Activation of Ras in Vitro and in Intact Fibroblasts by the Vav Guanine Nucleotide Exchange Proteint ERICH GULBINS,1* K

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Activation of Ras In Vitro and in Intact Fibroblasts by the Vav Guanine Nucleotide Exchange Proteint ERICH GULBINS,1* K. MARK COGGESHALL,lt CLAIRE LANGLET,1 GOTTFRIED BAIER,1 NATHALIE BONNEFOY-BERARD,l PAUL BURN,2 ALFRED WITTINGHOFER,3 SHULAMIT KATZAV,4 AND AMNON ALTMAN' Division of Cell Biology, La Jolla Institute for Allergy and Immunology, La Jolla, Califomia 920371; Department ofBiology, Pharmaceutical Research-New Technologies, F. Hoffinann-LaRoche Ltd., CH-4002 Basel, Switzerland2; Department of Structural Biology, Max Planck Institute for Molecular Physiology, Dortmund, Germany3; and Lady Davis Institute, Jewish General Hospital, Montreal, Quebec H3T 1E2, Canada4 Received 28 September 1993/Returned for modification 18 October 1993/Accepted 2 November 1993

We recently identified Vav, the product of the vav proto-oncogene, as a guanine nucleotide exchange factor (GEF) for Ras. Vav is enzymatically activated by lymphocyte antigen receptor-coupled protein tyrosine kinases or independently by diglycerides. To further evaluate the physiological role ofVav, we assessed its GDP-GTP exchange activity against several Ras-related proteins in vitro and determined whether Vav activation in transfected NIH 3T3 fibroblasts correlates with the activity status of Ras and mitogen-activated protein (MAP) kinases. In vitro translated purified Vav activated by phorbol myristate acetate (PMA) or phosphorylation with recombinant p56kk displayed GEF activity against Ras but not against recombinant RacI, RacHI, Ral, or RhoA proteins. Expression of vav or proto-vav in stably transfected NIH 3T3 cells led to a -10-fold increase in basal or PMA-stimulated Ras exchange activity, respectively, in total-cell lysates and Vav immunoprecipitates. Elevated GEF activity was paralleled in each case by a significant increase in the proportion of active, GTP-bound Ras. PMA had a minimal effect on the low Ras.GTP level in untransfected control fibroblasts but increased it from 20 to 37% in proto-vav-transfected cells. vav-transfected cells displayed a constitutively elevated Ras.GTP level (35%), which was not increased further by PMA treatment. MAP kinases, known downstream intermediates in Ras-dependent signaling pathways, similarly exhibited increased basal or PMA-stimulated activity in Vav-expressing cells by comparison with normal NIH 3T3 cells. These results demonstrate a physiologic interaction between Vav and its target, Ras, leading to MAP kinase activation.

