PIKfyve, a Mammalian Ortholog of Yeast Fab1p Lipid Kinase, Synthesizes 5-Phosphoinositides

Abstract

One or more free hydroxyls of the phosphatidylinositol (PtdIns) head group undergo enzymatic phosphorylation, yielding phosphoinositides (PIs) with key functions in eukaryotic cellular regulation. Two such species, PtdIns 5-P and PtdIns 3,5-P2, have now been identified in mammalian cells, but their biosynthesis remains unclear. We have isolated a novel mammalian PI kinase, p235, whose exact substrate specificity remained to be determined (Shisheva, A., Sbrissa, D., and Ikonomov, O. (1999)Mol. Cell. Biol. 19, 623–634). Here we report that recombinant p235 expressed in COS cells, like the authentic p235 in adipocytes, displays striking specificity for PtdIns over PI substrates and generates two products identified as PtdIns 5-P and PtdIns 3,5-P2 by HPLC analyses. Synthetic PtdIns 3-P substrates were also converted to PtdIns 3,5-P2 but to a substantially lesser extent than PtdIns isolated from natural sources. Important properties of the p235 PI 5-kinase include high sensitivity to nonionic detergents and relative resistance to wortmannin and adenosine. By analyzing deletion mutants in a heterologous cell system, we determined that in addition to the predicted catalytic domain other regions of the molecule are critical for the p235 enzymatic activity. HPLC resolution of monophosphoinositide products, generated by p235 immune complexes derived from lysates of 3T3-L1 adipocytes acutely stimulated with insulin, revealed essentially the same PtdIns 5-P levels as the corresponding p235 immune complexes of resting cells. However, the acute insulin action resulted in an increase of a wortmannin-sensitive PtdIns 3-P peak, suggestive of a plausible recruitment of wortmannin-sensitive PI 3-kinase(s) to p235. In conclusion, mouse p235 (renamed here PIKfyve) displays a strong in vitro activity for PtdIns 5-P and PtdIns 3,5-P2 generation, implying PIKfyve has a key role in their biosynthesis. One or more free hydroxyls of the phosphatidylinositol (PtdIns) head group undergo enzymatic phosphorylation, yielding phosphoinositides (PIs) with key functions in eukaryotic cellular regulation. Two such species, PtdIns 5-P and PtdIns 3,5-P2, have now been identified in mammalian cells, but their biosynthesis remains unclear. We have isolated a novel mammalian PI kinase, p235, whose exact substrate specificity remained to be determined (Shisheva, A., Sbrissa, D., and Ikonomov, O. (1999)Mol. Cell. Biol. 19, 623–634). Here we report that recombinant p235 expressed in COS cells, like the authentic p235 in adipocytes, displays striking specificity for PtdIns over PI substrates and generates two products identified as PtdIns 5-P and PtdIns 3,5-P2 by HPLC analyses. Synthetic PtdIns 3-P substrates were also converted to PtdIns 3,5-P2 but to a substantially lesser extent than PtdIns isolated from natural sources. Important properties of the p235 PI 5-kinase include high sensitivity to nonionic detergents and relative resistance to wortmannin and adenosine. By analyzing deletion mutants in a heterologous cell system, we determined that in addition to the predicted catalytic domain other regions of the molecule are critical for the p235 enzymatic activity. HPLC resolution of monophosphoinositide products, generated by p235 immune complexes derived from lysates of 3T3-L1 adipocytes acutely stimulated with insulin, revealed essentially the same PtdIns 5-P levels as the corresponding p235 immune complexes of resting cells. However, the acute insulin action resulted in an increase of a wortmannin-sensitive PtdIns 3-P peak, suggestive of a plausible recruitment of wortmannin-sensitive PI 3-kinase(s) to p235. In conclusion, mouse p235 (renamed here PIKfyve) displays a strong in vitro activity for PtdIns 5-P and PtdIns 3,5-P2 generation, implying PIKfyve has a key role in their biosynthesis. Phosphorylated species of phosphatidylinositol (PtdIns) 1The abbreviations used are: PtdIns, phosphatidylinositol; PI, phosphoinositide; P, phosphate; P2, bisphosphate; P3, trisphosphate; GroPIns, glycerophosphorylinositol; K, kinase; HPLC, high