Phosphatidylinositol 5-Phosphate Biosynthesis Is Linked to PIKfyve and Is Involved in Osmotic Response Pathway in Mammalian Cells (2002)

The cellular functions, regulation and enzymology of phosphatidylinositol (PtdIns) 5-P, the newest addition to the family of phosphoinositides (PI), are still elusive. Whereas a kinase that uses PtdIns-5-P as an intracellular substrate has been assigned, a kinase that produces it remained to be identified. Here we report that PIKfyve, the enzyme found to synthesize PtdIns-5-P in vitroand PtdIns-3,5-P2 in vitro and in vivo, is responsible for PtdIns-5-P production in a cellular context. Evidence is based on examination of two groups of cell types by two independent approaches. First, [32P]orthophosphate-labeled cells (Sf9, 3T3-L1 fibroblasts, and 3T3-L1 adipocytes) that show a high pressure liquid chromatography (HPLC)-detectable peak of the PtdIns-5-P head group at basal conditions demonstrated a 20–50% increase in radioactive PtdIns-5-P amounts upon expression of PIKfyveWT. Second, cell types (HEK293), in which the basal levels of radioactive PtdIns-5-P were undetectable by HPLC head group analysis, demonstrated higher in vitro type II PIP kinase-directed conversion of the endogenous PtdIns-5-P pool into PtdIns-4,5-P2, when induced to express PIKfyveWT. Conversely, a decrease by 60% in the conversion of PtdIns-5-P to PtdIns-4,5-P2 was associated with induced expression of the dominant-negative kinase-deficient PIKfyveK1831E mutant in HEK293 cells. When 3T3-L1 fibroblasts and 3T3-L1 adipocytes were subjected to osmotic shock, levels of PtdIns-5-P measured by both approaches were found to decrease profoundly upon a hypo-osmotic stimulus. Together, these results identify PIKfyve as an enzyme responsible for PtdIns-5-P biosynthesis and indicate a role for PtdIns-5-P in osmotic response pathways in mammalian cells. The cellular functions, regulation and enzymology of phosphatidylinositol (PtdIns) 5-P, the newest addition to the family of phosphoinositides (PI), are still elusive. Whereas a kinase that uses PtdIns-5-P as an intracellular substrate has been assigned, a kinase that produces it remained to be identified. Here we report that PIKfyve, the enzyme found to synthesize PtdIns-5-P in vitroand PtdIns-3,5-P2 in vitro and in vivo, is responsible for PtdIns-5-P production in a cellular context. Evidence is based on examination of two groups of cell types by two independent approaches. First, [32P]orthophosphate-labeled cells (Sf9, 3T3-L1 fibroblasts, and 3T3-L1 adipocytes) that show a high pressure liquid chromatography (HPLC)-detectable peak of the PtdIns-5-P head group at basal conditions demonstrated a 20–50% increase in radioactive PtdIns-5-P amounts upon expression of PIKfyveWT. Second, cell types (HEK293), in which the basal levels of radioactive PtdIns-5-P were undetectable by HPLC head group analysis, demonstrated higher in vitro type II PIP kinase-directed conversion of the endogenous PtdIns-5-P pool into PtdIns-4,5-P2, when induced to express PIKfyveWT. Conversely, a decrease by 60% in the conversion of PtdIns-5-P to PtdIns-4,5-P2 was associated with induced expression of the dominant-negative kinase-deficient PIKfyveK1831E mutant in HEK293 cells. When 3T3-L1 fibroblasts and 3T3-L1 adipocytes were subjected to osmotic shock, levels of PtdIns-5-P measured by both approaches were found to decrease profoundly upon a hypo-osmotic stimulus. Together, these results identify PIKfyve as an enzyme responsible for PtdIns-5-P biosynthesis and indicate a role for PtdIns-5-P in osmotic response pathways in mammalian cells. It is now well recognized that dynamic changes in the phosphorylation status of intracellular phosphatidylinositol (PtdIns) 1The abbreviations