INTRODUCTION

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Ras-GRF2 (GRF2) is a widely expressed guanine nucleotide exchange factor (GEF) that stimulates the release of bound guanine nucleotide by the low-molecular-weight G protein Ras (6, 10). GRF2 stimulates the conversion of Ras from its GDP-bound state into a GTP-bound activated conformation. The Ras-binding domain of GRF2, which catalyzes the activation of Ras, is located in its carboxyl-terminal region and is approximately 40% identical to the Ras GEF domain of the Saccharomyces cerevisiae Cdc25 gene product (10). GRF2 is a bifunctional GEF. In addition to its activity on Ras, it binds to the small G protein Rac through its Dbl homology (DH) domain (11).

By virtue of its two distinct GEF activities, GRF2 is a potent activator of two different mitogen-activated protein kinases which function downstream of Rac and Ras (11). They are, respectively, the stress-activated protein kinase (SAPK) and the extracellular signal-regulated protein kinase (ERK). The brain-specific protein Ras-GRF1 (GRF1) has a domain structure similar to that of GRF2 (5, 17), and recently its DH domain was demonstrated to possess Rac GEF activity (14). The Son-of-sevenless gene product (Sos) also has been demonstrated to function as a Rac GEF (16). The frequent coupling of Ras and Rac GEF activities into a single polypeptide may reflect a strict requirement for the coordination of Ras and Rac effector pathways.

GRF2 has not been subjected to three-dimensional structural analysis, but inspection of its primary sequence and functional studies suggest that it is a modular protein composed of discrete functional domains (10, 11). It contains, in amino-to-carboxyl-terminal order, a pleckstrin homology (PH) domain, a coiled-coil, an ilimaquinone (IQ) motif, a DH domain, a second PH domain, a Ras exchanger motif (REM), a PEST region (rich in the amino acids proline, glutamate, serine, and threonine) that contains a candidate destruction box (DB), and, finally, the Cdc25 domain (10). Based on the solved structure of the REM and Cdc25 regions of the Son-of-sevenless (Sos) protein, it is likely the REM and Cdc25 regions of GRF2, and indeed of all Ras GEFs, interact to form a stable Ras-binding domain (3). DH domains, including that of GRF2, are flanked on their COOH sides by a PH domain. In Sos this neighboring PH domain may stabilize the Rac-binding region in the DH domain (18) and may be affected by the lipid products of phosphatidylinositol 3'-kinase (16). Hence, both the Cdc25 and DH classes of the GEF domain are augmented and perhaps regulated by a neighboring noncatalytic domain. However, the intra- and intermolecular interactions involving the various noncatalytic domains in GRF2 have not been determined.

Activation of the SAPK and ERK pathways by GRF2 is stimulated by calcium influx, and this requires the IQ motif of GRF2, which is required to maintain the calcium-dependent binding of calmodulin (CaM) to GRF2 (10, 11). GRF1-mediated activation of ERK is also regulated in a similar fashion (12). However, in contrast to what is shown by the signaling data, GRF1 Ras-specific GEF activity does not appear to be stimulated by the binding of calmodulin in vitro. In fact, some studies have shown that this association inhibits the Cdc25 domain (2). Therefore, the precise role of CaM in the regulation of GRF2 and GRF1 and their CaM-binding site(s) remain to be determined. Activated GRF1 is phosphorylated on serine/threonine (15) and tyrosine (14), suggesting that phosphorylation may play a direct role in the regulation of the GRFs. The recently described Ras-GRP protein is another Ras GEF. It contains calcium and diacylglycerol binding sites and like the other Ras GEFs appears to be regulated by translocation to the plasma membrane (9).

In this study, we addressed structure-function relationships in GRF2. Deleted and truncated versions of GRF2 were constructed and analyzed for their abilities to bind CaM, to activate Ras in vitro and in vivo, and to stimulate the ERK pathway. Our data indicate that CaM binding does not correlate with Ras-ERK activation, which is contrary to the previously held belief that CaM binding activates GRF-mediated signaling. This analysis indicates that GRF2 is a modular protein subject to complex regulatory mechanisms involving its noncatalytic domains.