pressure liquid chromatography; GST, glutathione S- transferase; HA, hemagluttinin.1The abbreviations used are: PtdIns, phosphatidylinositol; PI, phosphoinositide; P, phosphate; P2, bisphosphate; P3, trisphosphate; GroPIns, glycerophosphorylinositol; K, kinase; HPLC, high pressure liquid chromatography; GST, glutathione S- transferase; HA, hemagluttinin. are an attribute of eukaryotes, where they regulate diverse cellular processes such as membrane ruffling, secretion, vesicular trafficking, insulin-mediated membrane translocation of GLUT4 glucose transporters, cell adhesion, chemotaxis, DNA synthesis, and cell cycle (for recent reviews see Refs.1Liscovitch M. Cantley L.C. 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Although the inositol head group of PtdIns contains five candidate phosphorylation positions, the hydroxyl groups only at positions D-3, D-4, and D-5 are found to be phosphorylated intracellularly, separately, or in all possible combinations, resulting in 7 phosphoinositide (PI) species, i.e. PtdIns 3-P, PtdIns 4-P, PtdIns 5-P, PtdIns 3,4-P2, PtdIns 4,5-P2, PtdIns 3,5-P2, and PtdIns 3,4,5-P3 (3Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Crossref PubMed Scopus (575) Google Scholar, 8Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1310) Google Scholar). A wide spectrum of phosphoinositide kinases with broad or more restricted substrate specificity are responsible for their biosynthesis. These enzymes are in the of their specificity for a PI PI and PI J.C. Boronenkov I.V. Parker G.J. Anderson R.A. Adv. Enzyme Regul. 1996; 36: 115-140Crossref PubMed Scopus (83) Google Scholar, 3Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Crossref PubMed Scopus (575) Google Scholar, 4Toker A. Cantley L.C. Nature. 1997; 387: 673-676Crossref PubMed Scopus (1221) Google Scholar, 7Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (837) Google Scholar, 8Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1310) Google Scholar). PI the phosphorylation of PtdIns or phosphorylated PtdIns and PtdIns at the of the inositol PI are also by all substrate specificity of PtdIns, but of PI the phosphorylated PtdIns and are now PtdIns D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1310) Google Scholar). In of phosphorylated PtdIns, but of PtdIns, be by the enzymatic activity of of PI as J.C. Boronenkov I.V. Parker G.J. Anderson R.A. Adv. Enzyme Regul. 1996; 36: 115-140Crossref PubMed Scopus (83) Google and D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1310) Google Scholar, A. Curr. Opin. Cell Biol. 1998; PubMed Scopus Google to more their substrate specificity Cantley L.C. Nature. 1997; PubMed Scopus Google Scholar). by a of enzymes now as PI D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1310) Google Scholar, A. Curr. Opin. Cell Biol. 1998; PubMed Scopus Google 2Loijens J.C. Boronenkov I.V. Parker G.J. Anderson R.A. Adv. Enzyme Regul. 1996; 36: 115-140Crossref PubMed Scopus (83) Google and I.V. Anderson R.A. J. Biol. 1995; Full Text Full Text PDF PubMed Scopus Google for phosphorylated PtdIns J.C. Boronenkov I.V. Parker G.J. J. O. Anderson R.A. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Cantley L.C. C.L. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). a substantially lesser the enzymes are of PtdIns to PtdIns 5-P J. Cantley L.C. C.L. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). an enzymatic activity of PtdIns has been in mammalian the PtdIns 5-P species are found at high to that of PI 3-P Cantley L.C. Nature. 1997; PubMed Scopus Google Scholar). We have identified a novel mammalian p235, whose in A. O. Cell. Biol. PubMed Scopus Google Scholar). p235 A. O. Cell. Biol. PubMed Scopus Google with a 3-P A. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, J. A. Nature. 1998; PubMed Scopus Google Scholar, Cell. 1998; Full Text Full Text PDF PubMed Scopus Google a Biochem. 1998; Full Text PDF PubMed Scopus Google and a PI domain J.C. Boronenkov I.V. Parker G.J. Anderson R.A. Adv. Enzyme Regul. 