Induction of rac-guanine nucleotide exchange activity of Ras-GRF1/CDC25(Mm) following phosphorylation by the nonreceptor tyrosine kinase Src.

Ras-GRF1/CDC25(Mm) has been implicated as a Ras-guanine nucleotide exchange factor (GEF) expressed in brain. Ras-GEF activity of Ras-GRF1 is augmented in response to Ca(2+) influx and G protein betagamma subunit (Gbetagamma) stimulation. Ras-GRF1 also acts as a GEF toward Rac, but not Rho and Cdc42, when activated by Gbetagamma-mediated signals. Tyrosine phosphorylation of Ras-GRF1 is critical for the induction of Rac-GEF activity as evidenced by inhibition by tyrosine kinase inhibitors. Herein, we show that the nonreceptor tyrosine kinase Src phosphorylates Ras-GRF1, thereby inducing Rac-GEF activity. Ras-GRF1 transiently expressed with v-Src was tyrosine-phosphorylated and showed significant GEF activity toward Rac, but not Rho and Cdc42, which was comparable with that induced by Gbetagamma. In contrast, Ras-GEF activity remained unchanged. The recombinant c-Src protein phosphorylated affinity-purified glutathione S-transferase-tagged Ras-GRF1 in vitro and thereby elicited Rac-GEF activity. Taken together, tyrosine phosphorylation by Src is sufficient for the induction of Rac-GEF activity of Ras-GRF1, which may imply the involvement of Src downstream of Gbetagamma to regulate Ras-GRF1.

The GDP/GTP state of low molecular weight GTP-binding proteins is regulated by guanine nucleotide exchange factors (GEFs) 1 and GTPase-activating proteins. GEFs, when activated by upstream signals, stimulate GDP/GTP exchange reaction of a distinct set of GTP-binding proteins, leading to the accumulation of the active GTP-bound form within the cell. Activation of GEFs occurs by diverse mechanisms including phosphorylation, direct interaction with other proteins, and in some cases, subsequent translocation to the plasma membrane.
The catalytic domain conserved among GEFs that target Ras is designated the CDC25 homology domain, which was originally identified as a region in the Saccharomyces cerevisiae Cdc25 protein essential for the function as an upstream regulator of Ras (1). To date, mSos1/2 (2, 3), Ras-GRF1/2 (4 -8), and Ras-GRP (9,10) have been characterized as a mammalian Ras-GEF containing the CDC25 homology domain. Particularly, the role of mSos in the regulation of Ras downstream of a variety of receptors including tyrosine kinase-type, cytokine, and G protein-coupled receptors has been clarified in detail. Virtually all types of receptors trigger the activation of specific tyrosine kinases, which in turn phosphorylate the receptor subunit itself and/or downstream molecules. Subsequently, a diverse array of proteins become associated with the tyrosinephosphorylated receptor, thereby stimulating multiple signaling pathways. Recruitment of mSos complexed with the adaptor protein Grb2 to the tyrosine-phosphorylated receptor at the plasma membrane is considered to be a crucial step for the onset of Ras activation (11).
Ras-GRFs contain various motifs in addition to the C-terminally located CDC25 homology domain, including the N-terminal pleckstrin homology (PH) domain, an IQ motif, a Dbl homology (DH) domain contiguous to the second PH domain (4 -7). Ras-GRF1 is reported to regulate Ras activity in response to Ca 2ϩ influx, for which binding of calmodulin to the IQ motif is crucial (12). On the other hand, Ca 2ϩ -responsive, Ras-independent as well as constitutive, Ras-dependent pathways downstream of Ras-GRF1 were shown to direct Raf and extracellular signal-regulated kinase activities (13). G protein ␤␥ subunits (G␤␥) also stimulate Ras-GEF activity of Ras-GRF1 through serine phosphorylation (14,15). Binding of G␤␥ to the N-terminal PH domain of Ras-GRF1 was demonstrated (16), although it is unclear whether the binding is critical for G␤␥ induction of Ras-GEF activity. Recently, oligomerization of Ras-GRFs through their DH domain was reported (13). Oligomerization seems to be required for biological functions, because a mutation within the DH domain that abolishes oligomerization rendered Ras-GRFs incapable of inducing transformation of NIH 3T3 cells. Ras-GRF1 is expressed exclusively in brain, suggesting an important role in brain-specific signaling. Disruption of Ras-GRF1, in fact, resulted in defects in brain functions such as memory consolidation (17). In contrast, Ras-GRF2 is expressed not only in brain but also in several other tissues such as lung and spleen, suggesting a role distinct from that of Ras-GRF1 (8).
