DIRECT STIMULATION OF VAV GUANINE-NUCLEOTIDE EXCHANGE ACTIVITY FOR RAS BY PHORBOL ESTERS AND DIGLYCERIDES

We recently identified Vav as a Ras-activating guanine nucleotide exch ange factor (GEF) stimulated by a T-cell antigen receptor-coupled prot ein tyrosine kinase (PTK). Here, we describe a novel, protein kinase-i ndependent alternative pathway of Vav activation. Phorbol ester, 1,2-d iacylglycerol, or ceramide treatment of intact T cells, Vav immunoprec ipitates, or partially purified Vav generated by in vitro translation or COS-1 cell transfection stimulated the Ras exchange activity of Vav in the absence of detectable tyrosine phosphorylation. GEF activity o f gel-purified Vav was similarly stimulated by phorbol myristate aceta te (PMA). Stimulation was resistant to PTK and protein kinase C inhibi tors but was blocked by calphostin, a PMA and diacylglycerol antagonis t. In vitro-translated Vav lacking its cysteine-rich domain, or mutate d at a single cysteine residue within this domain (C528A), was not sti mulated by PMA but was fully activated by p56(lck). This correlated wi th increased binding of radiolabeled phorbol ester to COS-1 cells expr essing wild-type, but not C528A-mutated, Vav. Thus, Vav itself is a PM A-binding and -activated Ras GEF. Recombinant interleukin-1 alpha stim ulated Vav via this pathway, suggesting that diglyceride-mediated Vav activation may couple PTK-independent receptors which stimulate produc tion of lipid Second messengers to Ras in hematopoietic cells.