Inhibition of Phosphatidylinositol-4-phosphate 5-Kinase Iα 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. PIPKI␣ was found to accumulate transiently on forming phagosomes. To test the functional involvement of PIPKI␣ 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 PIP5KI␣ 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 Fc␥ 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 PIPKI␣ activity is involved in the actin remodeling that is a prerequisite for efficient phagocytosis. PIPKI␣ appears to contribute to the transient changes in PIP 2 levels that are associated with, and likely required for, the recruitment and regulation of actinmodulating proteins.

Membrane Ruffling Requires Coordination between Type Iα Phosphatidylinositol Phosphate Kinase and Rac Signaling

“…Briefly, PIPKI␣ and Rac were cotransfected at a ratio of 1:3 (PIPKI/Rac) or as indicated with LipofectAMINE Plus (Invitrogen). The cells were then processed for immunofluorescence by fixation in 4% paraformaldehyde, permeabilized, and washed (15). Primary antibodies (PIPKI polyclonal antibodies or anti-Myc 9E10) were diluted in 3% IgG free bovine serum albumin (Jackson Immunoresearch).…”

“…PIPKI Constructs-The introduction of the human PIPKI␣ cDNA into the pET28b vector (Novagen) for Escherichia coli expression and pcDNA3 pFLAG vectors for mammalian expression has been described previously (15,32).…”

“…Overexpression of PIPKI, but not PIPKII isoforms, in fibroblasts results in decreased stress fiber formation and formation of highly dynamic actin structures termed actin foci and comets (12)(13)(14). In fibroblasts and macrophages, overexpression of kinase-dead PIPKI␣ and PIPKI␤ mutants do not induce actin foci and comets, indicating that PIP 2 production is a necessary for remodeling of the actin cytoskeleton (12,15).…”

Phosphoinositides in Constitutive Membrane Traffic

“…Thus the degree of subcompartmentalization of PtdIns(4,5)P 2 in the plasma membrane is currently uncertain. PtdIns(4,5)P 2 is generated during the process of fusion of regulated secretory vesicles or synaptic vesicles with the plasma membrane (66,219), at the locations where actin rearrangements occur (59,67,404), and is required for clathrin-mediated endocytosis (67,104,161,171,262). Although required for vesicle fusion at the plasma membrane and for homotypic fusion of yeast vacuoles in vitro (222), the role of PtdIns(4,5)P 2 in vesicle fusion is not known precisely.…”

PI5KI-dependent signals are critical regulators of the cytolytic secretory pathway

“…[12][13][14][15] Increasing evidences indicate that PI5KIs, providing spatially and functionally distinct PIP2 pools, guarantee the regulation of specific cellular events: PI5KI␥, for instance, is required for focal adhesion and synaptic vesicle trafficking, 16,17 PI5KI␤ for constitutive receptor endocytosis, 18 whereas PI5KI␣ acts in the regulation of processes dependent on local changes of actin dynamics, such as membrane ruffling and phagocytosis. 19,20 In this report, we took advantage of the fusion protein consisting of GFP and the pleckstrin homology (PH) domain of PLC␦1, that is known to bind PIP2 with high affinity and specificity, [21][22][23] to monitor the dynamics and function of PIP2 during the cytotoxic event. We present evidences of localized changes in the concentration of PIP2 at the cytolytic synapse area where it is required for granule exocytosis.…”