Mechanisms through which Sos-1 coordinates the activation of Ras and Rac

A major mechanism of signal transduction by receptor tyrosine kinases (RTKs)^* involves the activation of small GTPases, among which Ras and Rac are pivotal (Scita et al., 2000). There is evidence for hierarchical organization of small GTPases in signal transduction pathways (Van Aelst and D'Souza-Schorey, 1997; Hall, 1998; Scita et al., 2000). In addition, regulators of small GTPase activity, such as the guanine nucleotide exchange factor (GEF) son of sevenless (Sos-1), appear to control different events in the signaling cascade (for review see Bar-Sagi and Hall, 2000; Schlessinger, 2000; Scita et al., 2000). A scenario can thus be envisioned in which coordinated and/or sequential activation of small GTPases is achieved through the action of the same regulator at multiple steps. The molecular mechanisms involved, however, remain yet to be understood. Sos-1 is a well-known Ras-GEF (for review see Bar-Sagi, 1994; Schlessinger, 2000), whose specific catalytic activity is encoded by its Cdc25-like domain (Bar-Sagi, 1994). Elegant biochemical and genetic studies have clarified how the proline-rich COOH-terminal tail of Sos-1 interacts with the SH3-containing adaptor molecule Grb2 (Bar-Sagi, 1994). Grb2 in turn displays a SH2 domain responsible for the recruitment of the Grb2–Sos-1 complex to active, autophosphorylated RTKs (Lowenstein et al., 1992; Ceresa and Pessin, 1998). The relocalization of the complex to the plasma membrane is thought to be sufficient for Sos-1 to catalyze the exchange of guanine nucleotides on Ras, which is also present at the membrane, with ensuing activation of this small GTPase.

Active Ras (Ras-GTP) triggers a number of signaling cascades, among which is the one connecting Ras to Rac, a member of the Rho subfamily of small GTPases. There is evidence that Sos-1 is also active at this step. First, Sos-1 contains a Dbl homology domain in tandem with a Pleckstrin homology domain, a module responsible for GEF activity on Rho GTPases (Cerione and Zheng, 1996; Nimnual et al., 1998). Second, and most importantly, Sos-1 has been shown to form a tricomplex in vivo with two signaling molecules, Eps8 (Fazioli et al., 1993) and E3b1 (also known as Abi-1) (Shi et al., 1995; Biesova et al., 1997). E3b1 contains a SH3 domain and binds Sos-1 (Scita et al., 1999; Fan and Goff, 2000). In addition, E3b1 binds to the SH3 domain of Eps8 (Biesova et al., 1997). Thus E3b1 acts as a scaffold protein, which holds together Sos-1 and Eps8. The tricomplex Sos-1–E3b1–Eps8 (S/E/E8) is endowed with Rac GEF activity in vitro (Scita et al., 1999). Thus, the sum of the above observation raises the possibility that Sos-1 might function at different step in the signaling cascade, acting as a Ras-GEF and a Rac-GEF, respectively. A number of outstanding questions need clarification, however, before such a model could be accepted: does a trimeric S/E/E8 complex exist under physiological conditions? How is the specificity of Sos-1 directed toward Ras or Rac? Does the putative dual function of Sos-1 provide a mechanistic framework for the coordinated activation of Ras and Rac? The present studies were undertaken to elucidate these questions.

In previous studies, we showed that Sos-1, E3b1, and Eps8 could form a trimeric complex in vivo upon concomitant overexpression of the three proteins. However, we failed to detect the existence of an endogenous S/E/E8 complex (Scita et al., 1999). We reasoned that this could be due to the low efficiency of the immunoprecipitating antibodies used. We thus sought to exploit the availability of eps8^−/− fibroblasts to circumvent this problem. To this end, we performed immunoprecipitation experiments using eps8^−/− fibroblasts in which the expression of Eps8 was restored, to physiological levels, with an expression vector encoding a myc epitope-tagged Eps8 (−/− [Eps8myc] cells). We selected transfected clones in which the levels of expression of Eps8myc were very similar to those present in wild-type fibroblasts (Fig. 1 A). Endogenous Sos-1 and E3b1 could be detected in anti-myc immunoprecipitates from lysates of Eps8myc-reconstituted, but not from eps8^−/− fibroblasts (Fig. 1 B).