used are: PtdIns, phosphatidylinositol; GroPIns, glycerophosphorylinositol; HPLC, high pressure liquid chromatography; PI, phosphoinositide; K, kinase; HA, hemagglutinin; P, phosphate; P2, bisphosphate; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; HEK, human embryonic kidney 1The abbreviations used are: PtdIns, phosphatidylinositol; GroPIns, glycerophosphorylinositol; HPLC, high pressure liquid chromatography; PI, phosphoinositide; K, kinase; HA, hemagglutinin; P, phosphate; P2, bisphosphate; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; HEK, human embryonic kidney regulate diverse cellular processes such as cell signaling, membrane trafficking, cytoskeletal reorganization, DNA synthesis, and cell cycle. In mammalian cells, a wide spectrum of phosphoinositide kinases and phosphatases produce seven phosphorylated metabolites of PtdIns, known so far, i.e. PtdIns-3-P, PtdIns-4-P, PtdIns-5-P, PtdIns-3,4-P2, PtdIns-3,5-P2, PtdIns-4,5-P2, and PtdIns-3,4,5-P3 (for recent reviews see Refs. 1Fruman D.A. Rameh L.E. Cantley L.C. Cell. 1999; 97: 817-820Google Scholar, 2Anderson R.A. Boronenkov I.V. Doughman S.D. Kunz J. Loijens J.C. J. Biol. Chem. 1999; 274: 9907-9910Google Scholar, 3Toker A. Curr. Opin. Cell Biol. 1998; 10: 254-261Google Scholar, 4Vanhaesbroeck B. Leevers S.J. Ahmadi K. Timms J. Katso R. Driscoll P.C. Woscholski R. Parker P.J. Waterfield M.D. Annu. Rev. Biochem. 2001; 70: 535-602Google Scholar, 5Corvera S. Czech M.P. Trends Cell Biol. 1999; 8: 442-446Google Scholar, 6Majerus P.W. Kisseleva M.V. Norris F.A. J. Biol. Chem. 1999; 274: 10669-10672Google Scholar, 7Hughes W.E. Cooke Parker P.J. Biochem. J. Scholar, S. L.E. 2001; In phosphorylated metabolites of PtdIns, were found to as that when is the and such as the the of and the the and the (for recent reviews see Refs. S. A. Curr. Opin. Cell Biol. 1999; Scholar, S.D. Trends Biochem. Scholar, Curr. Opin. Cell Biol. 2001; Scholar, Cell. 2001; 817-820Google the of it is that the kinases and phosphatases responsible for and an role in cell Whereas the phosphatases and to the of the are well P.W. Kisseleva M.V. Norris F.A. J. Biol. Chem. 1999; 274: 10669-10672Google Scholar, 7Hughes W.E. Cooke Parker P.J. Biochem. J. Scholar, S. L.E. 2001; phosphoinositide kinases been for D.A. Rameh L.E. Cantley L.C. Cell. 1999; 97: 817-820Google Scholar, 2Anderson R.A. Boronenkov I.V. Doughman S.D. Kunz J. Loijens J.C. J. Biol. Chem. 1999; 274: 9907-9910Google Scholar, 3Toker A. Curr. Opin. Cell Biol. 1998; 10: 254-261Google Scholar, 4Vanhaesbroeck B. Leevers S.J. Ahmadi K. Timms J. Katso R. Driscoll P.C. Woscholski R. Parker P.J. Waterfield M.D. Annu. Rev. Biochem. 2001; 70: 535-602Google with mammalian into a of by the phosphorylation of groups in the and D.A. Rameh L.E. Cantley L.C. Cell. 1999; 97: 817-820Google Scholar, 2Anderson R.A. Boronenkov I.V. Doughman S.D. Kunz J. Loijens J.C. J. Biol. Chem. 1999; 274: 9907-9910Google Scholar, 3Toker A. Curr. Opin. Cell Biol. 1998; 10: 254-261Google Scholar, 4Vanhaesbroeck B. Leevers S.J. Ahmadi K. Timms J. Katso R. Driscoll P.C. Woscholski R. Parker P.J. Waterfield M.D. Annu. Rev. Biochem. 2001; 70: 535-602Google which the phosphorylation at in PtdIns, PtdIns-4-P, PtdIns-4,5-P2, and PtdIns-5-P, are into and and based on in vitro substrate and of are by of substrate of PtdIns, of and are in PtdIns, and to a in PtdIns, be by the of of known as type II PIP be phosphorylated by two of type Rameh L.E. J. Cantley L.C. J. Biol. Chem. 1998; and PIKfyve A. Cell. Biol. 1999; and A. J. Biol. Chem. 1999; 274: and in A. Cell Biol. 2001; which for PtdIns, are of to PtdIns-5-P in Rameh L.E. J. Cantley L.C. J. Biol. Chem. 1998; Scholar, A. Cell. Biol. 1999; Scholar, A. J. Biol. Chem. 1999; 274: It be that the in