We have used an extensive mutagenesis approach to study the specific role of the eight structural domains of Vav during both the activation and signaling steps of this Rac1 exchange factor. Our results indicate that several Vav domains (Dbl homology, pleckstrin homology, and zinc finger) are essential for all the biological activities tested, whereas others are required for discrete, cell type-specific biological effects. Interestingly, we have found that Vav domains have no unique functions. Thus, the calponin homology domain mediates the inhibition of Vav both in vitro and in vivo but, at the same time, exerts effector functions in lymphocytes upon receptor activation. The Vav SH2 and SH3 regions play regulatory roles in the activation of Vav in fibroblasts, mediating both its phosphorylation and translocation to the plasma membrane. In contrast, the Vav SH2 and SH3 regions act as scaffolding platforms in T-cells, ensuring the proper phosphorylation of Vav and the subsequent engagement of downstream effectors. We also provide evidence indicating that the zinc finger region exerts at least three different functional roles in Vav, aiding in the down-regulation of its basal activity, the engagement of substrates, and the induction of ancillary pathways required for cell transformation. Finally, the results obtained are consistent with a new regulatory model for Vav, in which the calponin homology region inhibits the basal activity of Vav through interactions with the zinc finger region.Vav proteins are phosphorylation-dependent exchange factors that catalyze the release of GDP from Rho/Rac family members, thereby facilitating their transition from the inactive (GDP-bound) to the active (GTP-bound) state (1). This activity is crucial for the coordination of developmental and mitogenic processes. Thus, the elimination of the vav gene results in impaired lymphoid development, lymphopenia, and defective immune responses in mice (2-5). Similarly, deletion of either vav2 or vav3 genes results in impaired signaling responses in activated B-cells (6 -8). It has also been demonstrated that the subversion on the normal activation/deactivation cycle of some members of the Vav family results in severe alterations of cell behavior, including tumorigenesis, changes in F-actin organization, and the acquisition of metastatic properties by transformed cells (1). Finally, the activation of Vav or Vav2 proteins by the Nef protein of the human immunodeficiency virus plays an essential role in the pathogenic cycle of this virus (9, 10).One important feature of this GEF family is the structural complexity of its members (1, 10) (see Fig. 1A). Mammalian and avian Vav proteins contain eight structural domains, including a calponin homology (CH) 1 region, an acidic (Ac) domain, the catalytic Dbl homology (DH) region, a pleckstrin homology (PH) domain, a zinc finger (ZF) region similar to those present in c-Raf and protein kinase C family members, and a SH2 domain flanked by two SH3 domains. Caenorhabditis elegans and Drosophila melanogas...

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␤2-chimaerin, a member of the GTPase-activating proteins for the small GTP-binding protein p21Rac, possesses a single cysteine-rich domain with high homology to those implicated in phorbol ester and diacylglycerol binding in protein kinase C (PKC) isozymes. We have expressed ␤2-chimaerin in Sf9 insect cells using the baculovirus expression system and determined that, like PKCs, ␤2-chimaerin binds phorbol esters with high affinity in the presence of phosphatidylserine as a cofactor. Scatchard plot analysis using the radioligand [ 3 H]phorbol 12,13-dibutyrate revealed a dissociation constant of 1.9 ؎ 0.2 nM for ␤2-chimaerin. Likewise, ␤2-chimaerin is a high affinity receptor for the bryostatins, a class of atypical PKC activators. A detailed comparison of structure-activity relations using several phorbol ester analogs revealed striking differences in binding recognition between ␤2-chimaerin and PKC␣. Although the diacylglycerol 1-oleoyl-2-acetylglycerol binds with similar potency to both ␤2-chimaerin and PKC␣, the mezerein analog thymeleatoxin has 56-fold less affinity for binding to ␤2-chimaerin. To establish whether ␤2-chimaerin responds to phorbol esters in cellular systems, we overexpressed ␤2-chimaerin in COS-7 cells and monitored its subcellular distribution after phorbol ester treatment. Interestingly, as described previously for PKC isozymes, ␤2-chimaerin translocates from cytosolic to particulate fractions as a consequence of phorbol ester treatment. Our results demonstrate