Inhibition of Phosphatidylinositol-4-phosphate 5-Kinase Iα Impairs Localized Actin Remodeling and Suppresses Phagocytosis

Actin polymerization drives the extension of pseudopods required for phagocytosis. Phosphatidylinositol 4,5-bisphosphate (PIP2) 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 PIP2from phosphatidylinositol 4-phosphate, is involved in the control of phagocytosis. PIPKIα was found to accumulate transiently on forming phagosomes. To test the functional involvement of PIPKIα 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 PIP5KIα 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 Fcγ receptors. Instead, expression of the inactive kinase diminished the accumulation of PIP2 and of F-actin in the phagosomal cup. These data suggest that PIPKIα activity is involved in the actin remodeling that is a prerequisite for efficient phagocytosis. PIPKIα appears to contribute to the transient changes in PIP2 levels that are associated with, and likely required for, the recruitment and regulation of actin-modulating proteins. Actin polymerization drives the extension of pseudopods required for phagocytosis. Phosphatidylinositol 4,5-bisphosphate (PIP2) 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 PIP2from phosphatidylinositol 4-phosphate, is involved in the control of phagocytosis. PIPKIα was found to accumulate transiently on forming phagosomes. To test the functional involvement of PIPKIα 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 PIP5KIα 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 Fcγ receptors. Instead, expression of the inactive kinase diminished the accumulation of PIP2 and of F-actin in the phagosomal cup. These data suggest that PIPKIα activity is involved in the actin remodeling that is a prerequisite for efficient phagocytosis. PIPKIα appears to contribute to the transient changes in PIP2 levels that are associated with, and likely required for, the recruitment and regulation of actin-modulating proteins. Phagocytosis is a fundamental component of the innate immune response in mammals. Macrophages and neutrophils have the ability to engulf invading microorganisms into a specialized compartment, known as the phagosome. Formation of phagosomes, where microbial killing occurs, is supported by the finely coordinated reorganization of the actin cytoskeleton, accompanied by remodeling of cellular membranes (1Allen L.A. Aderem A. Curr. Opin. Immunol. 1996; 8: 36-40Crossref PubMed Scopus (206) Google Scholar, 2Brown E.J. Bioessays. 1995; 17: 109-117Crossref PubMed Scopus (139) Google Scholar, 3Cox D. Greenberg S. Semin. Immunol. 2001; 13: 339-345Crossref PubMed Scopus (75) Google Scholar, 4Underhill D.M. Ozinsky A. Annu. Rev. Immunol. 2002; 20: 825-852Crossref PubMed Scopus (832) Google Scholar, 5Desjardins M. Huber L.A. Parton R.G. Griffiths G. J. Cell Biol. 1994; 124: 677-688Crossref PubMed Scopus (555) Google Scholar). This complex response is accomplished by the activation of a network of signaling and effector pathways, triggered by the engagement of the particle by phagocytic receptors. These include members of the Fcγ receptor (FcγR) 1The abbreviations used are: FcγR, Fcγ receptor; PIP2, phosphatidylinositol 4,5-bisphosphate; RBC, red blood cells; SRBC, sheep RBC; DMEM, Dulbecco's modified Eagle's medium; TRITC, tetramethylrhodamine-isothiocyanate; PLC, phospholipase C; GFP, green fluorescent protein; EGFP, enhanced GFP; GST, glutathione S-transferase; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PtdIns, phosphatidylinositol; DIC, differential interference contrast; PIPK, phosphatidylinositol-4-phosphate 