RAC-1 DEPENDENT STIMULATION OF THE JNK SAPK SIGNALING PATHWAY BY VAV/

The protein product of the human vav oncogene, Vav, exhibits a number of structural motifs suggestive of a role in signal transduction pathw ays, including a leucine-rich region, a plekstrin homology (PH) domain , a cysteine-rich domain, two SH3 regions, an SH2 domain, and a centra l Dbl homology (DH) domain. However, the transforming pathway(s) activ ated by Vav has not yet been elucidated. Interestingly, DH domains are frequently found in guanine nucleotide-exchange factors for small GTP -binding proteins of the Ras and Rho families, and it has been recentl y shown that, whereas Ras controls the activation of mitogen activated kinases (MAPKs), two members of the Rho family of small GTPases, Rac 1 and Cdc42, regulate activity of stress activated protein kinases (SA PKs), also termed c-jun N-terminal kinases (JNKs). The structural simi larity between Vav and other guanine nucleotide exchange factors for s mall GTP-binding proteins, together with the recent identification of biochemical routes specific for members of the Ras and Rho family of G TPases, prompted us to explore whether MAPK or JNK are downstream comp onents of the Vav signaling pathways. Using the COS-7 cell transient e xpression system, we have found that neither Vav nor the product of th e I,av proto-oncogene, proto-Vav, can enhance the enzymatic activity o f a coexpressed, epitope tagged MAPK. On the other hand, we have obser ved that, whereas proto-Vav can slightly elevate JNK/SAPK activity, on cogenic Vav potently activates JNK/SAPK to an extent comparable to tha t elicited by two guanine-nucleotide exchange factors for Rho family m embers, Dbl and Ost. We also show that point mutations in conserved re sidues within the cysteine rich and DH domains of Vav both prevent its ability to activate JNK/SAPK and render Vav oncogenically inactive. I n addition, we found that coexpression of the Rac-1 N17 dominant inhib itory mutant dramatically diminishes JNK/SAPK stimulation by Vav, as w ell as reduces the focus-forming ability of Vav in NIH3T3 murine fibro blasts. Taken together, these findings provide the first evidence that Rac-1 and JNK are integral components of the Vav signaling pathway.