The hematopoietically expressed product of the vav proto-oncogene, Vav, shared homology with guanine nucleotide releasing factors (GRFs) [also called guanosine diphosphate-dissociation stimulators (GDSs)] that activate Ras-related small guanosine triphosphate (GTP)-binding proteins. Human T cell lysates or Vav immunoprecipitates possessed GRF activity that increased after T cell antigen receptor (TCR)-CD3 triggering; an in vitro-translated Vav fragment that contained the putative GRF domain was also active. Vav-associated GRF stimulation after TCR-CD3 ligation paralleled its tyrosine phosphorylation; both were blocked by a protein tyrosine kinase (PTK) inhibitor. Vav also was a substrate for the p56lck PTK. Thus, Vav is a PTK-regulated GRF that may be important in TCR-CD3-initiated signal transduction through the activation of Ras.

ABSTRACT

We recently identified Vav, the product of the vav proto-oncogene, as a guanine nucleotide exchange factor (GEF) The three Ras proteins (Ha, Ki, and N) belong to the superfamily of small (molecular weight, 20,000 to 29,000) membrane-associated GTP-binding proteins that possess low intrinsic GTPase activity. These proteins cycle between an inactive GDP-bound state, and an active GTP-bound form (8,23,34,48). Recent studies indicated that Ras proteins represent essential intermediates in receptor-mediated growth and differentiation signaling pathways by coupling signals initiated by receptor or nonreceptor protein tyrosine kinases (PTKs) at the cell surface to cytoplasmic targets which coordinate signal transduction to the nucleus (61,71).Ras also participates in T-lymphocyte activation initiated by the antigen-specific T-cell receptor (TCR)-CD3 complex. Receptor ligation triggers a signaling cascade leading to T-cell activation and lymphokine production and proliferation (3,43,56,70 (59,61,71).The rate-limiting step in Ras activation is the exchange of bound GDP for GTP, which is catalyzed by guanine nucleotide exchange factors (GEFs [22,48]). Several yeast-derived exchange factors encoded by the CDC25, SDC25, and ste6 genes have been identified (10,19,37,38). Exchange activity has been described in extracts of human brain and placenta (25,36,72), rat PC12 pheochromocytoma (47), and Rat-1 (11) and murine NIH 3T3 (74) Immunoprecipitation and immunoblotting. Cells were lysed in 50 mM HEPES buffer (pH 7.5) containing 100 mM NaCl, 2% Nonidet P-40 (NP-40), phosphatase inhibitors (10 mM each NaF and Na3VO4), 10 mM EDTA, 10 mM sodium PPi, and protease inhibitors (20 ,ug each of aprotinin and leupeptin per ml). Lysates were subjected to immunoprecipitation (overnight at 4°C) with a rabbit anti-Vav peptide (residues 576 to 589) serum (29,41) or, as a control, normal rabbit Ig; formalin-fixed Staphylococcus aureus (Pansorbin; Calbiochem) was then added for 1 h. The presence of similar cell equivalents in all lysates was confirmed by determining protein concentrations (which varied by less than 10% among samples). Immunoprecipitates were washed five times in 10 mM HEPES (pH 7.5)-100 mM NaCl-2% NP-40-10 mM each NaF and Na3VO4-12 mM MgCl2-protease inhibitors, resuspended in the same buffer containing 1% NP-40 (exchange buffer), and subjected to exchange assays.This buffer was also used to lyse cells when exchange activity in total lysates was measured. For immunoblotting, Vav or MAP kinase immunoprecipitates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE; 7.5% polyacrylamide), transferred to Immobilon-P membranes, immunoblotted with Vav-specific (29) or MAP kinase-specific (see below) antibodies, and detected by using a chemiluminescence kit (ECL reagent; Amersham) and autoradiography.Preparation of affinity-purified Vav by in vitro translation or transient transfection. The human vav proto-oncogene cDNA was subcloned from the pSK115 plasmid (41) into the pTag/CMV-neo vector (5). Correc...