The three Ras proteins (Ha, Ki, and N) belong to the Ha-Ras protein (7, 60) and, conversely, its activation is superfamily of small (molecular weight, 20,000 to 29,000) inhibited by a transdominant inhibitory ras mutant (6, 60). membrane-associated GTP-binding proteins that possess Among the enzymes that are activated in T cells following low intrinsic GTPase activity. These proteins cycle between TCR-CD3 cross-linking are also the Raf-1 kinase (67) and an inactive GDP-bound state, and an active GTP-bound form mitogen-activated protein (MAP) kinase (58), two serine/ (8, 23, 34, 48). Recent studies indicated that Ras proteins threonine kinases that represent Ras-dependent downstream represent essential intermediates in receptor-mediated targets in signaling responses initiated by PTK receptors in growth and differentiation signaling pathways by coupling other cell types (59, 61, 71). signals initiated by receptor or nonreceptor protein tyrosine The rate-limiting step in Ras activation is the exchange of kinases (PTKs) at the cell surface to cytoplasmic targets bound GDP for GTP, which is catalyzed by guanine nucle- which coordinate signal transduction to the nucleus (61, 71). otide exchange factors (GEFs [22, 48]). Several yeast-de- Ras also participates in T-lymphocyte activation initiated rived exchange factors encoded by the CDC25, SDC25, and by the antigen-specific T-cell receptor (TCR)-CD3 complex. ste6 genes have been identified (10, 19, 37, 38). Exchange Receptor ligation triggers a signaling cascade leading to activity has been described in extracts of human brain and T-cell activation and lymphokine production and prolifera- placenta (25, 36, 72), rat PC12 pheochromocytoma (47), and tion (3, 43, 56, 70). Obligatory activation (40, 57) of receptor- Rat-1 (11) and murine NIH 3T3 (74) fibroblasts. This activity coupled PTKs of the Src family and/or ZAP-70/Syk family was stimulated by differentiation (47) or mitogenic (11, 74) (45, 70) is the earliest identifiable event (39) in this pathway. signals. Four recently isolated mammalian gene products, The potential importance of Ras in T-cell activation is i.e., smg.p21.GDS (53), Dbl (35, 62), the brain-specific indicated by several findings: first, T-cell stimulation with Ras-GRF (66), and the ubiquitous Sos (9, 12, 17), possess in ac- mitogenic anti-receptor antibodies or phorbol myristate vitro exchange activity toward Ras or related proteins. etate (PMA) stimulates Ras (24, 28); second, the interleu- Three other proteins, CDC25M, the murine homolog of kin-2 gene promoter is activated in T cells by an oncogenic Ras-GRF (16, 50), and human Bcr (62) and Ect2 (52), are candidate GEFs on the basis of their homology with proven or putative catalytic domains found in other GEFs (22). * Corresponding author. Phone: (619) 558-3500. Fax: (619) 558- 3525. We recently identified Vav, the 95,000-molecular-weight of the vav which is t Publication no. 86 from the La Jolla Institute for Allergy and product proto-oncogene selectively Immunology. expressed in hematopoietic cells (18, 41, 42), as a GEF that * Present address: Department of Microbiology, Ohio State Uni- mediates guanine nucleotide exchange on Ras in vitro (29) versity, Columbus, OH 43210. and whose enzymatic activity is regulated independently by 906 VOL. 14, 1994 Ras ACTIVATION BY Vav 907