Ras-GRP is expressed in nervous and hematopoietic cells (9,10). Besides the catalytic CDC25 homology domain, a pair of EF-hands, where Ca 2ϩ binds, and the diacylglycerol-binding C1 domain were identified in Ras-GRP. Whereas deletion of the C1 domain eliminated the transforming activity of Ras-GRP, the EF-hands seemed to be dispensable (9,10). Consistent with this, Ras⅐GTP formation was enhanced by a diacylglycerol analog in the presence of Ras-GRP, suggesting an involvement of Ras-GRP in diacylglycerol-mediated activation of the Ras pathway (9).
The Rho family of GTP-binding proteins consisting of Rho, Rac, and Cdc42 regulates various physiological responses through actin cytoskeleton rearrangements (18,19). Like Ras, Rho family proteins have been implicated as a molecular switch downstream of a wide variety of receptors. The activation of Rho family proteins in response to extracellular stimuli is thought to be directed by Rho family-specific GEFs. GEFs that act on Rho family proteins constitute the Dbl family, whose members possess DH and PH domains in tandem (20). The DH domain is responsible for catalysis, whereas the PH domain is believed to be implicated in membrane targeting and regulatory functions. Some Dbl family members are specific for an individual Rho family protein, whereas others act on all three subgroups. Although more than 20 Dbl family members have been identified, mechanisms underlying extracellular signaldependent regulation of Dbl family GEFs remain largely unknown.
Interestingly, mSos and Ras-GRFs possess the DH/PH domains as well, implying a role as a GEF for Rho family proteins. Indeed, mSos exhibits Rac-GEF activity when stimulated downstream of the Ras/phosphatidylinositol 3-kinase pathway, although Rac-GEF activity remains latent without upstream signals (21). Additionally, Ras-GRF1 functions as a Rac-GEF in response to signals mediated by G␤␥ (22), while Ras-GRF2 shows constitutive and Ca 2ϩ -stimulated Rac-GEF activity (23). Tyrosine phosphorylation of Ras-GRF1 has been implicated as a crucial step for the induction of Rac-GEF activity (22).
In this paper, we describe that Src elicits tyrosine phosphorylation of Ras-GRF1 in living cells and in a cell-free system, allowing Ras-GRF1 to act as a GEF for Rac. GEF activity toward other Rho family members, Rho and Cdc42, remained undetectable upon Src-dependent tyrosine phosphorylation. Ras-GEF activity was unaffected when phosphorylated by Src. Considering the observation that G␤␥ induces Rac-specific GEF activity of Ras-GRF1 in a tyrosine phosphorylation-dependent manner, Src may be involved in G protein-coupled receptor stimulation of Ras-GRF1.
Cell Culture and Transfection-Human embryonic kidney 293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum. Transfection of 293 cells with expression plasmids was carried out as described previously (24).
Purification of Recombinant GTP-Binding Proteins-Recombinant Rac1, RhoA, and Cdc42 were overexpressed in E. coli as a GST fusion protein and purified by a glutathione-Sepharose column. GST was subsequently removed by digestion with thrombin, and GTP-binding proteins were further purified as described (22). The recombinant Ha-Ras protein was described elsewhere (29).
