Rho and Rac family members participate in coordinated cellular responses to extracellular stimuli (11,28). Their action is important in promoting the formation of cytoskeletal structures, the activation of serine/threonine kinase cascades, and the induction of gene expression (11,28). Rho proteins are regulated by the binding of guanosine nucleotides (1). In quiescent cells, these GTPases are bound to GDP molecules and are in an inactive state. Stimulation of cells via a number of extracellular stimuli leads to the exchange of GDP by GTP, a transition that allows the acquisition of a conformation optimal for the binding to their effector molecules (1). Because the intrinsic GDP-GTP exchange rate of these GTPases is low under physiological conditions, the activation of these proteins during signal transduction requires the participation of enzymes generically known as guanosine nucleotide exchange factors (GEFs) (1). To date, two families of Rho GEFs have been identified. The first group is composed of Rho GDP dissociation stimulators, a family of proteins distantly related to the Cdc25 homology regions present in Ras GEFs (1). The second group comprises an extensive number of enzymes containing Dbl homology (DH) domains with catalytic activity exclusively directed towards Rho and Rac GTPases (4).Although Rho GEFs have been extensively characterized biochemically and oncogenically, little information is available regarding the mechanism by which they become activated during signal transduction. To date, the best example for the participation of a DH-containing protein in receptor-mediated cell signaling is perhaps the product of the vav proto-oncogene, a protein preferentially expressed in the hematopoietic system (2). In addition to the DH and Pleckstrin homology (PH) regions commonly found in Rho and Rac GEFs, Vav contains other structural motifs, including a calponin homology (CH) region, an acidic (Ac) motif, a zinc finger (ZF) domain, two SH3 regions, and one SH2 domain (2). Vav becomes tyrosinephosphorylated during the signaling of many membrane receptors, and binds to a number of cytoplasmic molecules via its SH2 and SH3 domains (2). Recently, biochemical experiments have demonstrated that the phosphorylation of Vav on tyrosine residues leads to the activation of its GDP/GTP exchange activity towards Rac-1 in vitro (7). In agreement with such observations, it has been shown that several elements of the Rac-1 pathway, including Rac-1 itself and JNK, are activated in vivo by wild type Vav protein upon tyrosine phosphorylation (7,26). Deletion of vav via gene targeting leads to decreased proliferation of prothymocytes (31), to defective positive and negative selection of immature T cells (15,27) and to ineffective functional responses of mature T and B cells (25, 31). In T lymphocytes, this phenotype is linked to abnormal actin clustering upon receptor engagement (10,14). Vav appears to provide therefore a direct connection between membrane receptors and Rac-1, a pathway that is essential for the generation ...

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Vav works as a GDP/GTP exchange factor for Rac GTPases, thereby facilitating the transition of these proteins from the inactive (GDP-bound) into the active (GTP-bound) state. The stimulation of Vav exchange activity during cell signaling is mediated by tyrosine phosphorylation. To understand the roles of phosphorylation in the regulation of Vav activity, we have initiated the characterization of the residues of Vav that are phosphorylated during signal transduction. Here we show that a Y-to-F mutation in one of these residues, Y174, leads to the oncogenic activation of Vav and to the enhancement of other Vav-mediated signals such as those for cytoskeletal reorganization, JNK activation, and stimulation of the nuclear factor of activated T cells. The effect induced by the Y174F mutation is further accentuated by mutations in residue Y142 or Y160. The Y174F mutation has no effect on the exchange activity of Vav in vitro but results in higher levels of phosphorylation in vivo. Using a phosphospecific antibody, we found that Y174 is phosphorylated following stimulation of mitogenic and antigenic receptors. This phosphorylation event is conserved in Vav-2 and Vav-3, the other two members of the Vav family. These results identify a previously unknown mechanism for the oncogenic activation of Vav and suggest that the activity of this exchange factor is modulated by two antagonistic phosphorylation events, one involved in Vav activation and a second one implicated in Vav inactivation.The Vav family is a novel group of signal transduction molecules with important roles in cell signaling and tumorigenesis (3). This family has four known members, three distributed in mammalian cells (Vav, Vav-2, and Vav-3) and one present in Caenorhabditis elegans (CelVav) (3, 23). Vav proteins are composed of seven different structural domains, including a calponin homology (CH) region, an acidic (Ac) domain, Dbl homology (DH) and pleckstrin homology (PH) regions, one zinc finger butterfly (ZF), and two SH3 regions flanking a single SH2 domain (3). The SH3 regions are not conserved in CelVav (31). At the biochemical level, Vav proteins promote the exchange of guanosine nucleotides in GTP-binding proteins of the Rho/Rac family, an action that facilitates the transition of these GTPases from their inactive (GDP-bound) to their active (GTP-bound) state (11,13,23,27). Activation of these GTPbinding proteins leads in turn to both cytoskeletal and mitogenic changes in the cell, as demonstrated by the ability of Vav proteins to induce membrane ruffles and lamellipodia (24, 27), to activate JNK-1 (10, 11), and to stimulate Rho/Rac-responsive transcriptional factors such as the nuclear factor of activated T cells (NF-AT) and 22,32). Despite their similar structures and biochemical activities, Vav family proteins show significant regulatory differences. Thus, while Vav is active preferentially on Rac-1, Rac-2, and RhoG, Vav-2 and Vav-3 target mainly RhoG and RhoA-like proteins (11,23,27). Moreover, whereas Vav is restricted to hematopoietic cells (...