5-kinase 1The abbreviations used are: FcγR, Fcγ receptor; PIP2, phosphatidylinositol 4,5-bisphosphate; RBC, red blood cells; SRBC, sheep RBC; DMEM, Dulbecco's modified Eagle's medium; TRITC, tetramethylrhodamine-isothiocyanate; PLC, phospholipase C; GFP, green fluorescent protein; EGFP, enhanced GFP; GST, glutathione S-transferase; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PtdIns, phosphatidylinositol; DIC, differential interference contrast; PIPK, phosphatidylinositol-4-phosphate 5-kinasefamily, which are expressed on the surface of macrophages and neutrophils, where they recognize IgG-opsonized particles (2Brown E.J. Bioessays. 1995; 17: 109-117Crossref PubMed Scopus (139) Google Scholar, 6Griffin Jr., F.M. Griffin J.A. Leider J.E. Silverstein S.C. J. Exp. Med. 1975; 142: 1263-1282Crossref PubMed Scopus (392) Google Scholar). Clustering of FcγR upon binding to IgG-coated particles activates Src family tyrosine kinases, which phosphorylate tyrosine residues of the receptors themselves (7Greenberg S. Chang P. Silverstein S.C. J. Exp. Med. 1993; 177: 529-534Crossref PubMed Scopus (168) Google Scholar, 8Greenberg S. Chang P. Silverstein S.C. J. Biol. Chem. 1994; 269: 3897-3902Abstract Full Text PDF PubMed Google Scholar). This in turn leads to the activation of additional protein and lipid kinases, including Syk, protein kinase C, and phosphatidylinositol 3′-kinase (9Kwiatkowska K. Sobota A. Bioessays. 1999; 21: 422-431Crossref PubMed Scopus (155) Google Scholar, 10Cox D. Chang P. Kurosaki T. Greenberg S. J. Biol. Chem. 1996; 271: 16597-16602Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 11Kiefer F. Brumell J., Al- Alawi N. Latour S. Cheng A. Veillette A. Grinstein S. Pawson T. Mol. Cell. Biol. 1998; 18: 4209-4220Crossref PubMed Google Scholar, 12Araki N. Johnson M.T. Swanson J.A. J. Cell Biol. 1996; 135: 1249-1260Crossref PubMed Scopus (774) Google Scholar, 13Cox D. Tseng C.C. Bjekic G. Greenberg S. J. Biol. Chem. 1999; 274: 1240-1247Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar) as well as of small GTPases like Rac1 and Cdc42 (14Cox D. Chang P. Zhang Q. Reddy P.G. Bokoch G.M. Greenberg S. J. Exp. Med. 1997; 186: 1487-1494Crossref PubMed Scopus (365) Google Scholar, 15Massol P. Montcourrier P. Guillemot J.C. Chavrier P. EMBO J. 1998; 17: 6219-6229Crossref PubMed Scopus (198) Google Scholar).Formation of a phagosome in response to FcγR stimulation involves the extension of membranous protrusions (pseudopods) around the adherent particle (1Allen L.A. Aderem A. Curr. Opin. Immunol. 1996; 8: 36-40Crossref PubMed Scopus (206) Google Scholar, 2Brown E.J. Bioessays. 1995; 17: 109-117Crossref PubMed Scopus (139) Google Scholar). Concomitantly, a cup-shaped structure rich in F-actin grows beneath the pseudopods (1Allen L.A. Aderem A. Curr. Opin. Immunol. 1996; 8: 36-40Crossref PubMed Scopus (206) Google Scholar, 2Brown E.J. Bioessays. 1995; 17: 109-117Crossref PubMed Scopus (139) Google Scholar, 16Greenberg S. Burridge K. Silverstein S.C. J. Exp. Med. 1990; 172: 1853-1856Crossref PubMed Scopus (87) Google Scholar). F-actin polymerization and pseudopod extension are both required for efficient phagocytosis, but are functionally distinct and regulated by different pathways (12Araki N. Johnson M.T. Swanson J.A. J. Cell Biol. 1996; 135: 1249-1260Crossref PubMed Scopus (774) Google Scholar, 13Cox D. Tseng C.C. Bjekic G. Greenberg S. J. Biol. Chem. 1999; 274: 1240-1247Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). It is not known exactly how the deposition of F-actin at the site of phagosome formation is controlled, but