To determine whether E3b1 mediates the interaction between Eps8 and Sos-1, as it would be expected according to the tricomplex model, we performed coimmunoprecipitation experiments under conditions in which the association between Eps8 and E3b1 was disrupted. The binding site of E3b1 to the SH3 domain of Eps8 was previously mapped to the amino acid sequence, PPPPPVDYTEDEE, where the D and the Y residues are critical for efficient binding (Mongiovi et al., 1999). Thus, a peptide encompassing this region should specifically disrupt the Eps8–E3b1 association. Indeed, no E3b1could be recovered in anti-myc immunoprecipitates from lysates of Eps8myc-reconstituted cells, when the immunoprecipitation was performed in the presence of an excess of the competing peptide. The association was, however, preserved when a control peptide, bearing a Y→A substitution and unable to bind to Eps8 (Mongiovi et al., 1999), was used (Fig. 1 C). Similarly, no Sos-1 could be recovered in anti-myc immunoprecipitates in the presence of the competing, but not of the control, peptide (Fig. 1 C). Thus, under physiological conditions, the coimmunoprecipitation of Eps8 and Sos-1 depends on the integrity of the Eps8–E3b1 interaction, pointing to the existence of a physiological S/E/E8 complex. It cannot be formally excluded that Eps8, E3b1, and Sos-1 associate after cell lysis, thus allowing coimmunoprecipitation. However, we have previously demonstrated (Scita et al., 2001) that the three endogenous proteins also colocalize in vivo in dynamic actin structures. Thus, the sum of our results strongly argues in favor of the existence of a physiological S/E/E8 complex.

Similarly, it cannot be formally excluded that other interactors of the SH3 domain of Eps8, besides E3b1, mediate the formation of the endogenous trimeric complex. This is, however, unlikely since no coimmunoprecipitation between Eps8 and Sos-1 could be detected when the two proteins were overexpressed in cell lines with relative low levels of E3b1 (Scita et al., 1999; unpublished data). Moreover, neither RN-tre, nor JIK, two other known interactors of the SH3 domain of Eps8 (Biesova et al., 1997; Mongiovi et al., 1999), were able to bind to Sos-1 (unpublished data).

Sos-1 has been shown to be part of a signaling complex with Grb2, which mediates the activation of Ras upon RTK stimulation. Our finding that, under physiological conditions, Sos-1 also participates in a complex with Eps8 and E3b1 raises the question of the physical and functional relationships between these two Sos-1–containing complexes. To gain insight into this issue, we initially mapped the region of E3b1 responsible for the interaction with Sos-1 by using a series of deletion mutants of E3b1 fused to glutathione S-transferase (GST). Native Sos-1 could be efficiently recovered onto GST-E3b1 full length (Fig. 2 A, amino acids [aa] 2–480). Further mapping revealed that the SH3 domain of E3b1 (aa 416–480) was necessary and sufficient for binding (Fig. 2 A).

We have previously shown that E3b1 binds to the proline-rich, COOH-terminal tail of Sos-1 (aa 1131–1333) (Scita et al., 1999), which also binds to the SH3 of Grb2 (Li et al., 1993; Cussac et al., 1994). Thus, Grb2 and E3b1 might compete for binding to Sos-1. This hypothesis was validated by a series of experiments performed both in vitro and in vivo. We could demonstrate that (a) the SH3 of Grb2, but not of Eps8, efficiently competed with the SH3 of E3b1 for binding to the proline-rich COOH-terminal tail of Sos-1 in vitro (Fig. 2 B); (b) the Sos-1 peptide VPVPPPVPPRRR, known to constitute a Grb2-SH3 binding site (Li et al., 1993; Cussac et al., 1994), competed equally well the binding of the COOH-terminal tail of Sos-1 to the SH3s of Grb2 or E3b1 (Fig. 2 C); (c) the binding constants of the interaction between the proline rich COOH-terminal tail of Sos-1 and the SH3 domains of Grb2 and E3b1 were very similar (Fig. 2 D); (d) overexpression of Grb2 abolished the ability of GST-E3b1 to bind to native Sos-1 (Fig. 3 A); and (e) overexpression of E3b1 significantly decreased the coimmunoprecipitation between Grb2 and Sos-1 (Fig. 3 B). Thus, Sos-1 binds either E3b1 or Grb2, in a mutually exclusive fashion, suggesting that two distinct pools of Sos-1 exist in the cells.