TCR-CD3-coupled PTKs (29) or by phorbol esters and (SDS-PAGE; 7.5% polyacrylamide), transferred to Immo- diglycerides (30). Vav shares homology with exchange do- bilon-P membranes, immunoblotted with Vav-specific (29) mains found in several proteins, namely, yeast CDC24, or MAP kinase-specific (see below) antibodies, and detected rodent CDC25Mm or Ras-GRF, and human Dbl, Bcr, and by using a chemiluminescence kit (ECL reagent; Amersham) CDC24Hs (1, 16, 22, 27, 66). Vav was implicated as a and autoradiography. signaling mediator because it contains SH3 and SH2 do- Preparation of affinity-purified Vav by in vitro translation mains that mediate interactions among signaling molecules or transient transfection. The human vav proto-oncogene and formation of multisubunit signaling complexes (15, 44) cDNA was subcloned from the pSK115 plasmid (41) into the and because it is rapidly and transiently phosphorylated on pTag/CMV-neo vector (5). Correct orientation and in-frame tyrosine after cross-linking antigen receptors on T or B cells cloning were confirmed by restriction enzyme digestion and (13, 14, 49), the high-affinity Fc receptor for immunoglobulin nucleotide sequencing of the 5' region, respectively. The vav E (IgE) on mast cells (49), or the c-kit receptor on hemato- plasmid was linearized with SspI, and an aliquot of the poietic stem cells (2). restriction digest was analyzed by electrophoresis in a 1.5% In the present study, we used affinity-purified Vav to agarose gel. The linearized DNA was phenol extracted and characterize in more detail its enzymatic activity toward precipitated with ethanol. A 2-,ug sample of DNA was used several Ras-related proteins and found that it is specific for for in vitro transcription by T7 RNA polymerase (40 U; Ras and, furthermore, that its in vitro exchange activity is Promega) in a buffer containing 50 mM Tris HCl (pH 7.9), inhibited by a dominant inhibitory Ras protein (RasSn-17). In 11 mM dithiothreitol, 0.1 mM EDTA, 100 jig of bovine addition, we investigated the physiological function of Vav serum albumin (BSA) per ml, 5% glycerol, 50 U of RNasin in fibroblasts expressing the products of vav or proto-vav. (Promega) per ml, and 2.5 mM each ribonucleotide. To We demonstrate that the expression and exchange activity increase the efficiency of the subsequent translation, the level of Vav in these cells correlates with the activity status RNA was capped with 5 mM P1-5'-(7-methyl)-guanosine-P3- of Ras and MAP kinases. These results demonstrate the 5'-guanosine triphosphate (Boehringer Mannheim). RNA interaction of Vav with Ras in intact cells, thereby establish- synthesis was performed for 4 h with further addition of T7 ing its physiological role as a Ras-specific GEF in T cells and RNA polymerase after 2 h. Following DNA digestion (15 potentially in other hematopoietic cells. min at 37°C) with 2 U of RQ1 DNase (Promega), the RNA was phenol extracted, ethanol precipitated, and resuspended MATERIALS AND METHODS in water, and an aliquot was analyzed by agarose gel electrophoresis. The RNA was then heated for 10 min at Cell lines and stimulation. K62 is a transformed NIH 3T3 67°C, cooled on ice, and incubated for 2 h at 25°C with a clone stably transfected with a proto-vav expression vector wheat germ extract supplemented with 300 ,uM amino acids under control of a murine sarcoma virus promoter (41). K49 (both from Promega), RNasin (40 U/ml), and potassium is a third-cycle human DNA-transfected clone of NIH 3T3 acetate (0.125 mM). Additionally, Vav was expressed in cells that was used to clone the vav oncogene (40a). Cells COS-1 cells by transient Lipofectin (GIBCO BRL)-mediated were grown in Dulbecco's modified Eagle's medium transfection of 5 x 106 cells with 25 jig of recombinant (GIBCO) supplemented with 10% fetal bovine serum (Irvine pTag/vav or control (pTag/CMV-neo) plasmid DNA. Cells Scientific), 10 mM N-2-hydroxyethylpiperazine-N'-2- were washed 48 h later and lysed in Tris-buffered saline ethanesulfonic acid (HEPES [pH 7.3]), 2 mM L-glutamine, 1 containing 1% NP40 and protease inhibitors. mM sodium pyruvate, 100 p,M nonessential amino acids, 100 To purify Vav, in vitro translation reactions were precip- U of penicillin per ml, 100 ,g of streptomycin per ml, and 50 itated by incubation for 2 h with 25 ,ul of a Ni2"-agarose bead ,M 3-mercaptoethanol. Cells were collected by brief suspension (Qiagen) by following the protocol supplied by trypsinization, washed twice in a 100 mM HEPES (pH the manufacturer. The beads were pelleted by centrifugation 7.3)-saline buffer, resuspended in the same buffer, and and washed six times in Tris-buffered saline containing 20 stimulated with PMA (10 ng/ml; Molecular Probes, Inc.) at mM imidazole, and bound proteins were eluted by increasing 37°C for 5 min. the imidazole concentration to 500 mM. Immunoblotting Immunoprecipitation