General and Versatile Autoinhibition of PLC Isozymes

Abstract

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.

WDR26 Functions as a Scaffolding Protein to Promote Gβγ-mediated Phospholipase C β2 (PLCβ2) Activation in Leukocytes

“…Upon PLC␤2 recruitment to negatively charged substrate membranes, the inhibitory linker is displaced from the active site by electrostatic repellence, leading to PLC␤2 activation. This hypothesis was supported by the findings that deletion of the linker region is sufficient to cause PLC␤2 activation in the absence of any stimuli (30). However, the PLC␤2 mutant with the linker deletion exhibited up to a 20-fold increase in basal activity, which is significantly larger than what we observed with the membranetargeted PLC␤2 (ϳ2-fold).…”

Membrane-induced Allosteric Control of Phospholipase C-β Isozymes

“…Deletion of the XY-linker of PLC-␤2 (PH-C2) and PLC-␤3 followed a similar method previously published for PLC-␤2 (15). PLC-␤2(PH-C2,⌬XY) lacks residues 470 -535, and PLC-␤3(⌬XY) lacks residues 472-585.…”

“…Purification schemes for PLC-␤3 and -␤2 followed previously published protocols (15,22). In brief, cell pellets were resuspended and lysed in buffer containing 50 mM HEPES, pH 8.0, 50 mM NaCl, 10 mM ␤-mercaptoethanol (␤-ME), 5% (v/v) glycerol, 15 mM imidazole, 0.1 mM EDTA, 0.1 mM EGTA, and 0.1 mg/ml PMSF.…”

“…Autoinhibition at Membranes-WH-15 is a unique substrate for quantification of PLC activity independent of potential allosterism mediated by membranes. We previously showed that a portion of the XY-linker inserted within the catalytic TIM barrel of PLC-␤ isozymes directly occludes the active site (15,22). Deletion of this "plug" elevates phospholipase activity at membranes, and activity is further increased by removal of the remaining portion of the XY-linker.…”

“…The two halves of the TIM barrel are connected by a region of variable length known as the XY-linker. This poorly conserved region nonetheless functions as a major regulatory element, and its removal from most PLCs results in constitutive phospholipase activity in vitro and in cells (15,16). We previously proposed that G protein activators recruit and orient PLC isozymes at membranes, leading to displacement of the XY-linker from the vicinity of the lipase active site (13).…”