To test this possibility in vivo, we set out to perturb the equilibrium between the Sos-1–E3b1 (S/E) and Sos-1–Grb2 (S/G) complexes, by increasing the levels of E3b1 within the cell. If the two complexes are functionally distinct, then the overexpression of E3b1, by competing the S/G interaction, should result in (a) reduced Sos-1 recruitment in vivo to active EGFR, (b) consequent reduction in Ras activation, as measured by reduced Ras-GTP levels, (c) reduced activation of Ras-dependent pathways, as measured by activation of MAPK, and finally (d) reduced proliferative response. All of these predictions were confirmed experimentally (Fig. 4, A–C).

We note that our mapping data are at variance with those recently reported by Fan and Goff (2000), who concluded that, in vivo, the interaction between Sos-1 and E3b1–Abi-1 is mediated by the NH₂-terminal portion of the latter. Consistently, Fan and Goff (2000) did not observe competition in vivo between Grb2 and E3b1 for binding to Sos-1. Although we cannot exclude that the NH₂-terminal region of E3b1–Abi-1 participates in binding, in vivo, we were not able to observe a high-affinity binding surface in that region by in vitro binding experiments (Fig. 2 A; Scita et al., 1999). Conversely, under our conditions of analysis, the efficient competition of the SH3 domains of Grb2 and E3b1 for binding to the same Sos-1 peptide, coupled to the readily detectable biological competition, strongly suggests that the SH3-mediated contact is the major determinant of the interaction between Sos-1 and E3b1.

The competition between E3b1 and Grb2 for the same binding site on Sos-1 implies that an individual Sos-1 molecule can only be part of one GEF complex at a time. However, different pools of Sos-1, either bound to Grb2 or to E3b1, could exist in vivo. We tried to gain insight into this issue. We showed that BIAcore measurements revealed similar kinetic parameters for the SH3-mediated S/G and S/E interactions (Fig. 2 D). However, when we measured the relative abundance of S/G and S/E complexes within the cell, we found a 10-fold excess of the former (Fig. 5 A), under conditions in which quantitative immunoprecipitation of the adaptor molecules, Grb2 and E3b1, was achieved (Fig. 5 A). Thus, the data indicate that two pools of Sos-1, associated with different adaptors, exist simultaneously in the cell. They further suggest that the pool of E3b1, available for binding to Sos-1, is reduced relative to that of Grb2, and may be rate-limiting. Indeed, overexpression of E3b1 resulted in increased PAK65 activity, an indicator of Rac activation (Manser et al., 1994) (Fig. 5 B), which was abrogated by a dominant negative Rac mutant (Fig. 5 B). On the other hand, overexpression of Sos-1 did not lead to increased PAK65 activity. An additional corollary of this hypothesis is that the overexpression of E3b1, by facilitating the formation of a trimeric complex with Eps8 and Sos-1, should directly lead to activation of Rac. To test this prediction E3b1 or a mutant of E3b1 (E3b1-DY), bearing alanine substitutions of the DY residues critical for the interaction with Eps8 (Mongiovi et al., 1999), were used to infect wild-type and eps8^−/− fibroblasts. The levels of GTP-bound Rac were then determined by in vitro binding assays using the immobilized CRIB domain of PAK65 (Manser et al., 1994). E3b1, but not the E3b1 mutant impaired in Eps8 binding, caused a readily detectable increase in Rac-GTP levels in wild type, but not in eps8^−/− fibroblasts (Fig. 5 C). Thus, the overexpression of E3b1 is sufficient to activate Rac and this effect is strictly dependent on Eps8.

The indispensability of E3b1 in the cascade of events leading to Rac activation and Rac-dependent actin reorganization was further analyzed at the biological level. We have previously shown that interference with E3b1 functions, by microinjection of anti-E3b1 antibodies, inhibited PDGF-induced ruffles (Scita et al. 1999). We also showed that in eps8^−/− cells, ruffling induced by PDGF and activated Ras, but not by activated Rac, was inhibited. Ruffling, however, could be restored upon reexpression of Eps8, but not of an Eps8 mutant unable to bind to E3b1 (Scita et al., 1999), suggesting that the Eps8–E3b1-based complex is implicated in the transmission of signal between Ras and Rac. Thus, one might predict that a mutant of E3b1 unable to associate to Eps8 should function as a dominant negative on Ras-induced Rac-dependent ruffling. To test this, we cotransfected the activated versions of either Ras or Rac, together with E3b1 or the E3b1DY mutant. RasV12-induced, but not RacQL-induced, ruffles were efficiently inhibited by the coexpression of E3b1DY, but not of E3b1 wild type (Fig. 6), supporting the contention that the ability of E3b1 to form a complex with Eps8 is required to transmit signals from Ras to Rac. In conclusion, our data show that two pools of Sos-1 exist in the cell, coupled respectively to Grb2 or E3b1. In addition, the availability of E3b1, but not of Sos-1, is indispensable and rate limiting in the pathway leading to Rac activation.

