ACTIVATION OF RAS IN-VITRO AND IN INTACT FIBROBLASTS BY THE VAV GUANINE-NUCLEOTIDE EXCHANGE PROTEIN

We recently identified Vav, the product of the vav proto-oncogene, as a guanine nucleotide exchange factor (GEF) for Ras. Vav is enzymatical ly activated by lymphocyte antigen receptor-coupled protein tryosine k inases or independently by diglycerides. To further evaluate the physi ological role of Vav, we assessed its GDP-GTP exchange activity agains t several Ras-related proteins in vitro and determined whether Vav act ivation in transfected NIH 3T3 fibroblasts correlates with the activit y status of Ras and mitogen-activated protein (MAP) kinases. In vitro translated purified Vav activated by phorbol myristate acetate (PMA) o r phosphorylation with recombinant p56(lck) displayed GEF activity aga inst Ras but not against recombinant RacI, RacII, Ral, or RhoA protein s. Expression of vav or proto-vav in stably transfected NIH 3T3 cells led to a similar to 10-fold increase in basal or PMA-stimulated Ras ex change activity, respectively, in total-cell lysates and Vav immunopre cipitates. Elevated GEF activity was paralleled in each case by a sign ificant increase in the proportion of active GTP-bound Ras. PMA had a minimal effect on low Ras. GTP level in untransfected control fibrobla sts but increased it from 20 to 37% in proto-vav-transfected cells. va v-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, si milarly exhibited increased basal or PMA-stimulated activity in Vav-ex pressing 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.