Expression of PI(4,5)P2-binding proteins lowers the PI(4,5)P2level and inhibits FcgammaRIIA-mediated cell spreading and phagocytosis.

We found that FcgammaRII-mediated cell spreading and phagocytosis were correlated with an increase of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] level in cells. During the spreading, a long-lasting elevation of PI(4,5)P(2) and concomitant actin polymerization occurred. Filopodia and lamellae of spreading cells were enriched in phosphatidylinositol 4-phosphate 5-kinase Ialpha (PIP5-kinase Ialpha) that colocalized with PI(4,5)P(2 )and actin filaments. Both spreading and phagocytosis were inhibited by expression of the C(374-440) fragment of PIP5-kinase Ialpha or the pleckstrin homology domain of phospholipase Cdelta(1 )(PLCdelta(1)-PH), two probes binding PI(4,5)P(2). These probes reduced the amount of PI(4,5)P(2) in the cells, evoked reorganization of the actin cytoskeleton and abolished PI(4,5)P(2) elevation during phagocytosis. Simultaneously, PLCdelta(1)-PH-GFP reduced the amount of PIP5-kinase Ialpha associated with the plasma membrane. In vitro studies demonstrated that PIP5-kinase Ialpha-GST bound PI(4,5)P(2), phosphatidylinositol 4-monophosphate, and less efficiently, phosphatidic acid. The data suggest that the PLCdelta(1)-PH domain, and possibly also the C(374-440) fragment, when expressed in cells, can compete with endogenous PIP5-kinase Ialpha for PI(4,5)P(2 )binding in the plasma membrane leading eventually to PI(4,5)P(2) depletion.