substrate that in cells, such as substrate and enzyme regulation the enzyme with the that the of be by of higher phosphorylated that biosynthesis and are with PtdIns-5-P is by two the responsible for PtdIns-5-P production in a cellular remained The of the addition to the was of on HPLC L.E. Cantley L.C. when in mammalian cells, PtdIns-5-P was found to a of L.E. Cantley L.C. cellular PtdIns-5-P is a substrate for type II L.E. Cantley L.C. far, has been to in fibroblasts in L.E. Cantley L.C. human A. cells A. Biochem. J. 2001; results were in S.D. J. Cell Biol. 1998; Scholar, Cooke A. Parker P.J. J. Biol. Chem. 1999; 274: mammalian cell types in A. J. Biol. Chem. 2001; J. Biol. Chem. 1999; 274: is known the intracellular regulation of In levels of PtdIns-5-P been to increase upon cells PtdIns-5-P was by A. Scholar, A. Biochem. J. 2001; the of PtdIns-5-P in the higher cells an role in cellular the intracellular regulation of and of PtdIns-5-P are to the enzyme in PtdIns-5-P biosynthesis and a regulation of PtdIns-5-P we cell in which radioactive PtdIns-5-P cell is by HPLC head group In mammalian cells, for radioactive PtdIns-5-P by HPLC A. J. Biol. Chem. 2001; were in vitro for type II conversion of PtdIns-5-P to report that PIKfyve is responsible for PtdIns-5-P biosynthesis and that the response in mammalian cells a in PtdIns-5-P the of PtdIns-5-P as a substrate for type II and in fibroblasts L.E. Cantley L.C. to been a we the intracellular synthesis, and regulation of of is to the that PtdIns-5-P is a of HPLC are to that of the that of the the was the the seven to be and the of conditions in the HPLC to of PtdIns-5-P L.E. Cantley L.C. When PtdIns-5-P was as a peak the of the the Rameh L.E. J. Cantley L.C. J. Biol. Chem. 1998; Scholar, A. J. Biol. Chem. 1999; 274: Scholar, L.E. Cantley L.C. Scholar, and amounts of PtdIns-5-P are to be the an for PtdIns-5-P intracellular has been based on the substrate of type II for PtdIns-5-P and the of PtdIns-4,5-P2 by HPLC A. The of was to the PIKfyve enzyme was responsible for PtdIns-5-P intracellular and to osmotic in PtdIns-5-P intracellular we both for of intracellular PtdIns-5-P, i.e. an HPLC head group of radioactive PtdIns-5-P cell with and the type II conversion of PtdIns-5-P to that higher PtdIns-5-P levels are associated with cell expression of kinase-deficient the for the of a decrease in PtdIns-5-P cellular production was upon hypo-osmotic in 3T3-L1 cells PtdIns-5-P as a in the osmotic response in mammalian role of PIKfyve in PtdIns-5-P cellular production has been by the a of a PIKfyve to the conversion of to PtdIns-5-P A. Cell. Biol. 1999; A. J. Biol. Chem. 1999; 274: in both in and mammalian cell types high levels of to intracellular PtdIns-5-P production Cooke A. Parker P.J. J. Biol. Chem. 1999; 274: Scholar, A. J. Biol. Chem. 2001; an increase of to be these PIKfyve has been in PtdIns-3,5-P2 in PtdIns-5-P the cell types so undetectable of radioactive PtdIns-5-P at conditions in in to HPLC head group Cooke A. Parker P.J. J. Biol. Chem. 1999; 274: Scholar, A. J. Biol. Chem. 2001; that of been PtdIns-5-P production an of PIKfyve which a conditions of PIKfyve in cells. in as an of the PIKfyve kinase S.D. J. Cell Biol. Scholar, A. Curr. Biol. indicate that be group of a PtdIns-5-P with of the pool cell PtdIns-5-P HPLC analysis, these was in the at in by two First, of cell types in which the of radioactive PtdIns-5-P peak upon HPLC head group was at and of the PtdIns-5-P conversion in cell types with undetectable levels of PtdIns-5-P HPLC demonstrated an increase of PtdIns-5-P production associated with expression of high levels of in both mammalian and cells, the in PtdIns-5-P is is in the by the a decrease of PtdIns-5-P levels by the expression of