Immunoprecipitation and Immunoblotting-Cells were dissolved in immunoprecipitation buffer (20 mM Tris-HCl (pH 7.5), 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 0.5% (v/v) Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 0.1 mM Na 3 VO 4 , 3 mM ␤-glycerophosphate), and the supernatant of centrifugation (15,000 ϫ g) for 10 min at 4°C was used as a cell lysate. The anti-FLAG antibody M2 (2 g) or the anti-Myc antibody 9E10 (2 g) was mixed with a rabbit antimouse Ig antibody conjugated to protein A-Sepharose (Amersham Pharmacia Biotech). Lysates were mixed gently with the antibody⅐protein A-Sepharose complex for 2 h at 4°C, and precipitates were washed four times with immunoprecipitation buffer. Precipitated proteins were subsequently separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was stained with the anti-phosphotyrosine antibody PY99 and a horseradish peroxidase-conjugated antimouse IgG antibody, followed by visualization by enhanced chemiluminescence detection reagents (NEN Life Science Products).
For [ 3 H]GDP binding assays, immunoprecipitated proteins were incubated with Rac1 (200 ng) or Ha-Ras (40 ng) at 30°C in exchange buffer supplemented with 2 mM DTT, 0.2 mg/ml bovine serum albumin, 1 mM ATP, and 1 M [ 3 H]GDP (1,265 TBq/mol). After incubation for specified periods, ice-cold wash buffer (10 mM Tris-HCl (pH 7.5), 10 mM MgCl 2 ) was added, and samples were filtered through a nitrocellulose membrane, which was subjected to extensive washing with wash buffer. Radioactivity remaining on the filter was quantitated by the liquid scintillation counter.
For After incubation for specified periods, radioactivity remaining in the complex was quantitated by filter binding assays as described above. Phosphorylated or unphosphorylated GST-Ras-GRF1 was obtained by preincubation of GST-Ras-GRF1 with or without the recombinant c-Src protein in the presence of 10 M nonradiolabeled ATP under conditions described above and subjected to GDP/GTP exchange assays.

RESULTS
Tyrosine Phosphorylation of Ras-GRF1 in v-Src-co-transfected and PDGF-stimulated Cells-As a first step to manifest a possible involvement of Src in the regulation of Ras-GRF1, Src-dependent tyrosine phosphorylation of Ras-GRF1 was examined by the use of a transient expression system in 293 cells. FLAG epitope-tagged full-length Ras-GRF1 was highly tyrosine-phosphorylated when co-expressed with v-Src as shown in Fig. 1A. The level of Ras-GRF1 tyrosine phosphorylation in response to v-Src as evaluated by immunoblotting using an anti-phosphotyrosine antibody was considerably higher than that induced by G␤␥ co-expression (data not shown). A similar construct consisting of the N-terminal 625 amino acids (N625), which contains all known regulatory motifs located within the N-terminal portion, was also tyrosine-phosphorylated in a v-Srcdependent manner (Fig. 1A). In addition, the Myc epitopetagged N-terminally truncated mutant ⌬N582, which contains only the C-terminally located CDC25 homology domain, and full-length Ras-GRF1 were tyrosine-phosphorylated by v-Src as illustrated in Fig. 1B. Overall, multiple tyrosine residues seemed to become tyrosine-phosphorylated upon co-expression of v-Src.
To show Src-dependent tyrosine phosphorylation of Ras-GRF1 under more physiological conditions, we examined the effect of PDGF because it is well known that Src acts downstream of the PDGF receptor (30,31). The ␤-type PDGF receptor was transiently expressed with FLAG-tagged Ras-GRF1 in 293 cells, which were subsequently stimulated with PDGF. As illustrated in Fig. 1C, PDGF stimulation induced tyrosine phosphorylation of Ras-GRF1, which was sensitive to the Srcspecific inhibitor PP2. In contrast, tyrosine phosphorylation of the PDGF receptor remained unaffected upon PP2 treatment. Therefore, Src may be implicated as a kinase responsible for tyrosine phosphorylation of Ras-GRF1.

