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Type II Phosphatidylinositol 4-Kinase β Is a Cytosolic and Peripheral Membrane Protein That Is Recruited to the Plasma Membrane and Activated by Rac-GTP

2002; Elsevier BV; Volume: 277; Issue: 48 Linguagem: Inglês

10.1074/jbc.m206860200

1083-351X

Yong Wei, Hui Sun, Masaya Yamamoto, Paweł Włodarski, Kaiko Kunii, Manuel Martínez, Barbara Baryłko, Joseph Albanesi, Helen L. Yin,

Pancreatic function and diabetes

Phosphoinositides have a pivotal role as precursors to important second messengers and asbona fide signaling and scaffold targeting molecules. Phosphatidylinositol 4-kinases (PtdIns 4-kinases or PI4Ks) are at the apex of the phosphoinsitide cascade. Sequence analysis revealed that mammalian cells contain two type II PtdIns 4-kinase isoforms, now termed PI4KIIα and PI4KIIβ. PI4KIIα was cloned first. It is tightly membrane-associated and behaves as an integral membrane protein. In this study, we cloned PI4KIIβ and compared the two isoforms by monitoring the distribution of endogenous and overexpressed proteins, their modes of association with membranes, their response to growth factor stimulation or Rac-GTP activation, and their kinetic properties. We find that the two kinases have different properties. PI4KIIβ is primarily cytosolic, and it associates peripherally with plasma membranes, endoplasmic reticulum, and the Golgi. In contrast, PI4KIIα is primarily Golgi-associated. Platelet-derived growth factor promotes PI4KIIβ recruitment to membrane ruffles. This effect is potentially mediated through Rac; overexpression of the constitutively active RacV12 induces membrane ruffling, increases PI4KIIβ translocation to the plasma membrane, and stimulates its activity. The dominant-negative RacN17 blocks plasma membrane association and inhibits activity. RacV12 does not boost the catalytic activity of PI4KIIα further, probably because it is constitutively membrane-bound and already activated. Membrane recruitment is an important mechanism for PI4KIIβ activation, because microsome-bound PI4KIIβ is 16 times more active than cytosolic PI4KIIβ. Membrane-associated PI4KIIβ is as active as membrane-associated PI4KIIα and has essentially identical kinetic properties. We conclude that PI4KIIα and PI4KIIβ may have partially overlapping, but not identical, functions. PI4KIIβ is activated strongly by membrane association to stimulate phosphatidylinositol 4,5-bisphosphate synthesis at the plasma membrane. These findings provide new insight into how phosphoinositide cascades are propagated in cells. Phosphoinositides have a pivotal role as precursors to important second messengers and asbona fide signaling and scaffold targeting molecules. Phosphatidylinositol 4-kinases (PtdIns 4-kinases or PI4Ks) are at the apex of the phosphoinsitide cascade. Sequence analysis revealed that mammalian cells contain two type II PtdIns 4-kinase isoforms, now termed PI4KIIα and PI4KIIβ. PI4KIIα was cloned first. It is tightly membrane-associated and behaves as an integral membrane protein. In this study, we cloned PI4KIIβ and compared the two isoforms by monitoring the distribution of endogenous and overexpressed proteins, their modes of association with membranes, their response to growth factor stimulation or Rac-GTP activation, and their kinetic properties. We find that the two kinases have different properties. PI4KIIβ is primarily cytosolic, and it associates peripherally with plasma membranes, endoplasmic reticulum, and the Golgi. In contrast, PI4KIIα is primarily Golgi-associated. Platelet-derived growth factor promotes PI4KIIβ recruitment to membrane ruffles. This effect is potentially mediated through Rac; overexpression of the constitutively active RacV12 induces membrane ruffling, increases PI4KIIβ translocation to the plasma membrane, and stimulates its activity. The dominant-negative RacN17 blocks plasma membrane association and inhibits activity. RacV12 does not boost the catalytic activity of PI4KIIα further, probably