the dominant-negative kinase-deficient PIKfyveK1831E PIKfyve is an found in A. Cell Biol. 2001; a basal PtdIns-5-P production has been to of the A. Biochem. J. 2001; we a that to PIKfyve in these cells. It to be by PtdIns-5-P conversion the of PtdIns-5-P production in cells S.D. J. Cell Biol. 1998; Scholar, Cooke A. Parker P.J. J. Biol. Chem. 1999; 274: is associated with in a substrate of In is to into PtdIns-5-P in vitro Cooke A. Parker P.J. J. Biol. Chem. 1999; 274: of the is the that the intracellular PIKfyve kinase two we a increase of both PtdIns-5-P and PtdIns-3,5-P2 production upon of 3T3-L1 adipocytes with A. J. Biol. Chem. 2001; was in the HEK293 cell we higher PtdIns-5-P levels by and an increase of the by HPLC cells induced to express A. J. Biol. Chem. 2001; and that intracellular regulate PIKfyve substrate is the that such regulation in the cellular is by recent substrate for upon of in PIKfyve, K. J. A. J. Biol. Chem. in the is the that PtdIns-5-P is profoundly upon hypo-osmotic in 3T3-L1 fibroblasts and these cells increase in PtdIns-5-P, a PtdIns-4,5-P2 we a of type II that be in Whereas to PtdIns-5-P in mammalian cells to the of a recent A. Biochem. J. 2001; in cells has demonstrated a and increase in PtdIns-5-P upon with for a of in the the PtdIns-4,5-P2 levels were to at the A. Biochem. J. 2001; a type II conversion of PtdIns-5-P in cell Together, these are with the that changes in PtdIns-5-P levels are in the cellular and be It is to that PtdIns-5-P, has a and is to with to cell osmotic of 3T3-L1 adipocytes is to the of and membrane cellular by and indicate that these two both and J. Biol. Chem. Scholar, Cell. Biol. 1999; Scholar, A. J. Biol. Chem. It be in to a role of PtdIns-5-P in the of osmotic and in cell dominant-negative kinase-deficient been to in 3T3-L1 adipocytes K. A. intracellular PtdIns-5-P production a role for PIKfyve in PtdIns-5-P be the enzymology of that of PtdIns-5-P, such a is the that we were to regulation in the in vitro PIKfyve kinase levels of PtdIns-5-P decrease to of PIKfyve, and PtdIns-3,5-P2 of type II and PtdIns-5-P such phosphatases been P.W. Kisseleva M.V. Norris F.A. J. Biol. Chem. 1999; 274: 10669-10672Google Scholar, 7Hughes W.E. Cooke Parker P.J. Biochem. J. Scholar, S. L.E. 2001; it be that hypo-osmotic in 3T3-L1 cells a in and PtdIns-4,5-P2 amounts to and results and the of the two PIKfyve a for the of PtdIns-5-P and both associated with the kinase of PIKfyve is with the for a hypo-osmotic of PIKfyve in vivo, it to be in we that PIKfyve kinase is responsible for biosynthesis of PtdIns-5-P, a was kinase remained elusive. The of PtdIns-5-P conversion to PtdIns-4,5-P2 by type II the of It is that PtdIns-5-P is a for an production of PtdIns-3,5-P2 that the of a The that hypo-osmotic of 3T3-L1 cells a decrease of PtdIns-5-P levels that PtdIns-5-P be a in response pathways in mammalian cells. It is now well recognized that dynamic changes in the phosphorylation status of intracellular phosphatidylinositol (PtdIns) 1The abbreviations used are: PtdIns, phosphatidylinositol; GroPIns, glycerophosphorylinositol; HPLC, high pressure liquid chromatography; PI, phosphoinositide; K, kinase; HA, hemagglutinin; P, phosphate; P2, bisphosphate; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; HEK, human embryonic kidney 1The abbreviations used are: PtdIns, phosphatidylinositol; GroPIns, glycerophosphorylinositol; HPLC, high pressure liquid chromatography; PI, phosphoinositide; K, kinase; HA, hemagglutinin; P, phosphate; P2, bisphosphate; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium; HEK, human embryonic kidney regulate diverse cellular processes such as cell signaling, membrane trafficking, cytoskeletal reorganization, DNA synthesis, and