1996; 36: 115-140Crossref PubMed Scopus (83) Google Scholar). the predicted of p235 and the mammalian PI and the that p235 be a or the of PtdIns phosphorylated or D-5 positions to be substrates of the p235 in the p235 found to PtdIns to A. O. Cell. Biol. PubMed Scopus Google whose exact remained to be p235 and in the with a A. Boronenkov I.V. Anderson R.A. Biol. Cell. 1995; PubMed Scopus Google whose in in the of has been now determined to be PtdIns 3,5-P2 J.C. J. Cell Biol. 1998; PubMed Scopus Google Scholar, Parker Curr. Biol. 1998; 8: Full Text Full Text PDF PubMed Google Scholar). of the to in the specificity of p235 for a inositol substrate and phosphorylation and a plausible of enzymatic activity by HPLC that the and we report here that the and the recombinant p235 to PtdIns to PtdIns 5-P and PtdIns 3,5-P2 in activity of the to remains acute cell with p235 the mammalian phosphoinositide with a enzymatic activity to PtdIns 5-P and PtdIns substrates were from the PtdIns, and from PtdIns 3-P from PtdIns 4-P, PtdIns 4,5-P2, and from PtdIns, PtdIns 4-P, PtdIns 5-P, and PtdIns from 4-P, and were from and were from from and from a recombinant the of p235 used the has been found to that of the p235 used in A. O. Cell. Biol. PubMed Scopus Google Scholar). a from and 5-P and PtdIns 3-P were a from of PI A. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google a from and insulin a from of a from the p235 in A. O. Cell. Biol. PubMed Scopus Google Scholar). of the in p235 were used to the role of regions for the p235 enzymatic activity. the domain the of the or the domain in the of the molecule were the deletion generated by the A. O. Cell. Biol. PubMed Scopus Google in with and of deletion generated by the in with resulting of and were the from the and Two were for the deletion the of the in with resulting two of and were and the of the In the the and with the of p235 by of all deletion by and from were at the with a derived from as we A. O. Cell. Biol. PubMed Scopus Google Scholar, A. Cell. Biol. PubMed Scopus Google Scholar). the the the and with and with the p235 deletion and of the mammalian of all mutants by for of 3T3-L1 mouse adipocytes were as A. J. M. J. Biol. Full Text PDF PubMed Google Scholar). were used 7 and the of the were to in and were at with or deletion mutants by the as we A. J. Cell Biol. 1995; Google Scholar). to the cell lysates were in and and and used for 3T3-L1 adipocytes, for to the were in the or of insulin for at in the were in at and the lysates were to with or p235 from 3T3-L1 adipocytes or the or p235 deletion or to the the were in lysates were by for in a at and with immune or for at in the of at were with in Nature. PubMed Scopus Google and by or to to their for activity. the were by the were with A. O. Cell. Biol. PubMed Scopus Google and at with or were with and a the were with with with with and with in the for at for HPLC and substrate PtdIns 3-P, PtdIns 4-P, PtdIns 5-P, PtdIns 3,4-P2, or PtdIns in by in a substrates and were as a they were in a in the with of and the were with of and with of of the resulting were and were by J. Cantley L.C. C.L. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, Anderson R.A. J. Biol. Full Text PDF PubMed Google as in the products were by in were with PI products, the the corresponding to that identified the were and at for with essentially as Cantley L.C. PubMed Scopus Google Scholar). and the the were at and in the same by the addition of by for at a products were in of and to of a HPLC with a and and a with an were a with and at a of a Cantley L.C. Nature. 1997; PubMed Scopus Google Scholar, J. Cantley L.C. C.L. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). of with for and and with to over to over to over and with for the phosphorylated PI, the as to the and to over and to over the and from and 4,5-P2, as and in at or 5-P were as HPLC PI, as also used as were and their for and and products with of liquid a liquid We have an of p235 immune complexes derived from 3T3-L1 adipocytes or COS to in an in vitro A. O. Cell. Biol. PubMed Scopus Google Scholar). However, p235 with the predicted catalytic regions of enzymes with for PtdIns substrates or p235 substrate specificity in more we have now PI species phosphorylated to a extent and at positions as substrates for p235. We used p235 immune complexes derived from two cell 3T3-L1 adipocytes and COS an of p235. in were a of a p235 the specificity and the of for p235, an p235 or cells, and cell lysates were with or were to to that expressed p235. in the with the a that with the and the of p235 identified in A. O. Cell. Biol. PubMed Scopus Google Scholar). p235 in of the and p235 and with of the p235 with the used in and A. O. Cell. Biol. PubMed Scopus Google Scholar). immune complexes isolated from 3T3-L1 lysates p235 were to a in the of PtdIns or PtdIns 3-P PtdIns or PtdIns 5-P PtdIns or PtdIns or revealed only the PtdIns substrate isolated from natural by the p235 and converted to two in the of the the PtdIns the with a with the phosphorylated PtdIns substrates in the of the and were only than with the and immune complexes from with substrate specificity to the p235. in of the lysates from the to PtdIns but the other substrates to an extent than that with of with the Here PtdIns converted to two products that in the of and the PtdIns that p235 displays a to PtdIns in used to the activity of other PI in and that p235 displays a strong enzymatic activity to However, the predicted catalytic of p235 displays to of enzymes that specificity or PI and or 2Loijens J.C. Boronenkov I.V. Parker G.J. Anderson R.A. Adv. Enzyme Regul. 1996; 36: 115-140Crossref PubMed Scopus (83) Google Scholar, 8Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1310) Google Scholar, A. Curr. Opin. Cell Biol. 1998; PubMed Scopus Google implying that have been phosphorylated by p235 enzymatic activity. the of the D-5 phosphorylation specificity of p235, the products from the in were from and by HPLC to a PtdIns and PtdIns 5-P Cantley L.C. Nature. 1997; PubMed Scopus Google Scholar, J. Cantley L.C. C.L. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). Cantley L.C. Nature. 1997; PubMed Scopus Google HPLC PtdIns 5-P with PtdIns 4-P, resulted in as PtdIns in PtdIns and PtdIns 5-P to as by the of 5-P or used as and of the by an HPLC in a generated by PI Cantley L.C. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google derived from as an of PI with the a of with the and the a 3-P by the enzymatic activity of recombinant PI A. J. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google to the and of products generated by p235 immune complexes of 3T3-L1 adipocytes stimulated with 3T3-L1 adipocytes were stimulated with and insulin for at as cell lysates were with immune or and to as in the to in the or of the products from the were HPLC a as A with the were for and of 3-P determined from a of PtdIns 3-P generated by PI are an of a and of their HPLC of a of five with in A are PtdIns and PtdIns from the of the by p235, the and products were the HPLC with 5-P or as with and with the were to in and the generated by of p235 immune complexes from the at a where the 5-P corresponding 5-P and only a in the of 5-P and that the generated by the authentic and expressed p235 PtdIns PtdIns 5-P, p235 an from the PtdIns substrate in than of the PtdIns 5-P the with as by their relative the of the and the of the the in from the to HPLC the J. Cantley L.C. C.L. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). In 3,4-P2, or from PtdIns or PtdIns 5-P with PI derived from 3T3-L1 as of the 3,5-P2 and from or and in the generated by the p235 immune complexes from the and as of PtdIns 3,5-P2 as of the p235 products that PtdIns 3-P be as substrate as However, as in and PtdIns 3-P a substrate for p235 enzymatic activity with PtdIns isolated from natural sources. PtdIns 3-P more by the of the in vitro with to or and substrate in a of In an to we PtdIns 3-P substrate from a Parker Curr. Biol. 1998; 8: Full Text Full Text PDF PubMed Google in an in vitro and an to increase the of PtdIns the used in the in and the to the and have been by the at of the in vitro p235 activity with the substrates in the immune complexes isolated from 3T3-L1 adipocytes PtdIns PtdIns 3-P substrate PtdIns 5-P and PtdIns 3,5-P2 products with PtdIns as a substrate