The role of the pleckstrin homology domain in membrane targeting and activation of phospholipase C beta(1)

Current studies involve an investigation of the role of the pleckstrin homo logy (PH) domain in membrane targeting and activation of phospholipase C be ta(1) (PLC beta(1)). Here we report studies on the membrane localization of the isolated PH domain from the amino terminus of PLC beta(1) (PLC beta(1) -PH) using fluorescence microscopy of a green fluorescent protein fusion pr otein. Whereas PLC beta(1)-PH does not localize to the plasma membrane in s erum-starved cells, it undergoes a rapid but transient migration to the pla sma membrane upon stimulation of cells with serum or lysophosphatidic acid (LPA). Regulation of the plasma membrane localization of PLC beta(1)-PH by phosphoinositides was also investigated. PLC beta(1)-PH was found to bind p hosphatidylinositol 3-phosphate most strongly, whereas other phosphoinositi des were bound with lower affinity. The plasma membrane localization of PLC beta(1)-PH induced by serum and LPA was blocked by wortmannin pretreatment and by LY294002. In parallel, activation of PLC beta by LPA was inhibited by wortmannin, by LY294002, or by the overexpression of PLC beta(1)-PH. Mic roinjection of beta gamma subunits of G proteins in serum-starved cells ind uced the translocation of PLC beta(1)-PH to the plasma membrane. These resu lts demonstrate that a cooperative mechanism involving phosphatidylinositol 3-phosphate and the G beta gamma subunit regulates the plasma membrane loc alization and activation of PLC beta(1)-PH.