membrane-associated molecules that regulate actin metabolism are likely involved in this process. Evidence from other systems suggests that Rho family GTPases and phospholipids are essential in F-actin polymerization and remodeling at the membrane. Of particular importance is phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane-associated lipid that can bind to, and alter the activity of, several proteins that are directly involved in controlling the architecture of the actin skeleton (17Toker A. Curr. Opin. Cell Biol. 1998; 10: 254-261Crossref PubMed Scopus (244) Google Scholar, 18Sechi A.S. Wehland J. J. Cell Sci. 2000; 113: 3685-3695Crossref PubMed Google Scholar). These include profilin (19Chaudhary A. Chen J., Gu, Q.M. Witke W. Kwiatkowski D.J. Prestwich G.D. Chem. Biol. 1998; 5: 273-281Abstract Full Text PDF PubMed Scopus (57) Google Scholar), cofilin (20Yonezawa N. Homma Y. Yahara I. Sakai H. Nishida E. J. Biol. Chem. 1991; 266: 17218-17221Abstract Full Text PDF PubMed Google Scholar), gelsolin (21Yu F.X. Sun H.Q. Janmey P.A. Yin H.L. J. Biol. Chem. 1992; 267: 14616-14621Abstract Full Text PDF PubMed Google Scholar), talin (22Tempel M. Goldmann W.H. Isenberg G. Sackmann E. Biophys. J. 1995; 69: 228-241Abstract Full Text PDF PubMed Scopus (57) Google Scholar), vinculin (23Gilmore A.P. Burridge K. Nature. 1996; 381: 531-535Crossref PubMed Scopus (454) Google Scholar), WASP (24Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (545) Google Scholar), and members of the ezrin/moesin/radixin family (25Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (508) Google Scholar).Recent studies of PIP2 metabolism during phagosome formation revealed that this phosphoinositide undergoes a focal and transient accumulation at the phagosomal cup, followed by a profound and sustained decrease (26Botelho R.J. Teruel M. Dierckman R. Anderson R. Wells A. York J.D. Meyer T. Grinstein S. J. Cell Biol. 2000; 151: 1353-1368Crossref PubMed Scopus (419) Google Scholar). These observations suggested that dynamic changes in the local availability of PIP2 are required for the assembly and subsequent remodeling of F-actin structures during phagosome formation. The aim of the present study was to elucidate the mechanism whereby PIP2 is regulated during phagocytosis and to evaluate its functional role in the engulfment process. Specifically, we examined the role that PIPKIα, a key enzyme in the biosynthesis of PIP2, plays in phagocytosis. This required the design and transfection of kinase-inactive forms of the enzyme. We report that heterologous expression of such mutants impairs phagocytosis, without significantly altering the basal actin architecture of macrophages. Phagocytosis is a fundamental component of the innate immune response in mammals. Macrophages and neutrophils have the ability to engulf invading microorganisms into a specialized compartment, known as the phagosome. Formation of phagosomes, where microbial killing occurs, is supported by the finely coordinated reorganization of the actin cytoskeleton, accompanied by remodeling of cellular membranes (1Allen L.A. Aderem A. Curr. Opin. Immunol. 1996; 8: 36-40Crossref PubMed Scopus (206) Google Scholar, 2Brown E.J. Bioessays. 1995; 17: 109-117Crossref PubMed Scopus (139) Google Scholar, 3Cox D. Greenberg S. Semin. Immunol. 2001; 13: 339-345Crossref PubMed Scopus (75) Google Scholar, 4Underhill D.M. Ozinsky A. Annu. Rev. Immunol. 