Rac-directed GEF Activity of Ras-GRF1 Was Induced following Tyrosine Phosphorylation by Co-expression of v-Src-On
the basis of our previous observations that G␤␥ induce Rac-GEF activity of Ras-GRF1 in a tyrosine phosphorylation-dependent manner, we next investigated the effect of Src-induced tyrosine phosphorylation on Rac-GEF activity. Myc epitopetagged full-length Ras-GRF1 was immunoprecipitated from cells transfected with various combinations of expression plasmids, followed by Rac-GEF assays carried out essentially as described previously (22). Binding of GDP to recombinant Rac1 and release of GDP from the Rac1⅐GDP complex under various conditions are shown in Fig. 2, A and B, respectively. Expression levels of Ras-GRF1 in individual transfectants were virtually the same as determined by immunoblotting (data not shown). As described previously, Ras-GRF1 recovered from G␤␥-co-expressing cells significantly enhances GDP binding to Rac1, whereas Ras-GRF1 exhibits virtually no Rac-GEF activity when immunoprecipitated from cells ectopically expressing Ras-GRF1 alone ( Fig. 2A). Ras-GRF1 from v-Src-co-expressing cells stimulated GDP binding to Rac1 like G␤␥-stimulated Ras-GRF1 ( Fig. 2A). Consistent with this, the release rate of Rac1bound GDP became enhanced following co-expression of v-Src as in the case of G␤␥ (Fig. 2B). Co-expression of G␤␥ and v-Src did not result in additive increase in Rac-GEF activity (data not shown).
To further investigate the activation of Rac1 within the cell, we employed a pull-down assay by the use of GST-PAK-CRIB as a probe (28). As shown in Fig. 2C, co-expression of Ras-GRF1 with v-Src resulted in synergistically enhanced formation of Rac⅐GTP. Fig. 3 shows the effect of Src-dependent tyrosine phosphorylation on GEF activity toward other Rho family members, RhoA and Cdc42. Relative amounts of GDP remaining com- plexed with respective GTP-binding proteins after incubation with v-Src-stimulated Ras-GRF1 for 60 min are shown. RhoA and Cdc42 were insensitive to Ras-GRF1 stimulated by coexpression of v-Src in contrast with Rac1. These results are analogous to previous observations that co-expression of G␤␥ conferred GEF activity toward Rac1, but neither RhoA nor Cdc42, to Ras-GRF1 (22).
Furthermore, the effect of v-Src on GEF activity toward Ha-Ras was examined (Fig. 4). Myc epitope-tagged Ras-GRF1 was immunoprecipitated and subjected to GDP binding assays for Ha-Ras. Ras-GRF1 exhibited constitutive Ras-GEF activity, which was not promoted by co-expression of v-Src in contrast to G␤␥ (14,15).

Tyrosine Phosphorylation of Ras-GRF1 by the Recombinant c-Src Protein in Vitro and Its Effect on Rac-GEF Activity-Fulllength Ras-GRF1 and its N-terminally deleted version ⌬N582
were yielded in E. coli as GST fusion proteins, which were partially purified by using standard glutathione-Sepharose column chromatography. These recombinant Ras-GRF1 proteins were subjected to in vitro kinase assays using the recombinant c-Src protein. As illustrated in Fig. 5A, both full-length and N-terminally deleted Ras-GRF1 were phosphorylated in a Srcdependent manner. Incorporation of 32 P into the GST protein was not detected under similar conditions (data not shown). Lower molecular weight proteins that were phosphorylated by the recombinant c-Src protein are degradation products. To further confirm tyrosine phosphorylation in vitro, reaction mixtures without radioactive ATP were subjected to SDS-PAGE and immunoblotting using an anti-phosphotyrosine antibody. As shown in Fig. 5B, both Ras-GRF1 constructs incubated in the presence of the recombinant c-Src protein were stained with an anti-phosphotyrosine antibody.