	MATERIALS AND METHODS

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Construction of Flag-tagged GRF2 deletions. The cloning of IQ, DH, and Cdc25 GRF2 mutants has been previously described (11). The GRF2 PH deletion mutants were generated by PCR. The PHn construct was obtained by deleting GRF2 gene codons 23 to 135 using a 5' primer containing an SpeI site and a 3' primer containing a BamHI site. The PCR product was digested with SpeI and BamHI and subcloned into SpeI-BamHI-digested pcDNA3-Flag-GRF2 cDNA. GRF2 gene codons 460 to 590 were deleted to generate the PHc mutant using a 5' primer containing a BamHI site and a 3' primer containing a KpnI site. The PCR product was digested with BamHI and ApaI and subcloned into BamHI-ApaI-digested pcDNA3-Flag-GRF2. PHn+c, with both PH domains deleted, was obtained by digesting each of the PHn and PHc constructs with BamHI and KpnI and ligating together the 343-bp PHn insert and the 8,010-bp fragment containing pcDNA3-FlagPHc. The REM-Cdc25 construct was generated by PCR amplification of codons 591 to 1189 of the GRF2 gene using a 5' primer containing a KpnI site, a Kozak sequence, and a Flag sequence and a 3' primer containing a stop codon and a BamHI site. The PCR product was digested with KpnI and BamHI and subcloned into KpnI-BamHI-digested pcDNA3. The constructs were confirmed by automated sequencing or enzymatic restriction analysis. The Cdc25 construct was constructed by amplifying nucleotides (nt) 1770 to 2799 by PCR using full-length GRF2 DNA as a template. A stop codon was added after nt 2799 by the addition of the sequence TCA in the 3' primer used for PCR. The PCR product was cloned into the pCR-BLunt II-TOPO PCR vector using the ZeroBlunt TOPO PCR cloning kit (Invitrogen), and the construct was verified by automated sequencing. The nt 1807 to 3570 of full-length pcDNA3-Flag-GRF2 were replaced by nt 1807 to 2799 (plus a stop codon) by digesting the PCR product in pCR-Blunt II-TOPO with EcoRI, and the 1.1-kb fragment was ligated to the 7-kb fragment produced by digestion of pcDNA3-Flag-GRF2 with EcoRI. A variety of restriction digests were performed to verify correct orientation of the 1.1-kb fragment.

Cell culture and transfections. 293T cells were maintained in Dulbecco's modified Eagle medium containing 10% fetal bovine serum, 4.5 g of L-glutamine/liter, 10 µM nonessential amino acids, 100 U of penicillin/ml, and 100 µg of streptomycin/ml. 293T cells grown in 10-cm-diameter dishes were transiently transfected by calcium phosphate precipitation as described previously (19).

In vitro guanine nucleotide exchange assays. 293T cells expressing wild-type (WT) GRF2 or mutant proteins were immunoprecipitated with anti-Flag antibodies as previously described (10). These immunoprecipitates were then used in exchange assays with bacterially purified, recombinant H-Ras as described by Fan et al. (11). For exchange assays measuring the effect of bound CaM, the WT Flag immunoprecipitates were washed in buffer with or without EGTA (1 mM) (to remove CaM) as described previously (10) and then used in exchange assays as described above.

