Mechanism of Phosphorylation-Induced Activation of Phospholipase C-γ Isozymes

Phospholipase C (PLC) isozymes regulate numerous cellular events including fertilization, proliferation, differentiation, and chemotaxis. PLCs hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) into the second messengers diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), which activate protein kinase C (PKC) and liberate intracellular calcium, respectively. Numerous transmembrane receptor (RTKs) and cytosolic tyrosine kinases recruit and phosphorylate PLC-γ isozymes (γ1 and γ2) to enhance their phospholipase activities, leading to ventricular contractility, angiogenesis, and cell motility among other processes. The molecular details linking phosphorylation and activation of PLC-γ isozymes remain poorly understood. The majority of PLC enzymes are auto-inhibited by a highly disordered and electronegative linker inserted between the two halves (X and Y box) of the catalytic TIM barrel. Recent data support a model of interfacial activation for most PLCs whereby the X/Y-linker is removed from the active site by electrostatic repulsion with lipid membranes, thus coupling localization and orientation of PLCs at substrate membranes with their activation. However, PLC-γ isozymes are structurally unique in having acquired several modular domains – a split PH domain, a tandem SH2 domain, and an SH3 domain – within the X/Y-linker. iv In this study, we investigate the roles of the highly elaborated X/Y-linker in regulating the phospholipase activity of PLC-γ isozymes. We identified the C-terminal SH2 (cSH2) domain within the X/Y-linker as the critical structural determinant in regulating the auto-inhibition of PLC-γ isozymes and demonstrate how tyrosine phosphorylation leads to phospholipase activity. Deletion of the cSH2 domain fully recapitulates the robust, constitutive phospholipase activity of PLC-γ isozymes lacking the entire X/Y-linker. Moreover, phosphorylation of PLC-γ1 at Tyr783 within the X/Ylinker promotes high affinity interactions with the cSH2 domain required for phospholipase activation. These interactions induce large conformational rearrangements that facilitate substrate access and subsequent hydrolysis. Consequently, the X/Y-linker distinctly regulates several aspects of PLC-γ activity: i) it auto-inhibits basal phospholipase activity of PLC-γ isozymes, ii) it is required for recruitment of PLC-γ isozymes to RTKs, and iii) phosphorylation of the X/Y-linker relieves this autoinhibition. Overall, PLC-γ isozymes elaborate upon a general mechanism of autoinhibition by the X/Y-linker found in most PLCs to couple tyrosine phosphorylation with phospholipase activation