and immunoblotting. Cells were with anti-Vav or anti-tag antibodies confirmed Vav expres- lysed in 50 mM HEPES buffer (pH 7.5) containing 100 mM sion in the eluate. Aliquots (5 ,ul [5% of total]) of Vav (or NaCl, 2% Nonidet P-40 (NP-40), phosphatase inhibitors (10 control eluate) were added to 145 pl of exchange buffer, and mM each NaF and Na3VO4), 10 mM EDTA, 10 mM sodium Vav was activated by a 10-min incubation at 37°C in the PPi, and protease inhibitors (20 ,ug each of aprotinin and absence or presence of PMA (10 ng/ml) or 200 ng of leupeptin per ml). Lysates were subjected to immunoprecip- recombinant p56'k (4) plus 10 mM Mgz+ and 10 jiM ATP. itation (overnight at 4°C) with a rabbit anti-Vav peptide Purified Vav was then assayed for Ras exchange activity. (residues 576 to 589) serum (29, 41) or, as a control, normal Exchange assay. Ras exchange activity of lysates or Vav rabbit Ig; formalin-fixed Staphylococcus aureus (Pansorbin; immunoprecipitates representing equal cell numbers was Calbiochem) was then added for 1 h. The presence of similar determined as described previously (29). Briefly, recombi- cell equivalents in all lysates was confirmed by determining nant Ha-Ras (0.9 jiM) was incubated for 90 min at 30°C with protein concentrations (which varied by less than 10% 12 jiM [3H]GDP (specific activity, 8 Ci/mmol; ICN) in 25 among samples). Immunoprecipitates were washed five mM Tris. HCl (pH 7.5)-100 mM KCl-0.01% BSA-0.2 mM times in 10 mM HEPES (pH 7.5)-100 mM NaCl-2% NP- dithiothreitol-12 mM EDTA. The [3H]GDP-Ras complex 40-10 mM each NaF and Na3VO4-12 mM MgCl2-protease was stabilized by addition of 22 mM MgCl2, and the mixture inhibitors, resuspended in the same buffer containing 1% was incubated for an additional 10 min. Labeled Ras-GDP NP-40 (exchange buffer), and subjected to exchange assays. complexes were immobilized on nitrocellulose membranes This buffer was also used to lyse cells when exchange (pore size, 0.45 ,im; Costar) by filtration of 30-,il aliquots per activity in total lysates was measured. For immunoblotting, well through a Bio-Dot apparatus (Bio-Rad) and washed with Vav or MAP kinase immunoprecipitates were resolved by 25 mM Tris (pH 7.5)-100 mM KCl-0.2 mM dithiothreitol-12 sodium dodecyl sulfate-polyacrylamide gel electrophoresis mM MgCl2. In a typical experiment, -100,000 cpm was 908 GULBINS ET AL. MOL. CELL. BIOL. bound to each filter piece. From the specific activity of Vav and anti-tag antibodies and its lack in control prepara- [3H]GDP and the concentration of Ras, and assuming stoi- tions were verified by immunoblotting (results not shown). chiometric [3H]GDP binding to Ras, this represents -12 The activity of purified Vav was assayed by measuring the pmol of bound Ras-GDP complex. The exchange reaction release of [3H]GDP from nitrocellulose-immobilized, was initiated by adding the filter pieces (4 by 4 mm) to 150 ,ul [3HTGDP-loaded Ras (29). This assay correlates well with of exchange assay buffer (29, 30) containing resuspended [a-3 P]GTP binding to immobilized or soluble Ras.GDP Vav immunoprecipitates or purified Vav plus 50 p,M GTP. complexes (30). Samples were incubated at 37°C, and aliquots were removed Unstimulated Vav had a low [3H]GDP-releasing activity at the indicated time points for liquid scintillation counting to above the nonspecific release, consistent with our findings determine the amount of released [3H]GDP. that Vav immunoprecipitates from resting cells possess low Ras activation. Washed cells were resuspended in phos- exchange activity (29) (see Fig. 4). The release of [3H]GDP phate-free Dulbecco's modified Eagle's medium containing in the presence of PMIA-treated Vav rose rapidly during the 10% dialyzed fetal bovine serum, labeled for 12 h with 1 mCi first 1 min of the assay and continued to increase up to 10 of 32Pi per ml, and activated with PMA as indicated. Cells min, the assay end point (Fig. 1A). At this time point, 28 and were lysed in 25 mM Tris HCl (pH 8.0)-1% NP-40-10 mM 65% of the filter-bound [3H]GDP at to (-100,000 cpm or -12 MgCl2-500 mM NaCl-0.5% sodium deoxycholate-protease pmol) was released from Ras by unstimulated or PMA- inhibitors. Nuclei were removed by centrifugation, and Ras stimulated purified Vav, respectively. These characteristics was immunoprecipitated by addition of rabbit anti-rat immu- compare favorably with the solution phase [3H]GDP release noglobulin-conjugated agarose beads coated with a rat anti- assay used by others (17, 35, 66). The more rapid increase in Ras monoclonal antibody (Y13-259; Oncogene Science, Inc.) background (nonspecific) [3H]GDP release made it difficult for 1 h at 4°C. Precipitates were washed seven times with to measure exchange activity reliably at later times. The lysis buffer, and labeled Ras-bound nucleotides were eluted validity of the [3H]GDP filter release assay