The existence of Sos-1 in two physically and functionally distinct pools suggests the hypothesis that its presence in a S/G or a S/E/E8 complex could dictate its Ras- or Rac-specific GEF activities, respectively. To explore this possibility, we used an in vitro assay that can score GEF activities in Sos-1–containing immunoprecipitates. Cells were transfected with either Sos-1 alone (SosTfx) or a combination of S/E/E8 (Triple Tfx) (Fig. 7 A). When Sos-1 was immunoprecipitated from SosTfx, it displayed Ras-GEF, but not Rac-GEF, activity (Fig. 7 B). Of note, little E3b1 and no Eps8 could be detected in Sos-1 immunoprecipitates (Fig. 7 A). Conversely, Grb2 was readily recovered from the same immunoprecipitates (unpublished data). We then used the Triple Tfx and coimmunoprecipitated Sos-1 with anti-Eps8 antibodies (Fig. 7, A and B). Under these conditions, all of the coimmunoprecipitated Sos-1 is present in the S/E/E8 tricomplex (Scita et al., 1999). Despite lower levels of Sos-1 in the coimmunoprecipitate (with respect to a Sos-1 immunoprecipitate from SosTfx), Rac-GEF activity was now present (Fig. 7 B). Conversely, no Ras-GEF activity could be detected (Fig. 7 B). The lack of Ras-GEF activity was not due to the low levels of Sos-1. To prove this point, we immunoprecipitated from SosTfx an amount of Sos-1 comparable to that present in anti-Eps8 immunoprecipitates from Triple Tfx (Fig. 7, A and B, lanes SosTfx1/10). Under these conditions, Ras-GEF activity was readily detectable (Fig. 7 B). The detection of Rac-specific GEF activity in anti-Eps8 immunoprecipitates strictly required the presence of Sos-1, since no GEF activity was detected in Eps8 immunoprecipitates, in the absence of coexpressed Sos-1 (Scita et al., 1999; unpublished data). Furthermore, no Rac-GEF activity could be detected in Eps8 immunoprecipitates from lysates of cells transfected with Sos-1, E3b1, and an Eps8 mutant that is impaired in its ability to bind E3b1 and thereby cannot form a trimeric complex (Scita et al., 1999).

At this stage, we cannot formally exclude that an unknown GEF, coprecipitating with Sos-1, is responsible for the observed activity. However, such a hypothetical protein should not only be associated with Sos-1, but also be active only in the presence of Eps8 and E3b1, which are both required for the formation of the trimeric complex endowed with Rac-specific GEF activity (Scita et al., 1999), making this possibility unlikely. It is reasonable, therefore, to propose that Sos-1 alone (or complexed with proteins, such as Grb2) is endowed with Ras-GEF activity. However, upon interaction with Eps8 and E3b1, its specificity is switched toward Rac.

A common feature of proteins endowed with GEF catalytic activity is their ability to bind to their specific nucleotide-depleted GTPase with relative high affinities. Thus, one might postulate that Sos-1 associates with the nucleotide-free form of Rac exclusively when engaged in the S/E/E8 complex. Purified and nucleotide depleted, GST-Rac and GST-Cdc42 proteins were therefore used to test their ability to interact with Sos-1. Native Sos-1, present in lysates of −/− [Eps8myc] fibroblasts, could be specifically recovered with nucleotide-depleted immobilized GST-Rac, but not with GST-Cdc42 or GST alone (Fig. 7 C). Conversely, no Sos-1 could be recovered with GST-Rac from lysates of eps8^−/− cells. Thus, Eps8 is required for the association between Sos-1 and nucleotide-free Rac. Furthermore, under conditions in which the Eps8–E3b1 or the E3b1–Sos-1 interactions were disrupted by specifically competing peptides, a reduction of >80% in the amount of Sos-1 bound to nucleotide-depleted Rac was observed (Fig. 7 C). This latter result strongly suggests that an intact S/E/E8 complex is required for binding to Rac, providing a molecular basis for the catalytic specificity of the complex.

