced transcriptional activation of the SRE promoter in a concentration-dependent manner (Fig. 7A), suggesting that both the Ras and Rap1 signaling cascades are utilized by oncogenic Src. Consistent with this result, cotransfection of SrcY527F with both RasN17 and RapN17 resulted in maximal inhibition of transcription even at the lowest concentrations of Rap and Ras inhibitor DNA (0.25 µg of each [Fig. 7A]). In addition, in contrast to Sin and Cas, which preferentially activated Rap1 but not Ras, we found that expression of the SrcY527F mutant resulted in increased GTP-bound levels of both Ras and Rap1 (Fig. 7B). Furthermore, as with the RasN17 inhibitor (Fig. 4A), we found that SrcY527F-mediated activation of ERK1,2 phosphorylation was also sensitive to the RapN17 inhibitor, whereas v-Raf-induced ERK phosphorylation was unaffected under the same conditions (Fig. 7C). Thus, these data suggest that the oncogenic SrcY527F mutant utilizes at least two separate cascades to ERK phosphorylation and SRE promoter activation, whereas signaling through ligand-activated Src is more directed and is primarily mediated by Rap1.

View larger version (45K): [in this window] [in a new window]   FIG. 7.   Signaling through the constitutively active SrcY527F mutant is mediated by both Ras and Rap. (A) 293 cells were transfected with SrcY527F in the presence of the Ras, Crk, and Rap inhibitors and in the presence of the SRE-luciferase reporter. Percent stimulation represents the average data from four experiments and was determined from samples expressing SrcY527F in the presence of empty vectors (pZipneo, pEBB, and pcDNA) that were used to express the inhibitors (dark gray bars). Actual activation was 79 ± 16-fold in the presence of Zipneo, 130 ± 28-fold for pEBB, and 40 ± 6.7-fold for pCDNA. The difference in the SrcY527F fold activation observed in the presence of the different vectors is likely due to promoter competition of the transfected plasmids. Luciferase activation in the presence of the different inhibitors is expressed as a percentage of the dark gray bar controls ± standard deviation. (B) Cell extracts expressing the pEVX vector or SrcY527F were incubated with GST-RalGD-RBD or GST-Raf-RBD; protein complexes were separated, transferred to membranes, and blotted with Rap- or Ras-specific antibodies, respectively. Protein bands were visualized using ECL. (C) 293 cell extracts expressing SrcY527F or v-Raf were assayed for ERK1,2 activation in the presence of the RapN17 inhibitor. SrcY527F or v-Raf (2 µg of each) and RapN17 (4 µg) expression plasmids were used. Cell lysates were analyzed as described for Fig. 4, using phospho-ERK- and ERK-specific antibodies.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Cas and Sin mediate Src-dependent transcriptional activation through the ERK MAP kinase. The Src-SH3 domain is important for c-Src regulation and signaling, and this domain is thought to participate in signaling specificity by determining the substrate selectivity of the enzyme (28). In this study we used the natural c-Src ligands Sin and Cas to activate Src and examine the mechanisms that mediate signaling under these conditions. In the experiments described above, we found that phosphorylated Sin and Cas act downstream of Src to activate transcription through an SRE-containing promoter. SRE-dependent transcriptional activation has been shown to depend on activation of Ras and the ERK1,2 MAP kinases. In our system, activation of transcription through the SRE was Ras independent and was instead mediated by the related protein Rap1. Our results for the first time implicate Rap1 in Src kinase signaling and show that this G protein acts downstream of phosphorylated Sin and Cas to activate ERK and SRE-dependent transcription.

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Src SH3-binding proteins differentially activate c-Src signaling. In the experiments described above, we found that although the different Sin and Cas proteins bound to and were phosphorylated by Src to similar levels, they elicited quantitatively different responses as measured by transcriptional activation. Despite these quantitative differences, both Sin and Cas mediated Src signaling through the activation of the Crk-Rap1-ERK1,2 signaling cascade.

c-Src signaling in response to ligand binding is mediated by Crk and Rap1, functioning upstream of ERK. The experiments presented above show that Cas and Sin can effectively activate the Crk-Rap1 signaling complex upstream of ERK and the SRE promoter and that this activation requires the binding of Crk to the phosphorylated YDVP motifs of Sin and Cas. Crk has been shown to bind to C3G through a Crk SH3 and C3G proline-rich motif interaction (82). Formation of the Crk-C3G complex in turn leads to Rap1 activation (34). In our experiments we found that both endogenous Crk and C3G associated with phosphorylated Sin and Cas proteins (Fig. 6A). These results suggest that phosphorylated Cas-Sin-induced activation of Rap1 is mediated by the Crk-C3G complex. It has been shown recently that Rap1 activation is mediated by different signaling pathways, involving second messengers such as calcium, diacylglycerol, and cyclic AMP (cAMP) (3, 32, 98). The formation of active, GTP-bound Rap1 is regulated by three different families of GEFs consisting of C3G, which regulates tyrosine kinase-induced Rap1 activation through Crk and Cbl (39, 69, 76), the guanine nucleotide-releasing proteins, which contain calcium and diacylglycerol motifs (25), and the Epac family of proteins, which are regulated by cAMP (22, 41). More recently, another protein family, the PDZ-GEFs, was characterized, members of which exhibit nucleotide exchange activity specific for Rap1 and -2 (41). Thus, we cannot exclude the possibility that other, newly discovered Rap1-specific GEFs are involved in Rap1 activation in our system. The involvement of different GEFs in Rap1 activation will be explored in future experiments.

Ligand-activated versus oncogenic Src signaling. In the experiments described above, we show that truncated Sin and Cas proteins mediate c-Src signaling through the Rap1 GTPase. These observations are important because (i) our results for the first time implicate a GTPase other than Ras in Src signaling, (ii) ERK activation can be mediated by small GTP proteins other than Ras, and (iii) the mode of Src activation (oncogenic versus ligand induced) may determine the signaling pathways activated by Src. The latter observation is particularly interesting given the fact that studies using constitutively active and oncogenic forms of Src have shown Src to act upstream of Ras (26, 51, 67). Consistent with this, our results show that signaling through the transforming allele SrcY527F is also Ras as well as Rap1 dependent (Fig. 7). In addition, a recent report by Hakak and Martin (35) showed that Cas mediates transcriptional activation of the Egr-1 SRE by v-Src and that this activation was through Grb2 and the Ras-MEK-ERK pathway. However, the increase in transcriptional activation was not dependent on the substrate region of Cas that contains the YXXP motifs, since deletion of this region resulted in levels of luciferase activity equivalent to that induced by full-length Cas. Consistent with this observation, a Crk-II SH3 mutant had no effect on luciferase activity, suggesting that the effect of Cas on v-Src-dependent transcriptional activation is mediated by a Ras-dependent mechanism which is different from the one we are describing.

View larger version (23K): [in this window] [in a new window]   FIG. 8.   Model for ligand-induced c-Src-dependent signal transduction leading to MAP kinase and transcriptional activation. Binding of the proline-rich motifs of Sin and Cas to the Src SH3 domain activates the c-Src tyrosine kinase activity, and the proteins become phosphorylated on tyrosine residues. Src-mediated phosphorylation of Sin and Cas results in the recruitment of the Crk-GEF signaling complex, which in turn activates Rap1 and ERK and induces SRE-mediated gene expression. On the other hand, mutations that disrupt the intramolecular interactions of Src, such as a point mutation on the C-terminal tyrosine 527, result in an open conformation of the enzyme, which then interacts nonspecifically with multiple cellular substrates. This, in turn, leads to the activation of at least two signaling cascades mediated by the Ras and Rap GTPases.