that ␤2-chimaerin is a novel target for the phorbol ester tumor promoters. The expansion of the family of phorbol ester receptors strongly suggests a potential for the "nonkinase" receptors as cellular mediators of the phorbol ester responses.The phorbol esters and related diterpenes are natural compounds that are used widely as tumor promoters in animal models. The search for receptors for the phorbol esters led to the identification of protein kinase C (PKC) 1 as their target (1-5). The complexity and heterogeneity in the biology of phorbol esters suggested the existence of multiple receptors, and 11 PKC isoforms have been identified so far: classic or calciumdependent isozymes (PKC ␣, ␤1, ␤2, and ␥), novel or calciumindependent isozymes (PKC ␦, ⑀, , , and ), and atypical isozymes (PKC and ) (6, 7). The last group is unresponsive to the phorbol esters. DAG, the postulated endogenous activator of PKC, competes with phorbol esters for binding to PKC in in vitro assays, although its binding potency is lower than that of the most commonly used phorbol esters (8). The cysteine-rich domains present at the NH 2 -terminal region of the PKCs are the binding sites for DAG/phorbol esters (9 -13). Each of these 50-or 51-amino acid domains possesses the motif HX 12 CX 2 CX 13/14 CX 2 CX 4 HX 2 CX 7 C, where H is histidine, C is cysteine, and X is any other amino acid. This motif is duplicated in tandem in both the classic and novel PKCs and is present only once in the atypical PKCs. The cysteine-rich domains have been implicated in the associati...

The members of the chimaerin family of Rac-GTPase-activating proteins possess a single C1 domain with high homology to those present in protein kinase C (PKC) isozymes. This domain in PKCs is involved in phorbol ester and diacylglycerol (DAG) binding. We previously have demonstrated that one of the chimaerin isoforms, ␤2-chimaerin, binds phorbol esters with high affinity. In this study we analyzed the properties of ␤2-chimaerin as a DAG receptor by using a series of conformationally constrained cyclic DAG analogues (DAG lactones) as probes. We identified analogs that bind to ␤2-chimaerin with more than 100-fold higher affinity than 1-oleoyl-2-acetylglycerol. The potencies of these analogs approach those of the potent phorbol ester tumor promoters. The different DAG lactones show some selectivity for this novel receptor compared with PKC␣. Cellular studies revealed that these DAG analogs induce translocation of ␤2-chimaerin from cytosolic (soluble) to particulate fractions. Using green fluorescent protein-fusion proteins for ␤2-chimaerin we determined that this novel receptor translocates to the perinuclear region after treatment with DAG lactones. Binding and translocation were prevented by mutation of the conserved Cys-246 in the C1 domain. The structural homology between the C1 domain of ␤2-chimaerin and the C1b domain of PKC␦ also was confirmed by modeling analysis. Our results demonstrate that ␤2-chimaerin is a high affinity receptor for DAG through binding to its C1 domain and supports the emerging concept that multiple pathways transduce signaling through DAG and the phorbol esters. Signaling in response to the second messenger diacylgycerol (DAG) is thought to proceed through the activation of protein kinase C (PKC) isozymes (1, 2). Binding of this lipid second messenger and its related analogs, the phorbol esters, occurs at the C1 domains (also called cysteine-rich regions or zinc fingers) present in the classical PKCs (PKC␣, ␤I, ␤II, and ␥) and novel PKCs (PKC␦, , , and ). This 50-to 51-aa domain, which is present in tandem in these PKC isozymes, possesses the motif HX 12 CX 2 CX n CX 2 CX 4 HX 2 CX 7 C, where H is histidine, C is cysteine, X is any other amino acid, and n is 13-14 (3-6). The phorbol ester receptor family has expanded with the discovery of the chimaerins. Unlike PKCs, the chimaerins do not possess a functional kinase domain but they are GTPase-activating proteins for Rac, a small GTP binding protein of the Ras superfamily (7). Four chimaerin isoforms (␣1-or n-, ␣2-, ␤1-, and ␤2-chimaerin) have been identified to date, all of them possessing a single C1 domain with approximately 40% homology to those present in PKCs (7-10). It is therefore predictable that the biological responses of the phorbol esters and those mediated by DAG signaling could involve the activation of PKC-independent pathways.We previously have reported that ␣1-and ␤2-chimaerin are indeed high affinity receptors for the phorbol esters and also for the bryostatins, macrocyclic lactones with antitumor properties (11,12). ...