cell cycle. In mammalian cells, a wide spectrum of phosphoinositide kinases and phosphatases produce seven phosphorylated metabolites of PtdIns, known so far, i.e. PtdIns-3-P, PtdIns-4-P, PtdIns-5-P, PtdIns-3,4-P2, PtdIns-3,5-P2, PtdIns-4,5-P2, and PtdIns-3,4,5-P3 (for recent reviews see Refs. 1Fruman D.A. Rameh L.E. Cantley L.C. Cell. 1999; 97: 817-820Google Scholar, 2Anderson R.A. Boronenkov I.V. Doughman S.D. Kunz J. Loijens J.C. J. Biol. Chem. 1999; 274: 9907-9910Google Scholar, 3Toker A. Curr. Opin. Cell Biol. 1998; 10: 254-261Google Scholar, 4Vanhaesbroeck B. Leevers S.J. Ahmadi K. Timms J. Katso R. Driscoll P.C. Woscholski R. Parker P.J. Waterfield M.D. Annu. Rev. Biochem. 2001; 70: 535-602Google Scholar, 5Corvera S. Czech M.P. Trends Cell Biol. 1999; 8: 442-446Google Scholar, 6Majerus P.W. Kisseleva M.V. Norris F.A. J. Biol. Chem. 1999; 274: 10669-10672Google Scholar, 7Hughes W.E. Cooke Parker P.J. Biochem. J. Scholar, S. L.E. 2001; In phosphorylated metabolites of PtdIns, were found to as that when is the and such as the the of and the the and the (for recent reviews see Refs. S. A. Curr. Opin. Cell Biol. 1999; Scholar, S.D. Trends Biochem. Scholar, Curr. Opin. Cell Biol. 2001; Scholar, Cell. 2001; 817-820Google the of it is that the kinases and phosphatases responsible for and an role in cell Whereas the phosphatases and to the of the are well P.W. Kisseleva M.V. Norris F.A. J. Biol. Chem. 1999; 274: 10669-10672Google Scholar, 7Hughes W.E. Cooke Parker P.J. Biochem. J. Scholar, S. L.E. 2001; phosphoinositide kinases been for D.A. Rameh L.E. Cantley L.C. Cell. 1999; 97: 817-820Google Scholar, 2Anderson R.A. Boronenkov I.V. Doughman S.D. Kunz J. Loijens J.C. J. Biol. Chem. 1999; 274: 9907-9910Google Scholar, 3Toker A. Curr. Opin. Cell Biol. 1998; 10: 254-261Google Scholar, 4Vanhaesbroeck B. Leevers S.J. Ahmadi K. Timms J. Katso R. Driscoll P.C. Woscholski R. Parker P.J. Waterfield M.D. Annu. Rev. Biochem. 2001; 70: 535-602Google with mammalian into a of by the phosphorylation of groups in the and D.A. Rameh L.E. Cantley L.C. Cell. 1999; 97: 817-820Google Scholar, 2Anderson R.A. Boronenkov I.V. Doughman S.D. Kunz J. Loijens J.C. J. Biol. Chem. 1999; 274: 9907-9910Google Scholar, 3Toker A. Curr. Opin. Cell Biol. 1998; 10: 254-261Google Scholar, 4Vanhaesbroeck B. Leevers S.J. Ahmadi K. Timms J. Katso R. Driscoll P.C. Woscholski R. Parker P.J. Waterfield M.D. Annu. Rev. Biochem. 2001; 70: 535-602Google which the phosphorylation at in PtdIns, PtdIns-4-P, PtdIns-4,5-P2, and PtdIns-5-P, are into and and based on in vitro substrate and of are by of substrate of PtdIns, of and are in PtdIns, and to a in PtdIns, be by the of of known as type II PIP be phosphorylated by two of type Rameh L.E. J. Cantley L.C. J. Biol. Chem. 1998; and PIKfyve A. Cell. Biol. 1999; and A. J. Biol. Chem. 1999; 274: and in A. Cell Biol. 2001; which for PtdIns, are of to PtdIns-5-P in Rameh L.E. J. Cantley L.C. J. Biol. Chem. 1998; Scholar, A. Cell. Biol. 1999; Scholar, A. J. Biol. Chem. 1999; 274: It be that the in substrate that in cells, such as substrate and enzyme regulation the enzyme with the that the of be by of higher phosphorylated that biosynthesis and are with PtdIns-5-P is by two the responsible for PtdIns-5-P production in a cellular remained The of the addition to the was of on HPLC L.E. Cantley L.C. when in mammalian cells, PtdIns-5-P was found to a of L.E. Cantley L.C. cellular PtdIns-5-P is a substrate for type II L.E. Cantley L.C. far, has been to in fibroblasts in L.E. Cantley L.C. human A. cells A. Biochem. J. 2001; results were in S.D. J. Cell Biol. 1998; Scholar, Cooke A. Parker P.J. J. Biol. Chem. 1999; 274: mammalian cell types in A. J. Biol. Chem. 2001; J. Biol. Chem. 1999; 274: is known the