SUMMARY Phospholipase C (PLC) isozymes are directly activated by heterotrimeric G proteins and Ras-like GTPases to hydrolyze phosphatidylinositol 4,5-bisphosphate into the second messengers diacylglycerol and inositol 1,4,5-trisphosphate. Although PLCs play central roles in myriad signaling cascades, the molecular details of their activation remain poorly understood. As described here, the crystal structure of PLC-β2 illustrates occlusion of the active site by a loop separating the two halves of the catalytic TIM barrel. Removal of this insertion constitutively activates PLC-β2 without ablating its capacity to be further stimulated by classical G protein modulators. Similar regulation occurs in other PLC members, and a general mechanism of interfacial activation at membranes is presented that provides a unifying framework for PLC activation by diverse stimuli.

Phospholipase C-⑀ (PLC-⑀) is a recently identified PLC isoform activated by subunits of heterotrimeric G proteins (G␣ 12 , G␣ 13 , and G␤␥) as well as by the low molecular weight GTPases, Rho and Ras. To define the enzymatic activity and substrate specificity of PLC-⑀ as well as its potential direct activation by Rho family GTPases, a major fragment of PLC-⑀ encompassing the catalytic core (EF-hand repeats through the tandem Ras-associating domains; ϳ118 kDa) was purified to near homogeneity and assayed after reconstitution under various conditions. Similar to the enzymatic profiles of previously purified PLC-␤ isozymes, the purified fragment of PLC-⑀ maximally hydrolyzed phosphatidylinositol 4-phosphate at a rate of ϳ10 mol/mg of protein/min, exhibited phospholipase activity dependent on the concentration of free calcium, and favored phosphatidylinositol 4,5-bisphosphate as substrate relative to other phosphoinositides. Furthermore, in mixed detergent phospholipid micelles, RhoA stimulated the phospholipase activity of the PLC-⑀ fragment in both a concentration-dependent and guanosine 5-O-(3-thiotriphosphate)-dependent manner. This activation was abolished by the deletion of a unique ϳ65 amino acid-insert within the catalytic core of PLC-⑀. Although Rac1 activated purified PLC-␤2 in a guanine nucleotide-dependent manner, Rac1 failed to promote guanine nucleotide-dependent activation of purified PLC-⑀. These results indicate that PLC-⑀ is a direct downstream effector for RhoA and that RhoAdependent activation of PLC-⑀ depends on a unique insert within the catalytic core of the phospholipase.Phospholipase C (PLC) 1 isozymes respond to a variety of extracellular signaling molecules to stimulate hydrolysis of PtdIns(4,5)P 2 into the second messengers, inositol(1,4,5) trisphosphate and diacylglycerol (1). The generation of these two second messengers triggers the release of intracellular Ca 2ϩ and stimulates protein kinase C isozymes, respectively. Five different isoforms of PLC have been identified, each with distinct mechanisms of activation. The PLC-␤ isozymes are directly activated by heterotrimeric G protein ␣ subunits of the G q family (2-4) and by G␤␥ subunits (5-7). More recently, these isozymes were shown to be activated by the small GTPase Rac (8 -11). Activation of receptor and cytosolic tyrosine kinases promotes activation of PLC-␥ via phosphorylation and translocation (12)(13)(14). The mechanisms by which extracellular stimuli regulate PLC-␦ (15, 16) and the recently identified PLC-(17, 18) are less understood, although both are believed to require the mobilization of Ca 2ϩ . The fifth family member of PLC isozymes, PLC-⑀, initially was identified in Caenorhabditis elegans as a Ras-binding protein (19). Mammalian PLC-⑀ subsequently was identified as a phospholipase C isozyme containing multiple Ras interaction domains (20 -22) including a CDC25 homology domain in the amino terminus and tandem Ras-associating (RA) domains at the carboxyl terminus. Coexpression of GTPase-deficient, constitutively active ...

Phospholipase C-⑀ (PLC-⑀) is a highly elaborated PLC required for a diverse set of signaling pathways. Here we use a combination of cellular assays and studies with purified proteins to show that activated RhoA and Ras isoforms directly engage distinct regions of PLC-⑀ to stimulate its phospholipase activity. Purified PLC-⑀ was activated in a guanine nucleotide-and concentration-dependent fashion by purified lipidated K-Ras reconstituted in PtdIns(4,5)P 2 -containing phospholipid vesicles. Whereas mutation of two critical lysine residues within the second Ras-association domain of PLC-⑀ prevented K-Ras-dependent activation of the purified enzyme, guanine nucleotidedependent activation by RhoA was retained. Activation of receptors by a variety of hormones, neurotransmitters, growth factors, and other extracellular signaling molecules promotes activation of inositol lipid-specific phospholipase C (PLC) 2 enzymes to catalyze hydrolysis of PtdIns(4,5)P 2 into diacylglycerol and inositol (1,4,5)-trisphosphate (1). These second messenger molecules are responsible for the stimulation of protein kinase C isozymes and the mobilization of intracellular Ca 2ϩ , respectively (2, 3). PLC-catalyzed depletion of cellular PtdIns(4,5)P 2 also provides regulation of many additional cellular events. For example, a broad range of proteins contain structural domains, including PH, FERM, ENTH, and PX domains, that specifically bind inositol lipids necessary for their function (4, 5), and PtdIns(4,5)P 2 -binding proteins are implicated in a broad range of cell signaling events including membrane trafficking, changes in the actin cytoskeleton, and ion channel and transporter function (5).The six PLC subfamilies (PLC-␤, -␥, -␦, -⑀, -, and -) all contain conserved X-and Y-regions, which fold cooperatively to form a catalytic triose-phosphate isomerase (TIM) barrel.

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