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.
Szymańska
Ewelina
E
Laboratory of Plasma Membrane Receptors, Nencki Institute of Experimental Biology, Warsaw, Poland.
Sobota
Andrzej
A
Czuryło
Edward
E
Kwiatkowska
Katarzyna
K
D000199:Actins / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000276:immunology*; D015153:Blotting, Western; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D006224:Cricetinae; D003599:Cytoskeleton; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006801:Humans; D008264:Macrophages / Q000276:immunology / Q000378:metabolism*; D018613:Microscopy, Confocal; D015778:Minor Histocompatibility Antigens; D010587:Phagocytosis / Q000502:physiology*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D054803:Phospholipase C delta / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D011554:Pseudopodia / Q000378:metabolism*; D017452:Receptors, IgG / Q000276:immunology*; D014162:Transfection
D000199:Actins; D015703:Antigens, CD; C080568:Fc gamma receptor IIA; D015778:Minor Histocompatibility Antigens; D018129:Phosphatidylinositol Phosphates; D017452:Receptors, IgG; C037178:phosphatidylinositol 4-phosphate; D017853:Phosphotransferases (Alcohol Group Acceptor); C402182:phosphatidylinositol phosphate 4-kinase; D054803:Phospholipase C delta