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 ...

Several proteins implicated in the regulation of cellular responses to mitogenic stimuli contain a common non-catalytic domain, SH2 (for src-homologous domain 2), that mediates their interaction with activated tyrosine protein kinases. Here we report that p95vav, a proto-oncogene product specifically expressed in cells of the haematopoietic system, contains an SH2 domain and is a substrate for tyrosine protein kinases. Exposure of quiescent NIH3T3 cells ectopically expressing p95vav to either epidermal or platelet-derived growth factors induces the rapid phosphorylation of this protein on tyrosine residues. Activation of the receptors for these growth factors by their cognate ligand results in their association with p95vav, a process mediated by its SH2 domain. In T cells, co-activation of the T-cell receptor and the accessory CD4 cell-surface protein also results in the phosphorylation of the endogenous p95vav protein in tyrosine residues. Phosphorylation of p95vav is rapid, transient and precedes the appearance of most other phosphotyrosine-containing proteins. In addition to the SH2 domain, p95vav contains structural motifs not found in other tyrosine kinase substrates. One such motif is a helix-loop-helix/leucine zipper-like domain which shares some sequence similarity with these motifs in the Myc and Max proteins. Deletion of the helix-loop-helix-like motif causes oncogenic activation of p95vav. These results indicate that p95vav is a new type of signal transduction molecule and suggest a possible role for this protein in the transduction of tyrosine phosphorylation signalling into transcriptional events.

We have shown recently that the azathioprine metabolite 6-Thio-GTP causes immunosuppression by blockade of GTPase activation in T lymphocytes. In the present study, we describe a new molecular mechanism by which 6-Thio-GTP blocks GTPase activation. Although 6-Thio-GTP could bind to various small GTPases, it specifically blocked activation of Rac1 and Rac2 but not of closely related Rho family members such as Cdc42 and RhoA in primary T cells upon stimulation with αCD28 or fibronectin. Binding of 6-Thio-GTP to Rac1 did not suppress Rac effector coupling directly but blocked Vav1 exchange activity upon 6-Thio-GTP hydrolysis, suggesting that 6-Thio-GTP loading leads to accumulation of 6-Thio-GDP-loaded, inactive Rac proteins over time by inhibiting Vav activity. In the absence of apoptosis, blockade of Vav-mediated Rac1 activation led to a blockade of ezrin-radixin-moesin dephosphorylation in primary T cells and suppression of T cell-APC conjugation. Azathioprine-generated 6-Thio-GTP thus prevents the development of an effective immune response via blockade of Vav activity on Rac proteins. These findings provide novel insights into the immunosuppressive effects of azathioprine and suggest that antagonists of the Vav-Rac signaling pathway may be useful for suppression of T cell-dependent pathogenic immune responses.