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

Abstract

We found that FcyRII-mediated cell spreading and phagocytosis were correlated with an increase of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] level in cells. During the spreading, a long-lasting elevation of PI(4,5)P2 and concomitant actin polymerization occurred. Filopodia and lamellae of spreading cells were enriched in phosphatidylinositol 4-phosphate 5-kinase Iα (PIP5-kinase Iα) that colocalized with PI(4,5)P2 and actin filaments. Both spreading and phagocytosis were inhibited by expression of the C374-440 fragment of PIP5-kinase Iα or the pleckstrin homology domain of phospholipase Cδ1 (PLCδ-PH), two probes binding PI(4,5)P2. These probes reduced the amount of PI(4,5)P2 in the cells, evoked reorganization of the actin cytoskeleton and abolished PI(4,5)P2 elevation during phagocytosis. Simultaneously, PLCδ1,-PH-GFP reduced the amount of PIP5-kinase Iα associated with the plasma membrane. In vitro studies demonstrated that PIP5-kinase Iα-GST bound PI(4,5)P2, phosphatidylinositol 4-monophosphate, and less efficiently, phosphatidic acid. The data suggest that the PLCδ1-PH domain, and possibly also the C374-440 fragment, when expressed in cells, can compete with endogenous PIP5-kinase Iα for PI(4,5)P2 binding in the plasma membrane leading eventually to PI(4,5)P2 depletion. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.