ERK1 in vitro kinase assays. 293T cells were transiently cotransfected with 4 µg of pJ3M-ERK1 (encoding myc epitope-tagged ERK1) and 4 µg of either pcDNA3 vector or pcDNA3-Flag-GRF2 constructs. After 48 h, the cells were starved for 18 h and then stimulated with 5 µM ionomycin for 5 min at 37°C, washed in phosphate-buffered saline (PBS), and then lysed in NP-40 lysis buffer (20 mM Tris-HCl [pH 7.5], 50 mM NaCl, 1% NP-40, 50 mM NaF, 10 mM sodium pyrophosphate, 1 mM sodium orthovanadate, 10 µg of aprotinin/ml, 0.1 mM AEBSF, and 10 µg of leupeptin/ml. Clarified lysates were incubated with 1 µg of 9E10 myc monoclonal antibody precoupled to 20 µl of goat anti-mouse agarose beads (Sigma) for 2 h at 4°C with gentle rotation. The phosphorylation of myelin basic protein (MBP) by immunoprecipitated ERK1 was performed as previously described (10).

CaM binding. 293T cells were transiently transfected with 8 µg of pcDNA3 or pcDNA3-Flag-GRF2 constructs. Forty-eight hours after transfection, the cells were washed in PBS and lysed in NP-40 lysis buffer. The lysates were clarified, precleared with antimouse agarose beads, and then immunoprecipitated with 3 µg of anti-Flag (M2) monoclonal antibody (Kodak) in the presence of 20 µl of anti-mouse agarose beads for 2 h at 4°C with gentle rotation. The immunoprecipitates were washed three times in NP-40 lysis buffer and then resuspended and boiled in 30 µl of Laemmli loading buffer. The samples were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose, and immunoblotted with anti-Flag (M2) or anti-CaM antibodies (Upstate Biotechnology Inc.).

Activated Ras pulldown assay. 293T cells were transiently transfected with 5 µg of pcDNA3 or pcDNA3-Flag-GRF2 constructs. Forty-eight hours after transfection, the cells were washed in HEPES-buffered saline (HBS) and lysed in a solution containing 25 mM HEPES (pH 7.5), 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, 10 mM MgCl₂, 25 mM NaF, 10 µg of aprotinin/ml, 10 µg of leupeptin/ml, 1 mM sodium orthovanadate, and 0.1 mM AEBSF. Levels of activated Ras in the lysate were determined as described previously (8, 11, 19a, 20) by using a glutathione S-transferase (GST) fusion protein containing the Ras-GTP-binding domain of Raf (Upstate Biotechnology Inc.).

	RESULTS

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In vitro and in vivo activity of GRF2 mutants. To investigate the function of the N-terminal region of GRF2, different deletion mutants, including one with a deletion of the entire N terminus, were made (Fig. 1). All these mutant proteins were expressed to similar levels in transiently transfected 293T cells (see below). To determine the effect of deleting different portions of the N terminus on Ras activation, WT GRF2 and the various mutants were compared for their abilities to catalyze in vitro nucleotide exchange of bound GDP for ³²P-labeled GTP on purified, recombinant Ras. Different GRF2 constructs expressed in 293T cells were immunoprecipitated with anti-Flag antibodies. The extensively washed immunoprecipitates were then tested for guanine nucleotide exchange activity in vitro with Ras. Figure 2 shows that all the different deletion mutants retained Ras GEF activity approximately equivalent to that of WT GRF2 with the exception of REM-Cdc25, whose activity was increased approximately 1.5-fold. These results indicate that GRF2 is a modular protein; the amino-terminal domains of GRF2 are not required for in vitro nucleotide exchange on Ras by the Cdc25 domain and may in fact impart a modest inhibitory effect.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   Schematic representation of the domain structures of GRF2 and of the constructs used in this report. Abbreviations are as defined in the text. cc, coiled-coil. The constructs were made using murine GRF2; the genetic sequences for the following mutants are missing the indicated codons: Cdc25, codons 934 to 1189; PHc, codons 460 to 590; PHn, codons 23 to 135; DH, codons 245 to 504; IQ, codons 205 to 229; PHn+c, codons 23 to 135 and codons 460 to 590. The REM-Cdc25 genetic sequence contains codons 591 to 1189.