Since S/G and S/E complexes are present in the cells simultaneously, then both Sos-1-dependent Ras- and Rac-GEF activities are present at the same time. The question remains as to how these two complexes, and the ensuing Ras and Rac activities, are coordinated to achieve propagation of signals. We therefore looked for evidence of differential regulation of the S/G and S/E complex upon RTK stimulation. Upon PDGF stimulation, we observed decreased coimmunoprecipitation between Grb2 and Sos-1 and consequently between PDGFR and Sos-1 (Fig. 8, A and B), which correlated with the appearance of a mobility-retarded form of Sos-1. This likely represents a hyperphosphorylated form of Sos-1, as previously demonstrated (Baltensperger et al., 1993; Cherniack et al., 1994; Pronk et al., 1994). Conversely, under identical conditions, the coimmunoprecipitation between E3b1 and Sos-1 was not affected (Fig. 8 A). Similarly, the stability of the endogenous trimeric complex, Sos-1/Eps8/E3b1 was not affected by treatment of cells with growth factors (Fig. 8 C).

The above data indicate that, as a consequence of activation of RTKs, the S/G complex is disrupted, whereas the S/E/E8 complex persists in the cell. This might lead to a transient peak in Ras activity vis a vis a more prolonged activation of Rac. Therefore, we measured the kinetic of RTK-induced activation of Ras and Rac. As shown in Fig. 8 D, activation of Ras was rapid and short lived, whereas activation of Rac was sustained over a longer period of time, compatible with the differential regulation of the corresponding activating complexes, S/G and S/E/E8.

Previously, we have demonstrated that the genetically engineered removal of Eps8 from mouse fibroblasts led to the abrogation of the activation of the small GTPase Rac by RTKs and/or activated Ras (Scita et al., 1999). Biochemically, this effect was mirrored by the absence of Rac-specific GEF activity in extracts of Eps8-null cells (Scita et al., 1999). Sos-1 might be the GEF involved, as witnessed by the observations that it forms a tricomplex with Eps8 and the scaffolding protein E3b1 in vivo and that this tricomplex is endowed with Rac-GEF activity in vitro (Scita et al., 1999). A model was therefore proposed in which Sos-1 acted as a dual GEF, involved in both Ras and Rac activation. In this study, after showing that a S/E/E8 complex can exist under physiological conditions, we investigated the molecular mechanisms through which Sos-1 coordinates the activation of these two small GTPases after RTK stimulation.

These results show that when Sos-1 is engaged in a multimolecular complex containing at least Eps8, E3b1, and Sos-1 itself (the contribution of other proteins cannot be excluded), it displays exclusive Rac specificity. This is mirrored biochemically by the finding that Sos-1 binds to nucleotide-depleted Rac only when its association with Eps8 and E3b1 is preserved. On the contrary, Sos-1 acts as a Ras-specific GEF when alone or in association with Grb2 (Fig. 7; Buday and Downward, 1993b). Thus, in light of these results and previous finding, we conclude that a S/G complex directs Ras activation through binding to RTKs, whereas a S/E complex directs Rac activation by entering into a tricomplex with Eps8 (Fig. 9).

The finding that Sos-1 participates in both Ras and Rac activation does not immediately explain the temporal sequence by which the activation of Ras precedes that of Rac. This latter concept, however, is largely based on the fact that ectopic expression of a constitutively active Ras mutant is sufficient to cause membrane ruffles, indicative of Rac activation (Bar-Sagi and Feramisco, 1986; Rodriguez-Viciana et al., 1997; Scita et al., 1999). Current thinking holds therefore that active Ras, through binding and activation of phosphatidylinositol 3-kinase, leads to the formation of phosphatidylinositol 3, 4, 5 triphosphate, which in turn regulates Rac-specific GEFs, like Vav-1, and possibly Sos-1 (Kotani et al., 1994; Rodriguez-Viciana et al., 1994, 1996, 1997; Han et al., 1998; Nimnual et al., 1998; Das et al., 2000) (Fig. 9).

