Second messengers generated from membrane lipids play a critical role in signaling and control diverse cellular processes. Despite being one of the most evolutionarily conserved of all the phosphoinositide-specific phospholipase C (PLC) isoforms, a family of enzymes responsible for hydrolysis of the membrane lipid phosphatidylinositol bisphosphate, the mechanism of PLC-␦1 activation is still poorly understood. Here we report a novel regulatory mechanism for PLC-␦1 activation that involves direct interaction of the small GTPase Ral and the universal calcium-signaling molecule calmodulin (CaM) with PLC-␦1. In addition, we have identified a novel IQ type CaM binding motif within the catalytic region of PLC-␦1 that is not found in other PLC isoforms. Binding of CaM at the IQ motif inhibits PLC-␦1 activity, while addition of Ral reverses the inhibition. The overexpression of various Ral mutants in cells potentiates PLC-␦1 activity. Thus, the Ral-CaM complex defines a multifaceted regulatory mechanism for PLC-␦1 activation.
Ral GTPases may be involved in calcium/calmodulinmediated intracellular signaling pathways. RalA and RalB are activated by calcium, and RalA binds calmodulin in vitro. It was examined whether RalA can bind calmodulin in vivo, whether RalB can bind calmodulin, and whether calmodulin is functionally involved in Ral activation. Yeast two-hybrid analyses demonstrated both Rals interact directly but differentially with calmodulin. Coimmunoprecipitation experiments determined that calmodulin and RalB form complexes in human platelets. In vitro pull-down experiments in platelets and in vitro binding assays showed endogenous Ral and calmodulin interact in a calcium-dependent manner. Truncated Ral constructs determined in vitro and in vivo that RalA has an additional calmodulin binding domain to that previously described, that although RalB binds calmodulin, its C-terminal region is involved in partially inhibiting this interaction, and that in vitro RalA and RalB have an N-terminal calciumindependent and a C-terminal calcium-dependent calmodulin binding domain. Functionally, in vitro Ral-GTP pull-down experiments determined that calmodulin is required for the thrombin-induced activation of Ral in human platelets. We propose that differential binding of calmodulin by RalA and RalB underlies possible functional differences between the two proteins and that calmodulin is involved in the regulation of the activation of Ral-GTPases.
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