because it is constitutively membrane-bound and already activated. Membrane recruitment is an important mechanism for PI4KIIβ activation, because microsome-bound PI4KIIβ is 16 times more active than cytosolic PI4KIIβ. Membrane-associated PI4KIIβ is as active as membrane-associated PI4KIIα and has essentially identical kinetic properties. We conclude that PI4KIIα and PI4KIIβ may have partially overlapping, but not identical, functions. PI4KIIβ is activated strongly by membrane association to stimulate phosphatidylinositol 4,5-bisphosphate synthesis at the plasma membrane. These findings provide new insight into how phosphoinositide cascades are propagated in cells. Phosphatidylinositol 4-kinases (PtdIns 1The abbreviations used for: PtdIns and PI4K, phosphatidylinositol 4-kinase; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP5K, PIP5-kinase; HA, hemagglutinin; GFP, green fluorescent protein; PDGF, platelet-derived growth factor; DTT, dithiothreitol; LSP, low speed pellet; HSP, high speed pellet; TGN, trans-Golgi network. 1The abbreviations used for: PtdIns and PI4K, phosphatidylinositol 4-kinase; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP5K, PIP5-kinase; HA, hemagglutinin; GFP, green fluorescent protein; PDGF, platelet-derived growth factor; DTT, dithiothreitol; LSP, low speed pellet; HSP, high speed pellet; TGN, trans-Golgi network.4-kinases, PI4Ks) are at the apex of the phosphoinositide cascade (1Balla T. Curr. Pharm. Des. 2001; 7: 475-507Crossref PubMed Scopus (47) Google Scholar). They phosphorylate phosphatidylinositol to generate phosphatidylinositol 4-phosphate (PIP), which is an immediate precursor of important signaling and scaffolding molecules such as phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate. In addition, genetic evidence inSaccharomyces cerevisiae suggests that PIP may also have a direct structural and functional role in its own right (2Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Crossref PubMed Scopus (286) Google Scholar, 3Foti M. Audhya A. Emr S.D. Mol. Biol. Cell. 2001; 12: 2396-2411Crossref PubMed Scopus (183) Google Scholar, 4Levine T.P. Munro S. Curr. Biol. 2002; 12: 695-704Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). There is increasing evidence that phosphoinositides are synthesized in a spatially and temporally defined manner to regulate signaling, cytoskeletal dynamics, and membrane trafficking (1Balla T. Curr. Pharm. Des. 2001; 7: 475-507Crossref PubMed Scopus (47) Google Scholar, 5Yin H.L. Janmey P.A. Annu. Rev. Physiol. 2003; (in press)PubMed Google Scholar, 6Di Fiore P.P. De Camilli P. Cell. 2001; 106: 1-4Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar).Multiple PI4Ks have been identified. Yeast has two dominant PtdIns 4-kinases, Pik1 and Stt4, that are functionally nonoverlapping (2Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Crossref PubMed Scopus (286) Google Scholar, 7Flanagan C.A. Schnieders E.A. Emerick A.W. Kunisawa R. Admon A. Thorner J. Science. 1993; 262: 1444-1448Crossref PubMed Scopus (171) Google Scholar). Pik1 is important for cell viability and Golgi-to-plasma membrane transport that appears to be regulated primarily by PIP and not by PIP2. In contrast, Stt4 maintains vacuole morphology and supplies PIP/PIP2 for actin cytoskeletal regulation. Mammalian homologs of Pik1 and Stt4 are called type III PtdIns 4-kinase β and PtdIns 4-kinase α (PI4KIIβ and PI4KIIα), respectively. They are found in the cytosol, Golgi, and endoplasmic reticulum, and neither is reported to be enriched in the plasma membrane or in lysosomes (8Wong K. Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 13236-13241Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 9Zhao X. Varnai P. Tuymetova G. Balla A. Toth Z.E. Oker-Blom C. Roder J. Jeromin A. Balla T. J. Biol. Chem. 2001; 276: 40183-40189Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar).Biochemical studies suggest that another class of PtdIns 4-kinases, designated as type II kinases, accounts for most of the PtdIns 4-kinase activity in the plasma membrane of mammalian cells (8Wong K. Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 13236-13241Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Because phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks), which convert PIP to PIP2, are enriched in the plasma membrane, and PIP2 is synthesized there in response to many stimuli (10Raucher D. Stauffer T. Chen W. Shen K. Guo S. York J.D. Sheetz M.P. Meyer T. Cell. 2000; 100: 221-228Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar, 11Holz R.W. Hlubek M.D. Sorensen S.D. Fisher S.K. Balla T. Ozaki S. Prestwich G.D. Stuenkel E.L. Bittner M.A. J. Biol. Chem. 2000; 275: 17878-17885Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 12Honda A. Nogami M. Yokozeki T. Yamazaki M. Nakamura H. Watanabe H. Kawamoto K. Nakayama K. Morris A.J. Frohman M.A. Kanaho Y. Cell. 1999; 99: 521-532Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar, 13Micheva K.D. Holz R.W. Smith S.J. J. Cell Biol. 2001; 154: 355-368Crossref PubMed Scopus (114) Google Scholar), the type II kinases may be the major source of PIP at this critical interface. Until recently, type II PtdIns 4-kinases have been identified only as biochemical entities that are distinguished from type III kinases based on their different sensitivities to inhibitors such as adenosine and wortmannin (1Balla T. Curr. Pharm. Des. 2001; 7: 475-507Crossref PubMed Scopus (47) Google Scholar, 14Fruman D.A. Meyers R.E. Cantley L.C. Ann. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1306) Google Scholar).We and another group (15Barylko B. Gerber S.H. Binns D.D. Grichine N. Khvotchev M. Sudhof T.C. Albanesi J.P. J. Biol. Chem. 2001; 276: 7705-7708Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 16Minogue S. Anderson J.S. Waugh M.G. dos S.M. Corless S. Cramer R. Hsuan J.J. J. Biol. Chem. 2001; 276: 16635-16640Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) recently cloned the first type II PtdIns 4-kinase, which is designated as PI4KIIα. Overexpression studies showed that PI4KIIα behaves like an integral membrane protein and is primarily organelle-associated (15Barylko B. Gerber S.H. Binns D.D. Grichine N. Khvotchev M. Sudhof T.C. Albanesi J.P. J. Biol. Chem. 2001; 276: 7705-7708Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 16Minogue S. Anderson J.S. Waugh M.G. dos S.M. Corless S. Cramer R. Hsuan J.J. J. Biol. Chem. 2001; 276: 16635-16640Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Database analyses reveal that humans have another type II PtdIns 4-kinase that has a unique N-terminal 100-amino acid sequence and a highly homologous (58% identical and 75% homologous) downstream sequence to that of PI4KIIα. This kinase, designated as PI4KIIβ, was cloned recently by Balla et al. (17Balla A. Tuymetova G. Barshishat M. Geiszt M. Balla T. J. Biol. Chem. 2002; 277: 20041-20050Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). According to this group, overexpressed PI4KIIβ colocalizes with overexpressed PI4KIIα in endosomal vesicles (17Balla A. Tuymetova G. Barshishat M. Geiszt M. Balla T. J. Biol. Chem. 2002; 277: 20041-20050Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Neither isoform is enriched obviously in the plasma membrane or the Golgi apparatus, and their endogenous distribution was not reported.The apparent low abundance of type II PI4Kα and PI4Kβ at the plasma membrane is surprising in view of previous biochemical data suggesting that they are enriched there (8Wong K. Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 13236-13241Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). This paradoxical finding also raises questions about whether PIP and PIP2 synthesis is coupled at the plasma membrane (18Anderson R.A. Boronenkov I.V. Doughman S.D. Kunz J. Loijens J.C. J. Biol. Chem. 1999; 274: 9907-9910Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar), and if so, how? One possibility that is modeled after yeast is that PIP is synthesized primarily in the Golgi, whereas PIP2 is synthesized primarily at the plasma membrane. The two processes are therefore not necessarily coupled (11Holz R.W. Hlubek M.D. Sorensen S.D. Fisher S.K. Balla T. Ozaki S. Prestwich G.D. Stuenkel E.L. Bittner M.A. J. Biol. Chem. 2000; 275: 17878-17885Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar,19Thorner J.W. Nat. Cell Biol. 2001; 3: E196-E198Crossref PubMed Scopus (7) Google Scholar). Another possibility is that PIP is synthesized in situat the plasma membrane by PtdIns 4-kinases that are recruited there by an as yet unidentified mechanism. There is already evidence for the plasma membrane and/or internal membrane recruitment of PIP5Ks and PI4KIIIβ by Rho (20Chatah N.E. Abrams C.S. J. Biol. Chem. 2001; 276: 34059-34065Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), Rac (21Hartwig J.H. Bokoch G.M. Carpenter C.L. Janmey P.A. Taylor L.A. Toker A. Stossel T.P. Cell. 1995; 82: 643-653Abstract Full Text PDF PubMed Scopus (606) Google Scholar, 22Ren X.-D. Bokoch G.M. Traynor-Kaplan A. Jenkins G.H. Anderson R.A. Schwartz M.A. Mol. Biol. Cell. 1996; 7: 435-442Crossref PubMed Scopus (199) Google Scholar), and Arf (12Honda A. Nogami M. Yokozeki T. Yamazaki M. Nakamura H. Watanabe H. Kawamoto K. Nakayama K. Morris A.J. Frohman M.A. Kanaho Y. Cell. 1999; 99: 521-532Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar, 23Godi A. Pertile P. Meyers R. Marra P. Di Tullio G. Iurisci C. Luini A. Corda D. De Matteis M.A. Nat. Cell Biol. 1999; 1: 280-287Crossref PubMed Scopus (449) Google Scholar, 24Jones D.H. Morris J.B. Morgan C.P. Kondo H. Irvine R.F. Cockcroft S. J. Biol. Chem. 2000; 275: 13962-13966Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) small GTPases.In this paper, we compared the intracellular localization of endogenous and overexpressed PI4KIIα and PI4KIIβ and examined the effect of growth factor stimulation and Rho family GTPases on their distribution and kinase activity. Our results are significantly different from those reported recently by Balla et al. (17Balla A. Tuymetova G. Barshishat M. Geiszt M. Balla T. J. Biol. Chem. 2002; 277: 20041-20050Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) for overexpressed PI4K. The differences and possible explanations are discussed at the end of this paper. Phosphatidylinositol 4-kinases (PtdIns 1The abbreviations used for: PtdIns and PI4K, phosphatidylinositol 4-kinase; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP5K, PIP5-kinase; HA, hemagglutinin; GFP, green fluorescent protein; PDGF, platelet-derived growth factor; DTT, dithiothreitol; LSP, low speed pellet; HSP, high speed pellet; TGN, trans-Golgi network. 1The abbreviations used for: PtdIns and PI4K, phosphatidylinositol 4-kinase; PIP, phosphatidylinositol 4-phosphate; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP5K, PIP5-kinase; HA, hemagglutinin; GFP, green fluorescent protein; PDGF, platelet-derived growth factor; DTT, dithiothreitol; LSP, low speed pellet; HSP, high speed pellet; TGN, trans-Golgi network.4-kinases, PI4Ks) are at the apex of the phosphoinositide cascade (1Balla T. Curr. Pharm. Des. 2001; 7: 475-507Crossref PubMed Scopus (47) Google Scholar). They phosphorylate phosphatidylinositol to generate phosphatidylinositol 4-phosphate (PIP), which is an immediate precursor of important signaling and scaffolding molecules such as phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 3,4-bisphosphate, and phosphatidylinositol 3,4,5-trisphosphate. In addition, genetic evidence inSaccharomyces cerevisiae suggests that PIP may also have a direct structural and functional role in its own right (2Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Crossref PubMed Scopus (286) Google Scholar, 3Foti M. Audhya A. Emr S.D. Mol. Biol. Cell. 2001; 12: 2396-2411Crossref PubMed Scopus (183) Google Scholar, 4Levine T.P. Munro S. Curr. Biol. 2002; 12: 695-704Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). There is increasing evidence that phosphoinositides are synthesized in a spatially and temporally defined manner to regulate signaling, cytoskeletal dynamics, and membrane trafficking (1Balla T. Curr. Pharm. Des. 2001; 7: 475-507Crossref PubMed Scopus (47) Google Scholar, 5Yin H.L. Janmey P.A. Annu. Rev. Physiol. 