and PtdIns from PtdIns 3-P and were by HPLC and that PtdIns 3-P substrates were converted to PtdIns 3,5-P2, that p235 has the to the of PtdIns 3,5-P2 from PtdIns PtdIns in a PtdIns as a substrate from a PtdIns 3-P in the substrate of PtdIns 3-P as p235 substrates be to their in the in vitro p235 activity. revealed of PtdIns 5-P and PtdIns 3,5-P2 by p235 over an of two phosphoinositide products from the PtdIns substrate PtdIns 5-P and PtdIns 3,5-P2 were by the of the that the p235 enzymatic activity responsible for the of products that the p235 displays specificity for and PtdIns 5-P as as PtdIns 3,5-P2 from PtdIns, PtdIns and PtdIns 3-P and the of p235 as a PI In addition to for PtdIns as a p235 PI 5-kinase from the PI in that the nonionic p235 activity p235 enzymatic activity also from of PtdIns by a activity in the of A and to and resistance to are of the PI D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1310) Google C.L. Cantley L.C. PubMed Scopus Google Scholar). However, p235 enzymatic activity to a PI O. M. J. Biol. Full Text PDF PubMed Google Scholar, M. A. PubMed Scopus Google Scholar). at a that PI D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1310) Google the enzymatic activity of and expressed p235 found at a at wortmannin of and the of p235 as a PI of p235 5-kinase 5-kinase expressed in or derived from 3T3-L1 adipocytes or p235 immune complexes were for in the of the and to a with PtdIns as a substrate in an as were as a with PtdIns in the at the to a products were by of generated PtdIns 5-P products were by from the of the the or by the from the the to five from the and recombinant p235, the with of were in a p235 expressed in or derived from 3T3-L1 adipocytes or p235 immune complexes were for in the of the and to a with PtdIns as a substrate in an as were as a with PtdIns in the at the to a products were by of generated PtdIns 5-P products were by from the of the the or by the from the the to five from the and recombinant p235, the with of were the enzymatic properties of p235, we sensitivity to the of p235 to PtdIns to PtdIns 5-P and PtdIns 3,5-P2 in the of and and or phosphorylated of PtdIns or the p235 enzymatic activity in the at a with PtdIns and or PI kinases J.C. Boronenkov I.V. Parker G.J. Anderson R.A. Adv. Enzyme Regul. 1996; 36: 115-140Crossref PubMed Scopus (83) Google Scholar). the sensitivity of p235 to the p235 enzymatic activity than over a wide all here the of products, the enzymatic activity in their as a are with the that p235 PI 5-kinase displays a of properties with the other PI In addition to a PI and p235 two other a 3-P at the and a in the of the molecule A. O. Cell. Biol. PubMed Scopus Google Scholar). the role of regions for p235 enzymatic we generated deletion mutants that the the with a of the or the predicted domain of mutants were cells, and the cell lysates were with of the predicted mutants by and A with the for deletion with the of the with the p235 complexes were for PI 5-kinase activity in the of PtdIns, and the generated products were by and in the activity of with to products with the activity of the However, p235 deletion mutants were expressed to levels in COS the that the in the of PtdIns we HPLC of the products in 5-P or These a of a with properties of PtdIns 5-P In the of products the with the immune complexes of or were identified as PtdIns 3-P the of their with the or 3-P and were a of PI activity of of the that p235 enzymatic activity an attribute of the of as as only of the at the of the the catalytic activity. p235 PI 5-kinase has been identified the of in and and the of in adipocytes and A. O. Cell. Biol. PubMed Scopus Google Scholar). These the that the p235 products, or are to acute insulin we the acute insulin of 3T3-L1 adipocytes p235 enzymatic activity and the levels of the products generated in 3T3-L1 cell has as a for the of insulin from to and an of insulin acute insulin of 3T3-L1 adipocytes, cell lysates were with or and the immune were to in the of PtdIns as a of the products revealed a increase of the generated in the p235 immune complexes derived from HPLC of the products from the