as a true mea- by incubation for 20 min at 25°C in 200 ,ul of 10 mM EDTA surement of Ras exchange activity is further indicated by the containing 25 ,uM each unlabeled GDP and GTP. The findings that only <7% of the radioactivity released from the supernatant was analyzed by high-pressure liquid chroma- filter in the presence of PMA-stimulated Vav was retained tography on HPLC an ion-exchange column (10 p,m silica; upon subsequent filtration through nitrocellulose (Fig. 1A, Vydac) run in a linear phosphate buffer gradient. GDP and inset), clearly demonstrating that it represents free GDP. GTP peaks were detected by measurement of A268 and The specific exchange activity of Vav toward Ras in this comparison with nucleotide standards, and labeled nucle- assay was further evaluated by examining the effect of a otides were determined by liquid scintillation counting, dominant negative Ras protein, Ras"-17, on the exchange which was then used to calculate the GTP/(GDP + GTP) reaction (Fig. 1B). This mutated Ras protein can block ratio. transformation and/or PTK receptor-dependent downstream MAP kinase assay. MAP kinases were immunoprecipitated signaling events (26, 68, 69, 73) and was recently found to (overnight at 4°C) from fibroblast lysates normalized to the effectively interfere with the Ras exchange activity of an- same protein concentration with 3 ,ug of anti-MAP kinase other GEF, i.e., mammalian Sos (12). In agreement with monoclonal antibody (Zymed) reactive with both Erkl and these findings, Ras"n17 blocked the exchange activity of Erk2 followed by agarose-conjugated goat anti-mouse Ig. PMA-stimulated purified Vav when added in solution at a The precipitates were washed four times in lysis buffer, -20:1 molar ratio relative to the filter-immobilized wild-type twice in Tris-buffered saline, and once in 25 mM HEPES (pH Ras (Fig. 1B). Addition of the same amount of soluble 7.4)-2 mM dithiothreitol-10 mM MgCl2 (kinase buffer). wild-type Ras had no inhibitory effect. Samples were resuspended in 32 pul of kinase buffer contain- Specificity of the in vitro exchange activity of Vav. Previous ing 20 ,ug of myelin basic protein (Sigma), and the reaction experiments demonstrated that Vav is activated both in was initiated by adding 10 ,uM [y- 2P]ATP (40 Ci/mmol; intact cells and in vitro by two independent mechanisms, Amersham). The samples were incubated at 30°C for 30 min, i.e., by tyrosine phosphorylation (29) or by treatment with the reaction was stopped with 8 ,ul of reducing 5 x SDS phorbol esters and diglycerides, such as diacylglycerol and sample buffer, and SDS-PAGE (12% polyacrylamide) and ceramide (30). To test the specificity of activated Vav, autoradiography were performed. several recombinant Ras-related small GTP-binding proteins in addition to Ha-Ras itself, i.e., RacI, RacIl, RhoA, and Ral, were tested as substrates for in vitro translated, purified RESULTS Vav following in vitro stimulation by either PMA or recom- Characteristics of the in vitro exchange activity of Vav. We binant p561ck. The basal activity of untreated purified Vav found recently that the enzymatic activity of Vav is stimu- against Ras was negligible. PMA- or p56lck-treated Vav, on lated by PMA via its cysteine-rich domain (30). Here, the other hand, displayed a -15-fold increase in Ras-di- PMA-stimulated Vav was subjected to a detailed kinetic rected exchange activity (Fig. 2). In contrast, the exchange analysis of its exchange activity on Ras. Affinity-purified activity of activated Vav against RhoA, Ral, RacI, or RaclI Vav generated by transient COS-1 cell transfection or in was barely above background levels. These results indicate vitro translation (see below) was used to demonstrate in a that Vav is a relatively Ras-specific GEF. rigorous manner that the observed effects represent direct GEF activity in Vav-expressing NIH 3T3 fibroblasts. To catalytic activity of Vav. The vav proto-oncogene cDNA establish the physiological relevance of the Ras-specific in was subcloned into pTag/CMV-neo, a modified pRc/CMV vitro exchange activity of Vav, we carried out a functional vector encoding tag epitopes that allow affinity purification analysis of vav- or proto-vav-transfected NIH 3T3 cells (41). and immunodetection of expressed proteins (5). This vector In contrast to T cells (24, 28), PMA treatment does not is suitable for in vitro transcription-translation or transient activate Ras in fibroblasts (21, 54, 55, 64). Thus, expression COS-1 cell transfection driven by the T7 or cytomegalovirus of the PMA-regulated Vav in NIH 3T3 cells enabled us to promoters, respectively. The presence of the corresponding compare the effects of PMA treatment on GEF and Ras 100,000-molecular-weight protein reactive with both anti- activities and to establish a potential cause-and-effect rela- VOL. 14, 1994 Ras ACf1VATION BY Vav 909