View larger version (18K): [in this window] [in a new window]   FIG. 2.   In vitro exchange activity of GRF2 and mutants. Assays were carried out 2 days after transfection of 293T cells with the indicated GRF2 construct. GRF2 was immunoprecipitated and used in an in vitro Ras exchange assay as described in Materials and Methods. Each bar shows the mean exchange activity and standard error of at least three experiments (duplicate readings in each experiment).

View larger version (60K): [in this window] [in a new window]   FIG. 3.   GTP-Ras in 293T cells expressing GRF and mutants. Assays were carried out 2 days after transfection of 293T cells with the indicated GRF2 construct. Lysate (1.5 mg) was incubated with 50 µg of GST-Ras-GTP binding domain beads for 30 min to pull down activated Ras. Upper panel, samples were Western blotted for Ras using the LAO45 pan-Ras monoclonal antibody, thereby allowing levels of GTP-bound Ras within the cells to be assessed; lower panel, the presence of GRF2 proteins was verified by Western blotting of lysates with Flag antibody. The data shown are representative of four experiments.

Effect of GRF2 deletion mutants on ERK1 signaling. To investigate whether the N-terminal region of GRF2 played a role in ERK1 signaling, in vitro kinase assays were performed. 293T cells were cotransfected with the GRF2 constructs and myc-tagged ERK1 mitogen-activated protein kinase. After serum deprivation for approximately 18 h, the cells were treated with the calcium ionophore ionomycin or were left untreated. The cells were then lysed immediately, and ERK1 was immunoprecipitated with anti-myc antibodies. The immunoprecipitates were analyzed and quantified for kinase activity using MBP as a substrate (Fig. 4). WT GRF2 and the mutant missing the amino-terminal PH domain (PHn) required stimulation with ionomycin to maximally activate ERK1 (Fig. 4, lanes 3 to 6), whereas the mutants missing the PH domain adjacent to the DH domain (PHc and PHn+c) were maximally activated under basal conditions (Fig. 4, lanes 9 and 10 and 11 and 12, respectively). Therefore, the PH domains of GRF2 are dispensable for ERK1 activation. The REM-Cdc25 protein was a potent activator of ERK1 and was more active (approximately 1.5-fold) than maximally stimulated WT GRF2 (Fig. 4, lanes 13 and 14). Activation of ERK1 by REM-Cdc25 was not stimulated by calcium and was maximal even under conditions of serum deprivation (Fig. 4). Despite its ability to efficiently activate Ras (Fig. 3) (10), the IQ-deleted GRF2 protein (IQ) was defective for basal and calcium-stimulated ERK activation (Fig. 4, lanes 7 and 8).

View larger version (61K): [in this window] [in a new window]   FIG. 4.   ERK1 in vitro kinase assay in 293T cells. Assays were carried out 3 days after transfection of 293T cells with the indicated GRF2 construct and myc-ERK1. Cells were serum starved for 18 h and then either left untreated () or treated with 5 µM ionomycin for 5 min at 37°C (+). Upper panel, Erk activity was determined by precipitating lysates with 9E10 myc antibody, followed by a kinase assay of the immune complex with MBP as the substrate; middle panel, equal expression of myc-ERK1 was confirmed by Western blotting of the anti-myc immunoprecipitates; lower panel, GRF2 protein expression was verified by the Western blotting of the lysates. The data shown are representative of three experiments. Autorad, autoradiography.

View larger version (38K): [in this window] [in a new window]   FIG. 5.   Partial trypsin proteolysis of GRF2 and the three PH deletion mutants. Assays were carried out 2 days after transfection of 293T cells with the indicated construct. GRF2 and the deletion mutants were isolated by anti-Flag immunoprecipitation (IP), and the immune complexes were treated with increasing amounts of trypsin (0, 1, 4, or 10 µg, from left to right) (Worthington Diagnostics) on ice for 15 min. The supernatant containing released fragments was separated by SDS-PAGE and blotted with purified anti-Cdc25 polyclonal antibodies. The data shown are representative of two experiments.