intracellular regulation of In levels of PtdIns-5-P been to increase upon cells PtdIns-5-P was by A. Scholar, A. Biochem. J. 2001; the of PtdIns-5-P in the higher cells an role in cellular the intracellular regulation of and of PtdIns-5-P are to the enzyme in PtdIns-5-P biosynthesis and a regulation of PtdIns-5-P we cell in which radioactive PtdIns-5-P cell is by HPLC head group In mammalian cells, for radioactive PtdIns-5-P by HPLC A. J. Biol. Chem. 2001; were in vitro for type II conversion of PtdIns-5-P to report that PIKfyve is responsible for PtdIns-5-P biosynthesis and that the response in mammalian cells a in PtdIns-5-P the of PtdIns-5-P as a substrate for type II and in fibroblasts L.E. Cantley L.C. to been a we the intracellular synthesis, and regulation of of is to the that PtdIns-5-P is a of HPLC are to that of the that of the the was the the seven to be and the of conditions in the HPLC to of PtdIns-5-P L.E. Cantley L.C. When PtdIns-5-P was as a peak the of the the Rameh L.E. J. Cantley L.C. J. Biol. Chem. 1998; Scholar, A. J. Biol. Chem. 1999; 274: Scholar, L.E. Cantley L.C. Scholar, and amounts of PtdIns-5-P are to be the an for PtdIns-5-P intracellular has been based on the substrate of type II for PtdIns-5-P and the of PtdIns-4,5-P2 by HPLC A. The of was to the PIKfyve enzyme was responsible for PtdIns-5-P intracellular and to osmotic in PtdIns-5-P intracellular we both for of intracellular PtdIns-5-P, i.e. an HPLC head group of radioactive PtdIns-5-P cell with and the type II conversion of PtdIns-5-P to that higher PtdIns-5-P levels are associated with cell expression of kinase-deficient the for the of a decrease in PtdIns-5-P cellular production was upon hypo-osmotic in 3T3-L1 cells PtdIns-5-P as a in the osmotic response in mammalian role of PIKfyve in PtdIns-5-P cellular production has been by the a of a PIKfyve to the conversion of to PtdIns-5-P A. Cell. Biol. 1999; A. J. Biol. Chem. 1999; 274: in both in and mammalian cell types high levels of to intracellular PtdIns-5-P production Cooke A. Parker P.J. J. Biol. Chem. 1999; 274: Scholar, A. J. Biol. Chem. 2001; an increase of to be these PIKfyve has been in PtdIns-3,5-P2 in PtdIns-5-P the cell types so undetectable of radioactive PtdIns-5-P at conditions in in to HPLC head group Cooke A. Parker P.J. J. Biol. Chem. 1999; 274: Scholar, A. J. Biol. Chem. 2001; that of been PtdIns-5-P production an of PIKfyve which a conditions of PIKfyve in cells. in as an of the PIKfyve kinase S.D. J. Cell Biol. Scholar, A. Curr. Biol. indicate that be group of a PtdIns-5-P with of the pool cell PtdIns-5-P HPLC analysis, these was in the at in by two First, of cell types in which the of radioactive PtdIns-5-P peak upon HPLC head group was at and of the PtdIns-5-P conversion in cell types with undetectable levels of PtdIns-5-P HPLC demonstrated an increase of PtdIns-5-P production associated with expression of high levels of in both mammalian and cells, the in PtdIns-5-P is is in the by the a decrease of PtdIns-5-P levels by the expression of the dominant-negative kinase-deficient PIKfyveK1831E PIKfyve is an found in A. Cell Biol. 2001; a basal PtdIns-5-P production has been to of the A. Biochem. J. 2001; we a that to PIKfyve in these cells. It to be by PtdIns-5-P conversion the of PtdIns-5-P production in cells S.D. J. Cell Biol. 1998; Scholar, Cooke A. Parker P.J. J. Biol. Chem. 1999; 274: is associated with in a substrate of In is to into PtdIns-5-P in vitro Cooke A. Parker P.J. J. Biol. Chem. 1999; 274: of the is the that the intracellular PIKfyve kinase two we a increase of both PtdIns-5-P and PtdIns-3,5-P2 production upon of 3T3-L1 adipocytes with A. J. Biol. Chem. 2001; was in the HEK293 cell we higher PtdIns-5-P levels by and an increase of