2002; 20: 825-852Crossref PubMed Scopus (832) Google Scholar, 5Desjardins M. Huber L.A. Parton R.G. Griffiths G. J. Cell Biol. 1994; 124: 677-688Crossref PubMed Scopus (555) Google Scholar). This complex response is accomplished by the activation of a network of signaling and effector pathways, triggered by the engagement of the particle by phagocytic receptors. These include members of the Fcγ receptor (FcγR) 1The abbreviations used are: FcγR, Fcγ receptor; PIP2, phosphatidylinositol 4,5-bisphosphate; RBC, red blood cells; SRBC, sheep RBC; DMEM, Dulbecco's modified Eagle's medium; TRITC, tetramethylrhodamine-isothiocyanate; PLC, phospholipase C; GFP, green fluorescent protein; EGFP, enhanced GFP; GST, glutathione S-transferase; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PtdIns, phosphatidylinositol; DIC, differential interference contrast; PIPK, phosphatidylinositol-4-phosphate 5-kinase 1The abbreviations used are: FcγR, Fcγ receptor; PIP2, phosphatidylinositol 4,5-bisphosphate; RBC, red blood cells; SRBC, sheep RBC; DMEM, Dulbecco's modified Eagle's medium; TRITC, tetramethylrhodamine-isothiocyanate; PLC, phospholipase C; GFP, green fluorescent protein; EGFP, enhanced GFP; GST, glutathione S-transferase; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; BSA, bovine serum albumin; PtdIns, phosphatidylinositol; DIC, differential interference contrast; PIPK, phosphatidylinositol-4-phosphate 5-kinasefamily, which are expressed on the surface of macrophages and neutrophils, where they recognize IgG-opsonized particles (2Brown E.J. Bioessays. 1995; 17: 109-117Crossref PubMed Scopus (139) Google Scholar, 6Griffin Jr., F.M. Griffin J.A. Leider J.E. Silverstein S.C. J. Exp. Med. 1975; 142: 1263-1282Crossref PubMed Scopus (392) Google Scholar). Clustering of FcγR upon binding to IgG-coated particles activates Src family tyrosine kinases, which phosphorylate tyrosine residues of the receptors themselves (7Greenberg S. Chang P. Silverstein S.C. J. Exp. Med. 1993; 177: 529-534Crossref PubMed Scopus (168) Google Scholar, 8Greenberg S. Chang P. Silverstein S.C. J. Biol. Chem. 1994; 269: 3897-3902Abstract Full Text PDF PubMed Google Scholar). This in turn leads to the activation of additional protein and lipid kinases, including Syk, protein kinase C, and phosphatidylinositol 3′-kinase (9Kwiatkowska K. Sobota A. Bioessays. 1999; 21: 422-431Crossref PubMed Scopus (155) Google Scholar, 10Cox D. Chang P. Kurosaki T. Greenberg S. J. Biol. Chem. 1996; 271: 16597-16602Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 11Kiefer F. Brumell J., Al- Alawi N. Latour S. Cheng A. Veillette A. Grinstein S. Pawson T. Mol. Cell. Biol. 1998; 18: 4209-4220Crossref PubMed Google Scholar, 12Araki N. Johnson M.T. Swanson J.A. J. Cell Biol. 1996; 135: 1249-1260Crossref PubMed Scopus (774) Google Scholar, 13Cox D. Tseng C.C. Bjekic G. Greenberg S. J. Biol. Chem. 1999; 274: 1240-1247Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar) as well as of small GTPases like Rac1 and Cdc42 (14Cox D. Chang P. Zhang Q. Reddy P.G. Bokoch G.M. Greenberg S. J. Exp. Med. 1997; 186: 1487-1494Crossref PubMed Scopus (365) Google Scholar, 15Massol P. Montcourrier P. Guillemot J.C. Chavrier P. EMBO J. 1998; 17: 6219-6229Crossref PubMed Scopus (198) Google Scholar). Formation of a phagosome in response to FcγR stimulation involves the extension of membranous protrusions (pseudopods) around the adherent particle (1Allen L.A. Aderem A. Curr. Opin. Immunol. 1996; 8: 36-40Crossref PubMed Scopus (206) Google Scholar, 2Brown E.J. Bioessays. 1995; 17: 109-117Crossref PubMed Scopus (139) Google Scholar). Concomitantly, a cup-shaped structure rich in F-actin grows beneath the pseudopods (1Allen L.A. Aderem A. Curr. Opin. Immunol. 