Role of CaM association in GRF2 activity. We reported previously that the association of CaM with GRF2 is dependent on an intact IQ domain and is calcium dependent (10). Since deletion of this domain impaired GRF2-mediated activation of ERK1 but not Ras, we sought to further examine the correlation of the GRF2-CaM complex with GRF2 activity. First, the effect of CaM on the in vitro GEF activity of GRF2 was examined. Second, the association of CaM with the various GRF2 variants was measured.

View larger version (19K): [in this window] [in a new window]   FIG. 6.   In vitro exchange activity of GRF2 with and without CaM. Assays were carried out 2 days after transfection of 293T cells with the indicated GRF2 construct. WT GRF2 immunoprecipitates were washed in buffer without EGTA or with 1 mM EGTA (to remove bound CaM). The washed immune complexes were used in an in vitro Ras exchange assay as for Fig. 2. Each bar shows the mean exchange activity and standard error of two experiments (duplicate readings in each experiment).

View larger version (27K): [in this window] [in a new window]   FIG. 7.   CaM binding to GRF2 or mutants in 293T cells. Assays were carried out 2 days after transfection of 293T cells with the indicated GRF2 construct. GRF2 and mutants were immunoprecipitated from 293T cell lysates using M2 anti-Flag antibodies. The washed immune complexes were Western blotted for CaM.

View larger version (19K): [in this window] [in a new window]   FIG. 8.   Cdc25 protein acts as a codominant inhibitor. Assays were carried out 2 days after transfection of 293T cells with the indicated GRF2 constructs. (a) Ras-GTP levels in 293T cells. Lysate (1.5 mg) was incubated with 50 µg of GST-Ras-GTP binding domain beads for 30 min to pull down activated Ras. Top panel, the presence of GRF2 proteins was verified by Western blotting of lysates with Flag antibody; bottom panel, the samples were Western blotted for Ras, thereby allowing levels of GTP-bound Ras within the cells to be assessed; middle panel, lysates were blotted for Ras to ensure equivalent levels of endogenous Ras between samples. (b) Quantitation of Ras-GTP levels from panel a. The amount of activated Ras was quantitated using a phosphorimager. (c) Ras-GTP levels in 293T cells. Top panel, the presence of GRF2 proteins was verified by Western blotting of lysates with Flag antibody. Lysate (1.5 mg) was incubated with 50 µg of GST-RBD beads for 30 min to pull down activated Ras. Middle panel, lysates were blotted for Ras to ensure equivalent levels of endogenous Ras between samples. Bottom panel, the complexes were Western blotted for Ras, thereby allowing levels of GTP-bound Ras within the cells to be assessed.

	DISCUSSION

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The primary sequence of GRF2 is indicative of a modular, multidomain protein and even suggests some of the protein-protein interactions in which it may participate (6, 10). Indeed, the physical interaction of GRF2 with CaM and Ras is not surprising given its IQ and Cdc25 domains. Furthermore, since the GRF2-CaM interaction is calcium dependent, it has been tempting to conclude that it accounts for the calcium-mediated activation of the ERK pathway by the Ras-GRF proteins. However, the findings in this report indicate that the regulation of GRF2 and its function in the calcium-mediated stimulation of Ras and ERK are complex and are yet to be fully defined.

GRF2 is certainly a modular protein, and its carboxyl-terminal GEF region is remarkably tolerant of the various domain deletions analyzed in this report. The Ras-binding and Ras GEF activities of GRF2 reside in the carboxyl REM-Cdc25 region (10, 11). The PH domains, DH domain, IQ sequence, and associated CaM are not required for these activities and therefore do not obviously contribute to the stabilization of the functional Ras GEF domain. The REM-Cdc25 fragment was consistently up to twice as active as WT GRF2. While we cannot conclude that this difference in activity is biologically significant, it may indicate that GRF2 is capable of modest activation through relief of inhibition caused by intramolecular determinants that lie outside the REM-Cdc25 region. Similar conclusions have also been made for Sos (7).