2003; (in press)PubMed Google Scholar, 6Di Fiore P.P. De Camilli P. Cell. 2001; 106: 1-4Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Multiple PI4Ks have been identified. Yeast has two dominant PtdIns 4-kinases, Pik1 and Stt4, that are functionally nonoverlapping (2Audhya A. Foti M. Emr S.D. Mol. Biol. Cell. 2000; 11: 2673-2689Crossref PubMed Scopus (286) Google Scholar, 7Flanagan C.A. Schnieders E.A. Emerick A.W. Kunisawa R. Admon A. Thorner J. Science. 1993; 262: 1444-1448Crossref PubMed Scopus (171) Google Scholar). Pik1 is important for cell viability and Golgi-to-plasma membrane transport that appears to be regulated primarily by PIP and not by PIP2. In contrast, Stt4 maintains vacuole morphology and supplies PIP/PIP2 for actin cytoskeletal regulation. Mammalian homologs of Pik1 and Stt4 are called type III PtdIns 4-kinase β and PtdIns 4-kinase α (PI4KIIβ and PI4KIIα), respectively. They are found in the cytosol, Golgi, and endoplasmic reticulum, and neither is reported to be enriched in the plasma membrane or in lysosomes (8Wong K. Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 13236-13241Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 9Zhao X. Varnai P. Tuymetova G. Balla A. Toth Z.E. Oker-Blom C. Roder J. Jeromin A. Balla T. J. Biol. Chem. 2001; 276: 40183-40189Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Biochemical studies suggest that another class of PtdIns 4-kinases, designated as type II kinases, accounts for most of the PtdIns 4-kinase activity in the plasma membrane of mammalian cells (8Wong K. Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 13236-13241Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Because phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks), which convert PIP to PIP2, are enriched in the plasma membrane, and PIP2 is synthesized there in response to many stimuli (10Raucher D. Stauffer T. Chen W. Shen K. Guo S. York J.D. Sheetz M.P. Meyer T. Cell. 2000; 100: 221-228Abstract Full Text Full Text PDF PubMed Scopus (580) Google Scholar, 11Holz R.W. Hlubek M.D. Sorensen S.D. Fisher S.K. Balla T. Ozaki S. Prestwich G.D. Stuenkel E.L. Bittner M.A. J. Biol. Chem. 2000; 275: 17878-17885Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, 12Honda A. Nogami M. Yokozeki T. Yamazaki M. Nakamura H. Watanabe H. Kawamoto K. Nakayama K. Morris A.J. Frohman M.A. Kanaho Y. Cell. 1999; 99: 521-532Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar, 13Micheva K.D. Holz R.W. Smith S.J. J. Cell Biol. 2001; 154: 355-368Crossref PubMed Scopus (114) Google Scholar), the type II kinases may be the major source of PIP at this critical interface. Until recently, type II PtdIns 4-kinases have been identified only as biochemical entities that are distinguished from type III kinases based on their different sensitivities to inhibitors such as adenosine and wortmannin (1Balla T. Curr. Pharm. Des. 2001; 7: 475-507Crossref PubMed Scopus (47) Google Scholar, 14Fruman D.A. Meyers R.E. Cantley L.C. Ann. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1306) Google Scholar). We and another group (15Barylko B. Gerber S.H. Binns D.D. Grichine N. Khvotchev M. Sudhof T.C. Albanesi J.P. J. Biol. Chem. 2001; 276: 7705-7708Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 16Minogue S. Anderson J.S. Waugh M.G. dos S.M. Corless S. Cramer R. Hsuan J.J. J. Biol. Chem. 2001; 276: 16635-16640Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar) recently cloned the first type II PtdIns 4-kinase, which is designated as PI4KIIα. Overexpression studies showed that PI4KIIα behaves like an integral membrane protein and is primarily organelle-associated (15Barylko B. Gerber S.H. Binns D.D. Grichine N. Khvotchev M. Sudhof T.C. Albanesi J.P. J. Biol. Chem. 2001; 276: 7705-7708Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 