1996; 8: 36-40Crossref PubMed Scopus (206) Google Scholar, 2Brown E.J. Bioessays. 1995; 17: 109-117Crossref PubMed Scopus (139) Google Scholar, 16Greenberg S. Burridge K. Silverstein S.C. J. Exp. Med. 1990; 172: 1853-1856Crossref PubMed Scopus (87) Google Scholar). F-actin polymerization and pseudopod extension are both required for efficient phagocytosis, but are functionally distinct and regulated by different pathways (12Araki N. Johnson M.T. Swanson J.A. J. Cell Biol. 1996; 135: 1249-1260Crossref PubMed Scopus (774) Google Scholar, 13Cox D. Tseng C.C. Bjekic G. Greenberg S. J. Biol. Chem. 1999; 274: 1240-1247Abstract Full Text Full Text PDF PubMed Scopus (326) Google Scholar). It is not known exactly how the deposition of F-actin at the site of phagosome formation is controlled, but membrane-associated molecules that regulate actin metabolism are likely involved in this process. Evidence from other systems suggests that Rho family GTPases and phospholipids are essential in F-actin polymerization and remodeling at the membrane. Of particular importance is phosphatidylinositol 4,5-bisphosphate (PIP2), a membrane-associated lipid that can bind to, and alter the activity of, several proteins that are directly involved in controlling the architecture of the actin skeleton (17Toker A. Curr. Opin. Cell Biol. 1998; 10: 254-261Crossref PubMed Scopus (244) Google Scholar, 18Sechi A.S. Wehland J. J. Cell Sci. 2000; 113: 3685-3695Crossref PubMed Google Scholar). These include profilin (19Chaudhary A. Chen J., Gu, Q.M. Witke W. Kwiatkowski D.J. Prestwich G.D. Chem. Biol. 1998; 5: 273-281Abstract Full Text PDF PubMed Scopus (57) Google Scholar), cofilin (20Yonezawa N. Homma Y. Yahara I. Sakai H. Nishida E. J. Biol. Chem. 1991; 266: 17218-17221Abstract Full Text PDF PubMed Google Scholar), gelsolin (21Yu F.X. Sun H.Q. Janmey P.A. Yin H.L. J. Biol. Chem. 1992; 267: 14616-14621Abstract Full Text PDF PubMed Google Scholar), talin (22Tempel M. Goldmann W.H. Isenberg G. Sackmann E. Biophys. J. 1995; 69: 228-241Abstract Full Text PDF PubMed Scopus (57) Google Scholar), vinculin (23Gilmore A.P. Burridge K. Nature. 1996; 381: 531-535Crossref PubMed Scopus (454) Google Scholar), WASP (24Miki H. Miura K. Takenawa T. EMBO J. 1996; 15: 5326-5335Crossref PubMed Scopus (545) Google Scholar), and members of the ezrin/moesin/radixin family (25Hirao M. Sato N. Kondo T. Yonemura S. Monden M. Sasaki T. Takai Y. Tsukita S. J. Cell Biol. 1996; 135: 37-51Crossref PubMed Scopus (508) Google Scholar). Recent studies of PIP2 metabolism during phagosome formation revealed that this phosphoinositide undergoes a focal and transient accumulation at the phagosomal cup, followed by a profound and sustained decrease (26Botelho R.J. Teruel M. Dierckman R. Anderson R. Wells A. York J.D. Meyer T. Grinstein S. J. Cell Biol. 2000; 151: 1353-1368Crossref PubMed Scopus (419) Google Scholar). These observations suggested that dynamic changes in the local availability of PIP2 are required for the assembly and subsequent remodeling of F-actin structures during phagosome formation. The aim of the present study was to elucidate the mechanism whereby PIP2 is regulated during phagocytosis and to evaluate its functional role in the engulfment process. Specifically, we examined the role that PIPKIα, a key enzyme in the biosynthesis of PIP2, plays in phagocytosis. This required the design and transfection of kinase-inactive forms of the enzyme. We report that heterologous expression of such mutants impairs phagocytosis, without significantly altering the basal actin architecture of macrophages.

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