The in vivo assay of GRF2-induced Ras-GTP formation gave results consistent with those obtained in vitro, indicating that the PH domains, IQ sequence, DB, and DH domain are dispensable for the constitutive activation of Ras which accompanies GRF2 expression in 293T cells. Activation of Ras by the DH mutant was expected since this protein is not diminished in its ability to stimulate ERK (11). This indicates that an ability to interact with Rac through the DH domain is not necessary for Ras-ERK signaling by GRF2. The PH domains of GRF2 are also not essential for GRF2 to access Ras and ERK. Since the PHc mutants were competent for Ras-ERK signaling and activated Ras to a greater extent than WT GRF2 in vivo, we conclude that the REM-Cdc25 portion of GRF2 may be repressed in a manner dependent on the PHc domain in unstimulated cells. The effects of deleting the DH and PHc domains in GRF2 contrast with those observed as a consequence of point mutations in the corresponding domains of GRF1 (13). In addition, deletion of the N-terminal PH domains in GRF2 and GRF1 has very different effects: GRF1 calcium-stimulated ERK activity is abolished, but there is little effect on GRF2 signaling (4) (Fig. 4). For GRF1, the effect of deleting the N-terminal PH domain results in a substantial redistribution of the protein from the particulate to the cytosolic fraction of cells (4). Therefore, this domain appears to be involved in targeting the protein to the membrane and appears to be required for maximal activation of Ras-ERK signaling. WT GRF2, on the other hand, is found predominantly localized in the cytosol of unstimulated cells, and further studies are aimed at identifying what factor(s) enable it to translocate to the cell periphery in response to calcium stimulation (10). Our present findings indicate that the REM-Cdc25 fragment contains the necessary localization determinants to access and activate the Ras-ERK pathway, at least when ectopically expressed in 293T cells.

Activation of Ras and activation of ERK are separable effects of GRF2. A similar resolution of these two activities has been observed for GRF1 (1). Two models, not mutually exclusive, are apparent. One, as proposed by Anborgh et al. (1) for GRF1, suggests that calcium-stimulated ERK activation by GRF1 is Ras independent. We suggest that for GRF2 it is Ras dependent. Both basal and calcium-stimulated modes of ERK activation by GRF2 are fully inhibited by N17 Ras, which targets the Cdc25 domains of Ras GEFs.

The effect of calcium on GRF2 may promote the assembly or proper orientation of a protein network able to couple with the ERK pathway. We have published other evidence in support of such a scaffolding or anchoring role for GRF2 in coupling Rac activation with stimulation of the SAPK pathway in 293T cells (11). In this model, GRF2 and perhaps other GEFs are not simply upstream activators of their target GTPases but may also determine effector interactions. For GRF2, the IQ sequence is required for the interaction of Ras-GTP with effectors ultimately required for activation of ERK. An alternative scheme, which does not exclude this kind of scaffolding model, is that in the absence of the IQ motif, GRF2 is able to bind and activate Ras in a futile manner such that GRF2 and the Ras-GTP produced are not properly localized, anchored, or oriented at the plasma membrane to physically couple to components of the ERK pathway.

Role of CaM in GRF2 regulation. CaM binding to GRF2 is clearly not required for Ras GEF activity in vitro (Fig. 6) or in vivo (Fig. 3). Since both the IQ and DH deletion mutants no longer associate with CaM and because we have not generated a minimal CaM-binding fragment of GRF2, we cannot conclude that the IQ motif is the sole binding site for CaM. Indeed, since the PH mutants and the Cdc25-deleted protein were severely impaired in their association with CaM, it is clear that optimal CaM association with GRF2 requires an intact amino-terminal region of GRF2. In contrast, the association of CaM with GRF1 is not perturbed by similar deletions in the amino terminus (4). We did not detect any interaction of CaM with the REM-Cdc25 protein, but this does not eliminate the possibility that in the context of native GRF2 such an interaction may occur, as suggested for Ras-GRF1 (2).