A Stimulus Protein EMA LiK 7/0TIP - 7/727, Ras + - FRI

Ral - + E 0

01 RhoA- + - Cl0 0 U + 0. Racl1 a a Rac2[ +

I 0 1000 2000 [3H]GDP Release (cpm) 0 2 4 6 8 10 12 FIG. 2. Specificity of the exchange activity of Vav. Aliquots (5 Time (min) pl) of affinity-purified, in vitro translated unstimulated Vav or Vav B treated for 10 min with PMA or recombinant p56kck were assayed against recombinant Ha-Ras or the other indicated proteins. Stimulation Ras [3H]GDP release was determined after 1 min. Similar results were obtained in three other experiments, including those with immunoprecipitated Vav from stimulated T cells. 0 N17

PMA N17 displayed an elevated basal exchange activity similar to that detected in their PMA-stimulated, proto-vav-transfected counterparts. This increased activity was not further ele- PMA wt vated by PMA treatment (Fig. 3). Similar results were obtained when the exchange activity of Vav immunoprecip- 1000ooo00 2000 3000 4000 5000 60060C 00 itates from these cells was analyzed (Fig. 4): proto-vav transfectants displayed low but PMA-stimulatable Vav ex- [3 HJGDP Release (cpm) change activity, whereas Vav immunoprecipitates from vav- FIG. 1. (A) PMA-stimulated Ras exchange activity of purified transfected fibroblasts displayed a constitutively elevated, Vav. Untreated or PMA-treated COS-1 cell-derived affinity-purified PMA-unresponsive GEF activity. These differences were Vav was subjected to a filter [3H]GDP release assay, and 30-ml seen even though K62 and K49 cells expressed similar aliquots were removed for liquid scintillation counting at the indi- amounts of the proto-vav or vav gene products, as evidenced cated times. Counts were multiplied by 5 to correct for the total by immunoblotting the immunoprecipitates with the Vav- reaction volume (150 pIl). Nonspecific release was determined in the specific antiserum (results not shown). The entire PMA- presence of a similarly purified preparation derived from COS-1 induced exchange activity from proto-Vav-expressing cell cells transfected with a control vector lacking vav cDNA (0). This lysates was immunodepleted by anti-Vav antibodies (Fig. 4), release did not differ significantly from that of a buffer control lacking any cellular material (reference 29 and results not shown). At to, 100,000 + 6,000 cpm (-12 pmol) of [3H]GDP was bound to each filter. The experiment was repeated twice with similar results. NIH 3T3 In the inset, an aliquot of the exchange reaction mixture was PMA Stim. cells

removed after 2 min to count [3H]GDP release from the filter - control (hatched bar). A similar aliquot was filtered through another nitrocellulose membrane, which was washed and counted (solid bar). (B) Inhibition of the exchange activity of PMA-stimulated Vav by a + control negative dominant Rasln'17 protein. In vitro translated purified Vav was incubated for 1 min with 270 pmol of recombinant Ras'n-17 or - onco-Vav wild-type Ras (representing a -20-fold excess over the filter- immobilized, [3H]GDP-bound Ras) prior to the exchange assay. + onco-Vav Similar results were obtained in another experiment. proto-Vav

tionship between the two. We therefore tested the exchange + proto-Vav activity in whole-cell lysates and Vav immunoprecipitates from control or transfected cells. Lysates of control fibro- 0 1000 2000 304100 blasts displayed low exchange activity, which did not in- [3H]GDP release (cpm) crease after PMA treatment. In contrast, lysates of proto- FIG. 3. Exchange activity in lysates of control or Vav-express- vav-transfected fibroblasts had a ca. twofold-higher basal ing NIH 3T3 fibroblasts. Cells were left untreated or stimulated for exchange activity, consistent with their high-level Vav ex- 5 min with 10 ng of PMA per ml prior to lysis. Total-cell lysates (106 pression (41), which was stimulated ca. eightfold by PMA cell equivalents) were assayed for their Ras exchange activity in a (Fig. 3). vav-transfected fibroblasts, on the other hand, 1-min assay as described in Materials and Methods. 910 GULBINS ET AL. MOL. CELL. BIOL.

