MOLECULAR AND CELLULAR BIOLOGY, Feb. 1994, p. 906-913 Vol. 14, No. 2 0270-7306/94/$04.00+0 Copyright © 1994, American Society for Microbiology 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.