Expression of PI(4,5)P-2-binding proteins lowers the PI(4,5)P-2 level and inhibits Fc gamma IRIIA-mediated cell spreading and phagocytosis

We found that Fc gamma RII-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 I alpha (PIP5-kinase I alpha) that colocalized with PI(4,5)P-2 and actin filaments. Both spreading and phagocytosis were inhibited by expression of the C374-440 fragment of PIP5-kinase I alpha or the pleckstrin homology domain of phospholipase C delta(1) (PLC delta(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, PLC delta(1)-PH-GFP reduced the amount of PIP5-kinase I alpha associated with the plasma membrane. In vitro studies demonstrated that PIP5-kinase 1 alpha-GST bound PI(4,5)P-2, phosphatidylinositol 4-monophosphate, and less efficiently, phosphatidic acid. The data suggest that the PLC delta(1)-PH domain, and possibly also the C374-440 fragment, when expressed in cells, can compete with endogenous PIP5-kinase I alpha. for PI(4,5)P-2 binding in the plasma membrane leading eventually to PI(4,5)P-2 depletion.