Our data and published literature highlight a much more complex scenario. First, it is difficult to conceive how Rac activation could be exclusively Ras dependent. In some cell lines, such as Swiss 3T3, RTK-induced Rac activation is apparently Ras independent (Ridley et al., 1992). In addition, even in those cell lines in which a dominant negative Ras mutant can suppress ruffle formation, the inhibition is never quantitative (a maximum of 65 ± 5% SEM inhibition of PDGF-induced ruffling was observed in mouse embryo fibroblasts microinjected with a Ras dominant negative mutant) (Scita, G., personal communication). Second, our kinetic data revealed that Rac activation persists long after the return to basal levels of Ras-GTP, a finding not immediately reconcilable with a simple and unique Ras→Rac pathway. It seems therefore that multiple mechanisms could lead to Rac activation from stimulated RTK (Fig. 9).

Nevertheless, signals appear to converge on a S/E/E8 complex, as suggested by the fact that genetic and biochemical interference with this complex leads to abrogation of Rac activation induced by either active RTKs or dominant active Ras and phosphatidylinositol 3-kinase mutants (Scita et al., 1999). Our demonstration of differential regulation of the stability of the S/G and S/E complexes sheds new light on the molecular understanding of this circuitry. A dynamic interaction between Sos-1 and Grb2 after RTK stimulation has been previously reported (Buday and Downward, 1993a; Egan et al., 1993; Rozakis-Adcock et al., 1993; Cherniack et al., 1994; Waters et al., 1995; Hu and Bowtell, 1996). Moreover, it is well established that upon EGF treatment, Sos-1 is rapidly phosphorylated, mainly as a consequence of MAPK activation (Cherniack et al., 1994; Rozakis-Adcock et al., 1995; Corbalan-Garcia et al., 1996; Porfiri and McCormick, 1996), leading to a decrease in its affinity for Grb2 (Corbalan-Garcia et al., 1996). Thus, the interaction between Sos-1 and Grb2 is dynamically regulated by RTK and might contribute to the rapid, but transient, activation of Ras after RTK stimulation (Fig. 8, A, B, and D). Conversely, the S/E/E8 complex is stable under the same conditions, and may account, at least in part, for the prolonged activation of Rac signaling (Fig. 8, A–D) and ruffling activity (Ridley et al., 1992; unpublished data) (Fig. 9 shows a proposed mode of action of Sos-1)

The shift in catalytic specificity of Sos-1, upon interaction with Eps8 and E3b1 does not exclude the possibility that additional mechanisms might participate in its action on Rac in vivo. This is supported by the observation that the Rac-specific GEF activity, detected in total cellular lysates and in the immunoprecipitated trimeric S/E/E8 complex, is not apparently affected by RTK activation (unpublished data). GTP-bound active Rac accumulates on the plasma membrane at sites where ruffling takes place (Kraynov et al., 2000). Similarly, upon growth factor stimulation, Eps8, E3b1, and Sos-1 are localized at the plasma membrane and colocalize with F-actin within membrane ruffles (Provenzano et al., 1998; Scita et al., 1999, 2001), whereas Grb2 cannot be detected in these structures (Scita et al. 2001). This provides a potential docking system for the Rac-based signaling machinery and argues that differential localization of adaptor molecules may further participate in the regulation of Sos-1 biological activities. Although more work will be needed to define the exact interplay of all components of the circuitry, our findings of differential regulation of the S/G and S/E complexes suggest an efficient mechanism for a wave-like propagation of signals in which upstream signaling is rapidly switched off after stimulation, whereas downstream signaling is still permitted (Fig. 9).

CMV-based eukaryotic expression vectors and GST fusion or His-tagged bacterial expression vectors were generated by recombinant PCR. Where indicated, epitope tagging (either myc or HA) was also obtained by recombinant PCR. The mutant of E3b1 (E3b1-DY), in which the D and Y residues were replaced by A, was generated by PCR-based site directed mutagenesis and cloned in the retroviral pBabe vector. All constructs were sequence verified. Details are available upon request. HA–Sos-1 was from Dr. D. Bar-Sagi (Yang et al., 1995). Antibodies were anti-Eps8 (Fazioli et al., 1993), anti-E3b1 (Biesova et al., 1997), and anti-EGFR sera (Di Fiore et al., 1990); rabbit polyclonals anti–Sos-1, anti-Grb2, and anti-ERK-1; anti-PAK65, anti-PDGFR (Santa Cruz Biotechnology); monoclonals anti-phosphoMAPK (New England BioLabs), anti–v-H-Ras (Oncogene Science), anti–Histidine (Sigma Aldrich), anti-BrdU (Becton and Dickinson), anti-HA11, and anti-myc 9E10 (Babco); and anti-Rac (Transduction Laboratories).

