Several isoforms of phospholipase C (PLC) are regulated through interactions with Ras superfamily GTPases, including Rac proteins. Interestingly, of two closely related PLC␥ isoforms, only PLC␥ 2 has previously been shown to be activated by Rac. Here, we explore the molecular basis of this interaction as well as the structural properties of PLC␥ 2 required for activation. Based on reconstitution experiments with isolated PLC␥ variants and Rac2, we show that an unusual pleckstrin homology (PH) domain, designated as the split PH domain (spPH), is both necessary and sufficient to effect activation of PLC␥ 2 by Rac2. We also demonstrate that Rac2 directly binds to PLC␥ 2 as well as to the isolated spPH of this isoform. Furthermore, through the use of NMR spectroscopy and mutational analysis, we determine the structure of spPH, define the structural features of spPH required for Rac interaction, and identify critical amino acid residues at the interaction interface. We further discuss parallels and differences between PLC␥ 1 and PLC␥ 2 and the implications of our findings for their respective signaling roles.Phosphoinositide-specific phospholipase C (PLC) 3 enzymes have been established as crucial signaling nodes involved in regulation of a variety of cellular functions via hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate. There are six major families of PLC enzymes (PLC␤, -␥, -␦, -⑀, -, and -) that share a common core of domains related to catalysis and are distinguished by family-specific regulatory regions (1-3). The two isoforms of the PLC␥ family, PLC␥ 1 and PLC␥ 2 , uniquely incorporate an array of domains comprising two SH2 domains, an SH3 domain, and an internal or "split" PH domain (spPH). spPHs represent a unique subclass of PH domains that are characterized by insertions of one or several autonomously folded protein modules encoded within the boundaries of PH domain sequences (4). This array also contains sites for phosphorylation by several receptor (e.g. epidermal growth factor and platelet-derived growth factor receptors) and nonreceptor tyrosine kinases. In addition to tyrosine phosphorylation, multiple protein-protein interactions (mainly mediated by SH2 and SH3 domains) contribute to PLC␥ activation and have an important role in localizing the enzyme to protein complexes in different cellular compartments (5, 6). However, the elucidation at the molecular level of how PLC␥ isoforms are regulated remains an area of intense study.Despite the common domain organization shared by the PLC␥ 1 and PLC␥ 2 isoforms, studies using gene-targeting approaches demonstrated that each has a distinct biological role (7,8). Different functions of PLC␥ 1 (essential role in embryonic development) and PLC␥ 2 (requirement for development and function of hematopoietic cells) to some degree reflect their different expression patterns and, in particular, the abundance of PLC␥ 2 in hematopoietic cells. However, studies of different cell types where both isoforms are present (e.g. platelets, macrophages/mo...

Background: Phospholipase C␥ 2 (PLC␥ 2 ) is stimulated by Rac GTPases through direct protein-protein interaction. Results: The Rac-PLC␥ 2 interaction markedly enhances B cell-receptor-mediated Ca 2ϩ mobilization and nuclear translocation of the Ca 2ϩ -regulated transcription factor NFAT in B cells. Conclusion: Rac-mediated stimulation of PLC␥ 2 activity amplifies B cell receptor-induced Ca 2ϩ signaling. Significance: A specific Rac-resistant PLC␥ 2 variant is used to determine the physiological cell signaling relevance of a functional Rac-PLC␥ 2 interaction in an appropriate cellular context.

We performed analyses of the molecular mechanisms involved in the regulation of phospholipase C␥2 (PLC␥2). We identified several regions in the PLC␥-specific array, ␥SA, that contribute to autoinhibition in the basal state by occlusion of the catalytic domain. While the activation of PLC␥2 by Rac2 requires stable translocation to the membrane, the removal of the domains required for membrane translocation in the context of an enzyme with impaired autoinhibition generated constitutive, highly active PLC in cells. We further tested the possibility that the interaction of PLC␥2 with its activator protein Rac2 was sufficient for activation through the release of autoinhibition. However, we found that Rac2 binding in the absence of lipid surfaces was not able to activate PLC␥2. Together with other observations, these data suggest that an important consequence of Rac2 binding and translocation to the membrane is that membrane proximity, on its own or together with Rac2, has a role in the release of autoinhibition, resulting in interfacial activation.Phosphoinositide-specific phospholipase C (PLC)-catalyzed formation of the second messengers inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG) from its substrate phosphatidylinositol 4,5-bisphosphate (PIP 2 ) constitutes one of the major cell signaling responses (3, 36). There are six families of PLC enzymes (PLC␤, ␥, ␦, ε, , and ) consisting of 13 isoforms in humans. Enzymes from each PLC family are uniquely integrated into complex signaling networks through diverse regulatory mechanisms and contribute to the regulation of a variety of biological functions. Despite this diversity, some common principles of their regulation at the molecular level have been proposed for several PLC families (16). However, such mechanistic concepts need to be tested further for each of the families and, in particular, for PLC␥ enzymes, which represent a branch separate from all other PLC families (22).One of the two members of the PLC␥ family, PLC␥2, is most highly expressed in cells of the hematopoietic system and plays a key role in the regulation of the immune response. The regulatory interactions that control PLC␥2 in B cells have been well characterized and involve phosphorylation on critical tyrosine residues by Src and Tec family kinases; similar types of regulatory interactions have been described for the regulation of PLC␥1 in T-cell responses (2). Both PLC␥ enzymes can be activated in response to growth factor stimulation (26, 37). A number of studies of the ubiquitously expressed PLC␥1 in different cell types support the critical importance of tyrosine phosphorylation for PLC␥ activation. In addition to the regulatory mechanisms that are shared by the two PLC␥ enzymes, the Rac GTPases Rac1, Rac2, and Rac3 have been specifically implicated in PLC␥2 regulation that is not dependent on tyrosine phosphorylation of this enzyme (29). However, like stimulation via tyrosine kinase-linked receptors, Rac2 also leads to membrane translocation (29). Several studies suggest signali...

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