16Minogue S. Anderson J.S. Waugh M.G. dos S.M. Corless S. Cramer R. Hsuan J.J. J. Biol. Chem. 2001; 276: 16635-16640Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Database analyses reveal that humans have another type II PtdIns 4-kinase that has a unique N-terminal 100-amino acid sequence and a highly homologous (58% identical and 75% homologous) downstream sequence to that of PI4KIIα. This kinase, designated as PI4KIIβ, was cloned recently by Balla et al. (17Balla A. Tuymetova G. Barshishat M. Geiszt M. Balla T. J. Biol. Chem. 2002; 277: 20041-20050Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). According to this group, overexpressed PI4KIIβ colocalizes with overexpressed PI4KIIα in endosomal vesicles (17Balla A. Tuymetova G. Barshishat M. Geiszt M. Balla T. J. Biol. Chem. 2002; 277: 20041-20050Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Neither isoform is enriched obviously in the plasma membrane or the Golgi apparatus, and their endogenous distribution was not reported. The apparent low abundance of type II PI4Kα and PI4Kβ at the plasma membrane is surprising in view of previous biochemical data suggesting that they are enriched there (8Wong K. Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 13236-13241Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). This paradoxical finding also raises questions about whether PIP and PIP2 synthesis is coupled at the plasma membrane (18Anderson R.A. Boronenkov I.V. Doughman S.D. Kunz J. Loijens J.C. J. Biol. Chem. 1999; 274: 9907-9910Abstract Full Text Full Text PDF PubMed Scopus (239) Google Scholar), and if so, how? One possibility that is modeled after yeast is that PIP is synthesized primarily in the Golgi, whereas PIP2 is synthesized primarily at the plasma membrane. The two processes are therefore not necessarily coupled (11Holz R.W. Hlubek M.D. Sorensen S.D. Fisher S.K. Balla T. Ozaki S. Prestwich G.D. Stuenkel E.L. Bittner M.A. J. Biol. Chem. 2000; 275: 17878-17885Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar,19Thorner J.W. Nat. Cell Biol. 2001; 3: E196-E198Crossref PubMed Scopus (7) Google Scholar). Another possibility is that PIP is synthesized in situat the plasma membrane by PtdIns 4-kinases that are recruited there by an as yet unidentified mechanism. There is already evidence for the plasma membrane and/or internal membrane recruitment of PIP5Ks and PI4KIIIβ by Rho (20Chatah N.E. Abrams C.S. J. Biol. Chem. 2001; 276: 34059-34065Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar), Rac (21Hartwig J.H. Bokoch G.M. Carpenter C.L. Janmey P.A. Taylor L.A. Toker A. Stossel T.P. Cell. 1995; 82: 643-653Abstract Full Text PDF PubMed Scopus (606) Google Scholar, 22Ren X.-D. Bokoch G.M. Traynor-Kaplan A. Jenkins G.H. Anderson R.A. Schwartz M.A. Mol. Biol. Cell. 1996; 7: 435-442Crossref PubMed Scopus (199) Google Scholar), and Arf (12Honda A. Nogami M. Yokozeki T. Yamazaki M. Nakamura H. Watanabe H. Kawamoto K. Nakayama K. Morris A.J. Frohman M.A. Kanaho Y. Cell. 1999; 99: 521-532Abstract Full Text Full Text PDF PubMed Scopus (688) Google Scholar, 23Godi A. Pertile P. Meyers R. Marra P. Di Tullio G. Iurisci C. Luini A. Corda D. De Matteis M.A. Nat. Cell Biol. 1999; 1: 280-287Crossref PubMed Scopus (449) Google Scholar, 24Jones D.H. Morris J.B. Morgan C.P. Kondo H. Irvine R.F. Cockcroft S. J. Biol. Chem. 2000; 275: 13962-13966Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar) small GTPases. In this paper, we compared the intracellular localization of endogenous and overexpressed PI4KIIα and PI4KIIβ and examined the effect of growth factor stimulation and Rho family GTPases on their distribution and kinase activity. Our results are significantly different from those reported recently by Balla et al. (17Balla A. Tuymetova G. Barshishat M. Geiszt M. Balla T. J. Biol. Chem. 2002; 277: 20041-20050Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) for overexpressed PI4K. The differences and possible explanations are discussed at the end of this paper.