the by HPLC cells induced to express A. J. Biol. Chem. 2001; and that intracellular regulate PIKfyve substrate is the that such regulation in the cellular is by recent substrate for upon of in PIKfyve, K. J. A. J. Biol. Chem. in the is the that PtdIns-5-P is profoundly upon hypo-osmotic in 3T3-L1 fibroblasts and these cells increase in PtdIns-5-P, a PtdIns-4,5-P2 we a of type II that be in Whereas to PtdIns-5-P in mammalian cells to the of a recent A. Biochem. J. 2001; in cells has demonstrated a and increase in PtdIns-5-P upon with for a of in the the PtdIns-4,5-P2 levels were to at the A. Biochem. J. 2001; a type II conversion of PtdIns-5-P in cell Together, these are with the that changes in PtdIns-5-P levels are in the cellular and be It is to that PtdIns-5-P, has a and is to with to cell osmotic of 3T3-L1 adipocytes is to the of and membrane cellular by and indicate that these two both and J. Biol. Chem. Scholar, Cell. Biol. 1999; Scholar, A. J. Biol. Chem. It be in to a role of PtdIns-5-P in the of osmotic and in cell dominant-negative kinase-deficient been to in 3T3-L1 adipocytes K. A. intracellular PtdIns-5-P production a role for PIKfyve in PtdIns-5-P be the enzymology of that of PtdIns-5-P, such a is the that we were to regulation in the in vitro PIKfyve kinase levels of PtdIns-5-P decrease to of PIKfyve, and PtdIns-3,5-P2 of type II and PtdIns-5-P such phosphatases been P.W. Kisseleva M.V. Norris F.A. J. Biol. Chem. 1999; 274: 10669-10672Google Scholar, 7Hughes W.E. Cooke Parker P.J. Biochem. J. Scholar, S. L.E. 2001; it be that hypo-osmotic in 3T3-L1 cells a in and PtdIns-4,5-P2 amounts to and results and the of the two PIKfyve a for the of PtdIns-5-P and both associated with the kinase of PIKfyve is with the for a hypo-osmotic of PIKfyve in vivo, it to be in we that PIKfyve kinase is responsible for biosynthesis of PtdIns-5-P, a was kinase remained elusive. The of PtdIns-5-P conversion to PtdIns-4,5-P2 by type II the of It is that PtdIns-5-P is a for an production of PtdIns-3,5-P2 that the of a The that hypo-osmotic of 3T3-L1 cells a decrease of PtdIns-5-P levels that PtdIns-5-P be a in response pathways in mammalian cells. the of PtdIns-5-P as a substrate for type II and in fibroblasts L.E. Cantley L.C. to been a we the intracellular synthesis, and regulation of of is to the that PtdIns-5-P is a of HPLC are to that of the that of the the was the the seven to be and the of conditions in the HPLC to of PtdIns-5-P L.E. Cantley L.C. When PtdIns-5-P was as a peak the of the the Rameh L.E. J. Cantley L.C. J. Biol. Chem. 1998; Scholar, A. J. Biol. Chem. 1999; 274: Scholar, L.E. Cantley L.C. Scholar, and amounts of PtdIns-5-P are to be the an for PtdIns-5-P intracellular has been based on the substrate of type II for PtdIns-5-P and the of PtdIns-4,5-P2 by HPLC A. The of was to the PIKfyve enzyme was responsible for PtdIns-5-P intracellular and to osmotic in PtdIns-5-P intracellular we both for of intracellular PtdIns-5-P, i.e. an HPLC head group of radioactive PtdIns-5-P cell with and the type II conversion of PtdIns-5-P to that higher PtdIns-5-P levels are associated with cell expression of kinase-deficient the for the of a decrease in PtdIns-5-P cellular production was upon hypo-osmotic in 3T3-L1 cells PtdIns-5-P as a in the osmotic response in mammalian cells. The role of PIKfyve in PtdIns-5-P cellular production has been by the a of a PIKfyve to the conversion of to PtdIns-5-P A. Cell. Biol. 1999; A. J. Biol. Chem. 1999; 274: in both in and mammalian cell types high levels of to intracellular PtdIns-5-P production Cooke A. Parker P.J. J. Biol. Chem. 1999; 274: Scholar, A. J. Biol. Chem. 2001; an increase of to be these PIKfyve has been in PtdIns-3,5-P2 in PtdIns-5-P the cell types so