Moreover, the effect of tyrosine phosphorylation of recombinant full-length Ras-GRF1 on its Rac-GEF activity was assessed (Fig. 6). Consistent with the results obtained from in vivo phosphorylation experiments, recombinant Ras-GRF1, when tyrosine-phosphorylated by the recombinant c-Src protein in vitro, displayed GEF activity toward Rac1, whereas Ras-GRF1 incubated without the recombinant c-Src protein had virtually no Rac-GEF activity. The c-Src protein preparation had no GEF activity (data not shown). Taken together with these results, it is likely that Src directly phosphorylates Ras-GRF1, thereby conferring Rac-GEF activity. DISCUSSION Rho family GTP-binding proteins including Rho, Rac, and Cdc42 have been implicated as a molecular switch of a wide variety of signal transduction pathways (18,19). Activity of Rho family proteins is believed to be regulated through the action of Rho family-specific GEFs in response to extracellular stimulation. Although more than 20 proteins belong to the Dbl family of GEFs that target Rho family members (20), precise mechanisms underlying the coordinated regulation downstream of diverse receptors remain elusive.
Herein, we show that the nonreceptor tyrosine kinase Src directly phosphorylates Ras-GRF1, thereby inducing GEF activity toward Rac. In contrast to Src, the epidermal growth factor receptor did not phosphorylate Ras-GRF1, implying that Ras-GRF1 does not participate in its signaling pathways (32). However, another receptor-type tyrosine kinase PDGF receptor induced tyrosine phosphorylation of Ras-GRF1 through Src, at least in our reconstitution system. In addition, Src may mediate signals triggered by a certain set of nontyrosine kinase-type receptors. It is important to delineate the receptors that activate the Src/Ras-GRF1 pathway upon extracellular stimulation particularly in neuronal cells.
Recently, we found that co-expression of G␤␥ enabled Ras-GRF1 to act as a GEF toward Rac, although Rac-GEF activity of Ras-GRF1 was latent when recovered from unstimulated cells (22). Tyrosine phosphorylation of Ras-GRF1 itself was crucial for the induction of Rac-GEF activity by G␤␥, as evidenced by the effect of tyrosine kinase-specific inhibitors (22). However, tyrosine kinases responsible for Ras-GRF1 activation downstream of G␤␥ have not been identified. Considering our results presented in this paper, it is possible that Src may function downstream of G␤␥ to regulate Ras-GRF1. Indeed, activation of Src family kinases in response to G protein-coupled receptor stimulation was reported in various types of cells (33)(34)(35)(36)(37)(38)(39)(40). Moreover, G␤␥ overexpression leads to enhancement of Src kinase activity although only weakly (35). Particularly, Src has been implicated as a link between G protein-coupled receptors and the downstream Ras/extracellular signal-regulated kinase pathway (34,35) because tyrosine phosphorylation of specific proteins such as Shc is important for the activation of the Ras/extracellular signal-regulated kinase pathway following G␤␥ stimulation (41,42).
However, arguing against the notion that Src is responsible for G␤␥ induction of Rac-GEF activity of Ras-GRF1, Ras-GRF1 becomes phosphorylated by Src much more heavily than by G␤␥ in 293 cells, which may reflect distinct sets of phosphorylation sites. Therefore, Src may not represent a tyrosine kinase that primarily mediates G␤␥ signals to Ras-GRF1 even if respective phosphorylation sites are overlapping. Instead, the PH domain-containing tyrosine kinase Tec (43) may provide a link between G␤␥ and downstream molecules because its relatives Tsk and Btk interact with G␤␥, being stimulated in co-transfected cells and in vitro (44), and Tec is expressed in a variety of tissues in contrast to Tsk and Btk, which are expressed mainly in hematopoietic cells.