Immunoprecipitation and coimmunoprecipitation were performed as described (Fazioli et al., 1993). Briefly, cells were lysed on ice with buffer containing 1% Triton X-100, 50 mM Hepes (pH 7.5), 150 mM NaCl, 5 mM EGTA, 1.5 mM MgCl₂, 10% glycerol, 0.1 mM sodium orthovanadate, 2 mM phenylmethylphosphate fluoride, 2 mM sodium pyrophosphate 10 μg/ml aprotinin, and 10 μg/ml leupeptin. Immunoprecipitations were performed for 2 h at 4°C, and immunocomplexes were recovered by adsorption to protein A–Sepharose. After several washes with buffer containing 0.1% Triton X-100, 20 mM Hepes (pH 7.6), 10% glycerol, and 150 mM NaCl (washing buffer), immunoprecipiates were resolved be SDS-PAGE and analyzed by immunoblotting.

In vitro binding assays were performed as described (Fazioli et al., 1993). GST fusion proteins were purified by affinity chromatography on agarose-bound glutathione. His-tagged COOH-terminal tail of Sos-1 was purified by affinity chromatography on agarose-bound Ni. For the in vitro binding experiments, 5–60 pmol of purified immobilized GST fusion proteins, or wild-type GST, were incubated with total cellular proteins (1–2 mg), prepared as described above. After several washing with washing buffer, proteins were resolved by SDS-PAGE and analyzed by immunoblotting.

The in vitro binding to recombinant purified nucleotide-free GST-Cdc42 and GST-Rac was performed as described (Fan et al., 1998). Briefly, Rac1 and Cdc42, prepared as GST fusion proteins (20 μg) and immobilized on glutathione–agarose beads (∼1 mg protein/ml resin), were rendered free of nucleotide by incubation for 20 min at 23°C in nucleotide-depleting (20 mM Tris-HCl, pH 7.5, 20 mM NaCl, 5% glycerol, 1 mM dithiothreitol, 0.1% Triton X-100) supplemented with 10 mM EDTA. Clarified lysates were prepared from eps8^−/− or −/− [Eps8myc] cells in 20 mM Tris-Cl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1.0 mM PMSF, 10 μg/ml aprotinin, and 10 μg/ml leupeptin. After incubation with total cellular lysates for 3 h at 4°C, beads were washed extensively with nucleotide-depleting buffer. Bound Sos-1 was determined by immunoblotting with anti–Sos-1 antibody.

The levels of Ras-GTP and Rac GTP were measured by affinity precipitation using GST-RBD (Ras-binding domain) of Raf (Taylor and Shalloway, 1996) or GST-CRIB (Cdc42 and Rac Interactive Region) of PAK65 (Manser et al., 1994), respectively, as previously described (Scita et al., 1999). The activity of PAK65 was also measured as described (Manser et al., 1995). Whenever indicated, treatment was performed with EGF (100 ng/ml) or PDGF (20 ng/ml), after serum starvation, for the indicated lengths of time.

In vitro GEF activity toward Ras or Rac was performed as described (Scita et al., 1999). Data are the mean ± SEM of at least three independent experiments performed in triplicate. Results are expressed as the [³H]GDP released after 20 min relative to time 0, after subtracting the background counts released in control reactions (obtained in the presence of a mock immunoprecipitate).

Fibroblasts, seeded on gelatine, were microinjected with 100 ng/ml of an empty vector together with rabbit IgG or of an HA-tagged E3b1 expression vector followed by treatment with 10% serum. After 15 h, BrdU was added, three hours later the cells were fixed in 4% paraformaldheyde for 10 min, permeabilized in 0.1% Triton X-100 and 2% BSA for 10 min, blocked with 2% BSA for 30 min and stained to detect BrdU incorporation with anti-BrdU antibody, E3b1, or control microinjected cells with anti-E3b1 and anti-rabbit IgG, respectively.

We thank Dr. D. Bar-Sagi for the gift of HA-Sos-1; Giuseppina Giardina for technical assistance; Enrica Migliaccio, Pascale Romano, Veronica Raker, and members of the Di Fiore laboratory for helpful discussion. The microinjector Axiovert 100 (ZEISS) was donated by the Lattanzio family.

This work was supported by a grant from Associazione Italiana Ricerca sul Cancro.