The role of the pleckstrin homology domain in membrane targeting and activation of phospholipase Cβ1

Current studies involve an investigation of the role of the pleckstrin homology (PH) domain in membrane targeting and activation of phospholipase C beta(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 protein. Whereas PLC beta(1)-PH does not localize to the plasma membrane in serum-starved cells, it undergoes a rapid but transient migration to the plasma 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 phosphatidylinositol 3-phosphate most strongly, whereas other phosphoinositides 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. Microinjection of beta gamma subunits of G proteins in serum-starved cells induced the translocation of PLC beta(1)-PH to the plasma membrane. These results demonstrate that a cooperative mechanism involving phosphatidylinositol 3-phosphate and the G beta gamma subunit regulates the plasma membrane localization and activation of PLC beta(1)-PH.