Like Ras-GRF1, Rac-GEF activity of the proto-oncogene product Vav becomes evident upon phosphorylation by the tyrosine kinase Lck (45,46). Thus, activation of Dbl family GEFs through tyrosine phosphorylation may be a general mechanism of regulation, although Ras-GRF1 is a brain-specific protein, while Vav is primarily expressed in hematopoietic cells. Furthermore, Lck-dependent Rac-GEF activity of Vav is enhanced by phosphatidylinositol-3,4,5-trisphosphate, whereas phosphatidylinositol-4,5-bisphosphate inhibits the activity (47). The PH domain of Vav probably mediates the effects of phosphoinositides (47). In fact, the ␥ isoform of phosphatidylinositol 3-kinase, when activated by G␤␥ (48), yields phosphoinositides that activate the Rac-GEF Vav in transfected cells (49,50). It is obscure at present whether Rac-GEF activity of Ras-GRF1 is regulated similarly by substrates and products of phosphatidylinositol 3-kinase.
Ras-GEF activity of CDC25 homology domain-containing proteins is thought to be constitutive. For the activation of downstream pathways, targeting to the plasma membrane, which renders GEFs (including mSos and Ras-GRF1) accessible to Ras, is important (51)(52)(53). Membrane localization of mSos is achieved through sequential interaction with adaptors and receptors under physiological conditions. In contrast, GEF activity toward Rho family proteins appears to be regulated by changing the tertiary structure through phosphorylation or protein-protein interaction. For instance, Rac-GEF activity of Ras-GRF1 and Vav is directed through tyrosine phosphorylation as addressed above. Threonine phosphorylation also seems to be important for the regulation of GEF activity of Tiam1 (54,55). Additionally, p115RhoGEF is activated by G␣13 through direct interaction, which in turn modulates downstream Rhodependent events such as transcriptional activation of specific genes (56 -58). Likewise, PDZ-RhoGEF seems to be regulated through direct binding of G␣12 and G␣13 (59). GEF activity of PIX/Cool as well seems to be modulated by the binding of the N-terminal portion of p21-activated kinase (60 -62). Therefore, the analysis of structural alterations of Ras-GRF1 induced by Src-dependent tyrosine phosphorylation may help in understanding the detailed regulatory mechanisms underlying the induction of Rac-GEF activity.
G␤␥ stimulate multiple downstream signaling cascades, leading to the activation of Ras and Rho family GTP-binding proteins. For Ras activation, the mSos-dependent pathway has been characterized intensively as described above (42), where Src may be involved (34,35). In addition, G␤␥ signals enhance Ras-GEF activity of Ras-GRF1 through serine phosphorylation (14,15). G␤␥ trigger Rac activation also in Ras-GRF1-depend- ent and -independent fashions. G␤␥ induce Rac-GEF activity of Ras-GRF1 through tyrosine phosphorylation (22). On the other hand, G␤␥ activation of the Rac pathway in the absence of Ras-GRF1 was reported (63), although the mechanism whereby G␤␥ activate Rac remains obscure. In S. cerevisiae, the G␤␥ (Ste4⅐Ste18) signal that regulates cytoskeletal organization and cell polarity is relayed to the Rho family GTP-binding protein Cdc42 through Cdc24, a Dbl family GEF that acts on Cdc42. Direct binding of yeast G␤␥ to Cdc24 is reported (64,65), raising the possibility that a mammalian counterpart of Cdc24 may be regulated through the interaction with G␤␥ (66). Collectively, both Ras-GRF1-dependent and -independent pathways function downstream of G␤␥ to control Ras and Rac in Ras-GRF1-expressing cells. Taking into consideration that Ras-GRF1 is detected exclusively in brain, apparently redundant signaling networks may be critical for coordinated regulation of the GTP-binding proteins, particularly in brain-specific functions.