title: Inhibition of phosphatidylinositol-4-phosphate 5-kinase Ialpha impairs localized actin remodeling and suppresses phagocytosis.
abstract: Actin polymerization drives the extension of pseudopods required for phagocytosis. Phosphatidylinositol 4,5-bisphosphate (PIP(2)) is thought to play a central role in this process, because it interacts with several actin-regulatory proteins and undergoes acute and localized changes at sites of phagocytosis. We therefore studied whether phosphatidylinositol-4-phosphate 5-kinase (PIPK), the enzyme responsible for the generation of PIP(2) from phosphatidylinositol 4-phosphate, is involved in the control of phagocytosis. PIPKIalpha was found to accumulate transiently on forming phagosomes. To test the functional involvement of PIPKIalpha in particle engulfment, we generated a double mutant (D309N/R427Q) that lacks kinase activity. When ectopically expressed in cultured cells, this mutant is targeted to the plasma membrane and accumulates at the phagosomal cup during particle engulfment. Expression of PIP5KIalpha D309N/R427Q impaired phagocytosis in RAW264.7 macrophages and in engineered phagocytes generated by transfection of Fc receptors in Chinese hamster ovary cells. Inhibition of phagocytosis could not be attributed to defects in particle binding or receptor clustering, which was monitored using green fluorescent protein-tagged Fcgamma receptors. Instead, expression of the inactive kinase diminished the accumulation of PIP(2) and of F-actin in the phagosomal cup. These data suggest that PIPKIalpha activity is involved in the actin remodeling that is a prerequisite for efficient phagocytosis. PIPKIalpha appears to contribute to the transient changes in PIP(2) levels that are associated with, and likely required for, the recruitment and regulation of actin-modulating proteins.
authors.affiliation: Cell Biology Program, Hospital for Sick Children, and Department of Biochemistry University of Toronto, Ontario M5G 1X8, Canada.
authors.forename: Marc G
authors.initials: MG
authors.lastname: Coppolino
authors.forename: Renee
authors.initials: R
authors.lastname: Dierckman
authors.forename: Joost
authors.initials: J
authors.lastname: Loijens
authors.forename: Richard F
authors.initials: RF
authors.lastname: Collins
authors.forename: Mahmoud
authors.initials: M
authors.lastname: Pouladi
authors.forename: Jenny
authors.initials: J
authors.lastname: Jongstra-Bilen
authors.forename: Alan D
authors.initials: AD
authors.lastname: Schreiber
authors.forename: William S
authors.initials: WS
authors.lastname: Trimble
authors.forename: Richard
authors.initials: R
authors.lastname: Anderson
authors.forename: Sergio
authors.initials: S
authors.lastname: Grinstein
mesh_terms: D000199:Actins / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004247:DNA / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D049452:Green Fluorescent Proteins; D007074:Immunoglobulin G / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism; D051379:Mice; D008856:Microscopy, Fluorescence; D009154:Mutation; D010587:Phagocytosis; D010588:Phagosomes; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors*; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D013997:Time Factors; D014162:Transfection
chemical_list: D000199:Actins; D007074:Immunoglobulin G; D008164:Luminescent Proteins; D020033:Protein Isoforms; D049452:Green Fluorescent Proteins; D004247:DNA; D017853:Phosphotransferases (Alcohol Group Acceptor); C020077:1-phosphatidylinositol-4-phosphate 5-kinase