NIH 3T3 PMA - PMA stim. cells

- control MBP-* *- + control onco-Vav normal proto-vav onco-vav + onco-Vav FIG. 6. Constitutive and PMA-induced MAP kinase activity in proto-Vav control, proto-vav-, or vav-transfected NIH 3T3 fibroblasts. MAP kinase immunoprecipitates were assayed for myelin basic protein + proto-Vav (MBP) phosphorylation. Where indicated, the cells were stimulated proto-Vav for 5 min with 0 ng PMA per ml. The position of the substrate + (sup) (myelin basic protein) is indicated by the arrow. Similar results were two 0 2000 4000 6000 8000 obtained in additional experiments. [3H]GDP release (cpm) FIG. 4. Exchange activity of Vav immunoprecipitates from con- an increased Ras-GTP level (21%), consistent with the trol, proto-vav-, or vav-transfected NIH 3T3 fibroblasts. Vav immu- transformed phenotype of these cells (41). Nevertheless, noprecipitates (5 x 106 cell equivalents per group) from untreated PMA treatment caused a further increase in the amount of cells or PMA-stimulated cells were used to measure GEF activity (hatched bars). The supernatant remaining after Vav immunopre- active Ras to 35% (Fig. 5). The vav-transfected cells, on the cipitation from PMA-stimulated, proto-Vav-expressing cells (solid other hand, displayed a high basal activity of Ras (32% bar) was similarly assayed for its Ras exchange activity. Ras.GTP), which was not elevated significantly by PMA (Fig. 5). These results parallel the measurements of total or Vav-associated exchange activities in the same cells (Fig. 3 confirming that the cells do not express an endogenous and 4) and strongly suggest that increased Vav exchange PMA-sensitive GEF. activity is the cause of the elevated levels of GTP-bound Ras and MAP kinase activities in vav-transfected NIH 3T3 Ras. cells. Assuming that Vav represents a physiological Ras MAP kinases represent essential downstream intermedi- activator, conditions associated with elevated GEF activity ates in Ras-dependent signaling pathways (59). We wished to of Vav should also lead to activation of Ras and, possibly, determine, therefore, whether Vav expression and increased additional Ras-dependent intermediates that participate in Ras activity in the vav-transfected fibroblasts correlate with signal transduction pathways. We therefore determined the elevated MAP kinase activity. The enzymatic activity of activation status of Ras in control or Vav-expressing NIH MAP kinase immunoprecipitates from normal or transfected 3T3 cells (Fig. 5). A low level (9%) of active, GTP-bound Ras fibroblasts was determined in immune complex kinase as- was detected in untransfected control fibroblasts; PMA says by using myelin basic protein as a substrate. Unstimu- treatment caused a small increase, i.e., to 14% Ras.GTP, lated proto-Vav-expressing fibroblasts displayed increased which was not seen in another experiment (results not MAP kinase activity, which was more pronounced (i.e., ca. Stable of was with twofold higher) in vav-transfected cells. MAP kinase activity shown). expression proto-vav associated was stimulated by PMA in all three cell types, but the overall PMA-stimulated activity in cells expressing the vav or proto- NIH 3T3 vav products was ca. twofold higher than in normal, PMA- PMA stim. cells treated NIH 3T3 cells (Fig. 6). Immunoblotting of the same filters with anti-MAP kinase (Erkl and Erk2) antibodies - control indicated similar expression levels of these kinases in the different cell lines (results not shown). The ability of PMA to + control increase MAP kinase, but not Ras, activity in the onco-Vav- expressing fibroblasts is not surprising since MAP kinases - onco-Vav (20), as well as the Raf-1 kinase (46), which acts between Ras and MAP kinases in the same pathway, can also be activated + onco-Vav in fibroblasts by PMA via a Ras-independent, protein kinase i- C (PKC)-mediated pathway (59). proto-Vav DISCUSSION

+ proto-Vav Our studies identified Vav as a PTK-regulated Ras GEF i o 20 30 activated by several lymphocyte receptors known to be GTP/GDP+GTP (%) coupled to PTK activation, i.e., the TCR-CD3 (29) or interleukin-2 (32) receptors in T cells or surface Ig in B cells FIG. 5. Ras activation in control versus Vav-expressing NIH (31). More recently, we defined an alternative, PTK-inde- 3T3 cells. The bars show the percentage of [32P]GTP in total labeled pendent Vav activation pathway mediated by phorbol esters guanine nucleotides (GDP + GTP) present in Ras immunoprecipi- or diglycerides (30). Depletion of the majority of antigen tates from untreated NIH 3T3 fibroblasts or cells stimulated with from PMA for 5 min. Background counts eluted from control, normal receptor-stimulated Ras exchange activity lymphocyte rabbit Ig immunoprecipitates (<80 cpm, representing <15% of lysates by anti-Vav antibodies (29, 31) suggested that Vav specific counts) were subtracted from all samples. Each experiment may be a major contributor to Ras-stimulating GEF activity was repeated at least twice. under these conditions. Since, however, GDP-GTP ex- VOL. 14, 1994 Ras ACTIVATION BY Vav 911