undetectable of radioactive PtdIns-5-P at conditions in in to HPLC head group Cooke A. Parker P.J. J. Biol. Chem. 1999; 274: Scholar, A. J. Biol. Chem. 2001; that of been PtdIns-5-P production an of PIKfyve which a conditions of PIKfyve in cells. in as an of the PIKfyve kinase S.D. J. Cell Biol. Scholar, A. Curr. Biol. indicate that be group of a PtdIns-5-P with of the pool cell PtdIns-5-P HPLC analysis, these was in the at in by two First, of cell types in which the of radioactive PtdIns-5-P peak upon HPLC head group was at and of the PtdIns-5-P conversion in cell types with undetectable levels of PtdIns-5-P HPLC demonstrated an increase of PtdIns-5-P production associated with expression of high levels of in both mammalian and cells, the in PtdIns-5-P is is in the by the a decrease of PtdIns-5-P levels by the expression of the dominant-negative kinase-deficient PIKfyveK1831E PIKfyve is an found in A. Cell Biol. 2001; a basal PtdIns-5-P production has been to of the A. Biochem. J. 2001; we a that to PIKfyve in these cells. It to be by PtdIns-5-P conversion the of PtdIns-5-P production in cells S.D. J. Cell Biol. 1998; Scholar, Cooke A. Parker P.J. J. Biol. Chem. 1999; 274: is associated with in a substrate of In is to into PtdIns-5-P in vitro Cooke A. Parker P.J. J. Biol. Chem. 1999; 274: of the is the that the intracellular PIKfyve kinase two we a increase of both PtdIns-5-P and PtdIns-3,5-P2 production upon of 3T3-L1 adipocytes with A. J. Biol. Chem. 2001; was in the HEK293 cell we higher PtdIns-5-P levels by and an increase of the by HPLC cells induced to express A. J. Biol. Chem. 2001; and that intracellular regulate PIKfyve substrate is the that such regulation in the cellular is by recent substrate for upon of in PIKfyve, K. J. A. J. Biol. Chem. in the is the that PtdIns-5-P is profoundly upon hypo-osmotic in 3T3-L1 fibroblasts and these cells increase in PtdIns-5-P, a PtdIns-4,5-P2 we a of type II that be in Whereas to PtdIns-5-P in mammalian cells to the of a recent A. Biochem. J. 2001; in cells has demonstrated a and increase in PtdIns-5-P upon with for a of in the the PtdIns-4,5-P2 levels were to at the A. Biochem. J. 2001; a type II conversion of PtdIns-5-P in cell Together, these are with the that changes in PtdIns-5-P levels are in the cellular and be It is to that PtdIns-5-P, has a and is to with to cell osmotic of 3T3-L1 adipocytes is to the of and membrane cellular by and indicate that these two both and J. Biol. Chem. Scholar, Cell. Biol. 1999; Scholar, A. J. Biol. Chem. It be in to a role of PtdIns-5-P in the of osmotic and in cell dominant-negative kinase-deficient been to in 3T3-L1 adipocytes K. A. intracellular PtdIns-5-P production a role for PIKfyve in PtdIns-5-P be the enzymology of that of PtdIns-5-P, such a is the that we were to regulation in the in vitro PIKfyve kinase levels of PtdIns-5-P decrease to of PIKfyve, and PtdIns-3,5-P2 of type II and PtdIns-5-P such phosphatases been P.W. Kisseleva M.V. Norris F.A. J. Biol. Chem. 1999; 274: 10669-10672Google Scholar, 7Hughes W.E. Cooke Parker P.J. Biochem. J. Scholar, S. L.E. 2001; it be that hypo-osmotic in 3T3-L1 cells a in and PtdIns-4,5-P2 amounts to and results and the of the two PIKfyve a for the of PtdIns-5-P and both associated with the kinase of PIKfyve is with the for a hypo-osmotic of PIKfyve in vivo, it to be in In we that PIKfyve kinase is responsible for biosynthesis of PtdIns-5-P, a was kinase remained elusive. The of PtdIns-5-P conversion to PtdIns-4,5-P2 by type II the of It is that PtdIns-5-P is a for an production of PtdIns-3,5-P2 that the of a The that hypo-osmotic of 3T3-L1 cells a decrease of PtdIns-5-P levels that PtdIns-5-P be a in response pathways in mammalian cells. for the for the of and of the