change on Ras was assayed in vitro, it was important to GRF in the brain (66) or with Vav in hematopoietic cells (18, determine whether Vav functions as a physiological Ras- 29, 41, 42) indicates a certain redundancy in the expression specific GEF in the context of intact cells. of Ras exchange proteins. Such redundancy is shared by Several lines of evidence indicate that Vav itself mediates other signaling molecules, e.g., Ras proteins themselves a bona fide exchange activity on Ras. They include the use of (Ha, Ki, and N), Src family PTKs, and PKC isoenzymes, affinity-purified Vav, the kinetic and quantitative analysis of and may be important for cell-specific responses, coupling to Vav exchange activity and the demonstration that our assay distinct coexpressed receptors, and/or fine tuning of cellular measures the release of free [3H]GDP (Fig. 1A), the corre- signal transduction pathways. Vav may couple predomi- lation of the [3H]GDP filter release assay with measurements nantly PTK-independent hematopoietic cell receptors which of [a-32P]GTP binding to Ras.GDP complexes in solution induce diglyceride synthesis via stimulation of phospho- (30) used by others (12, 17, 35, 66), and, finally, the finding lipase C (63) and/or sphingomyelinase (51, 65) to the Ras that GEF activity of purified Vav is blocked by the trans- pathway. This notion is supported by our finding that Vav is dominant inhibitory Ras"-17 protein (Fig. 1B). RasIn'17 activated in intact cells by interleukin-1, or in vitro by can block transformation and PTK receptor-dependent ceramide (30), an interleukin-1-induced second messenger downstream signaling events (26, 68, 69, 73) and was re- (51). Other, PTK-dependent receptors, e.g., antigen or lym- cently found to effectively interfere with the Ras exchange phokine receptors (29, 31, 32), may activate Vav predomi- activity of another mammalian GEF, i.e., Sos (12), presum- nantly by tyrosine phosphorylation. Thus, the pathway of ably by competing with endogenous Ras for binding to GEF Vav, and potentially Ras, activation may be dictated by the proteins. Furthermore, the inability of Vav to catalyze activated receptor. Our findings documenting the specific guanine nucleotide exchange on four other small GTP- action of Vav on Ras both in vitro and in intact cells open the binding proteins (Fig. 2) indicates that Vav is a Ras-specific way for additional studies that will clarify the function and GEF. regulation of this hematopoietic GEF in growth and differ- Analysis of vav- or proto-vav-transfected and control entiation signaling pathways. fibroblasts demonstrated a correlation between total and Vav-associated GEF activity on one hand and Ras and MAP ACKNOWLEDGMENTS kinase activities on the other in intact NIH 3T3 cells. The of Vav to interact with We thank L. Giampa and E. Herbert for their dedicated technical ability autophosphorylated epidermal assistance, L. Feig for Ral and RhoA proteins, G. Bokoch for Racl or platelet-derived growth factor receptors on NIH 3T3 cells and Rac2 proteins, and T. Mustelin and T. Kawakami for critical in an SH2-dependent manner and to undergo tyrosine phos- reading of the manuscript. phorylation (14, 49), as well as the similar PMA-mediated This work was supported in part by NIH grant CA35299, the activation of Vav in T cells (30) and fibroblasts (Fig. 4), National Cancer Institute of Canada with funds from the Canadian suggests that the observed functional effects of Vav in NIH Cancer Society, Gemini Science, a German National Scholarship 3T3 cells, which do not normally express this protein, are Foundation/BASF fellowship (E.G.), and an ARC fellowship (N.B.- physiologically relevant. Therefore, our results lend strong B.). support to the notion that Vav is a physiological Ras activator and that Vav activation can lead not only to Ras ADDENDUM IN PROOF stimulation but also to activation of a known Ras-dependent signal transducer, i.e., MAP kinase. 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