in the of the insulin in the whose were to the PtdIns 5-P were in the and 3T3-L1 the levels of PtdIns 3-P to insulin and were wortmannin the in vitro PtdIns 5-P with wortmannin by the 5-P levels of the These that resting adipocytes a of p235 PI remains acute cell at with to acute insulin In a plausible of p235 with wortmannin-sensitive PI and the of the by acute insulin a that Here we report the enzymatic activity of the identified novel mammalian p235 A. O. Cell. Biol. PubMed Scopus Google Scholar). generates PtdIns 5-P and PtdIns 3,5-P2 and displays phosphorylation specificity of the inositol of PtdIns and PtdIns 3-P to for substrate and specificity of other inositol PtdIns 3,5-P2 more in a PtdIns substrate isolated from natural that PtdIns 3-P in a more than the PtdIns 3-P substrates in the p235 enzymatic activity. PtdIns 3,5-P2 from the substrate than a by p235 by the p235 PtdIns 3,5-P2 from PtdIns 3-P to extent the of an PtdIns 3-P to p235 PI 5-kinase implying a activity in and and the p235 PI 5-kinase to PI A. O. Cell. Biol. PubMed Scopus Google Scholar). p235 the with PtdIns 5-kinase and PtdIns (3Carpenter C.L. Cantley L.C. Curr. Opin. Cell Biol. 1996; 8: 153-158Crossref PubMed Scopus (575) Google 5-kinase and we to as an with a specificity for the five a In addition to substrate phosphorylation PIKfyve displays a of such as to nonionic for over and a relative resistance to wortmannin and A and and that are from PtdIns and PI kinases D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1310) Google Scholar). in the that enzymatic activity only the PIKfyve of the or of the molecule mutants deletion of than from the PIKfyve from the catalytic but from the at the the activity. In with are that a the predicted catalytic and a enzymatic activity A. O. Cell. Biol. PubMed Scopus Google A for a for of activity for the PI as of are of their enzymatic in the catalytic of the J.C. Boronenkov I.V. Parker G.J. Anderson R.A. Adv. Enzyme Regul. 1996; 36: 115-140Crossref PubMed Scopus (83) Google Scholar, 8Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1310) Google Scholar, Cantley L.C. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google K. Cantley L.C. J. Biol. Full Text PDF PubMed Google Scholar). A plausible of an for a to the PIKfyve to be that the an to and the molecule a with a of enzymatic activity. the deletion the of PIKfyve enzymatic the of of PIKfyve enzymatic activity from A. O. Cell. Biol. PubMed Scopus Google Scholar). that an of PI to all PIKfyve regions in and the products generated by PtdIns 5-P and PtdIns 3,5-P2 have been found to be Cantley L.C. Nature. 1997; PubMed Scopus Google Scholar, Biochem. J. 1997; PubMed Scopus Google Scholar, Parker Nature. 1997; PubMed Scopus Google Scholar). of the phosphorylated be in their D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1310) Google in the PI are at to PtdIns 5-P and PtdIns 3,5-P2 J. Cantley L.C. C.L. J. Biol. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar). However, the enzymes are responsible for the high levels of the two the in vitro PtdIns 5-P and PtdIns 3,5-P2 are generated with catalytic that PIKfyve the for PtdIns 5-P and PtdIns 3,5-P2 biosynthesis in that i.e. adipocytes, and other and the PIKfyve catalytic with PtdIns 3-P substrate that with PtdIns 3-P only a of in the of PtdIns 5-P and PtdIns 3,5-P2 be generated of substantially levels of PtdIns 3-P Cantley L.C. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). PtdIns and PtdIns functions are the remains to their in mammalian cell regulation. Two recent by J.C. J. Cell Biol. 1998; PubMed Scopus Google and Parker Curr. Biol. 1998; 8: Full Text Full Text PDF PubMed Google a of PtdIns or with a that generates PtdIns 3,5-P2 In the of the two J.C. J. Cell Biol. 1998; PubMed Scopus Google enzymatic activity has been possible to by in vitro in the Parker Curr. Biol. 1998; 8: Full Text Full Text PDF PubMed Google an in vitro PtdIns 3,5-P2 from PtdIns 3-P by a recombinant has been In the in vitro of at Parker Curr. Biol. 1998; 8: Full Text Full Text PDF PubMed Google PtdIns and mammalian PIKfyve strong specificity for the phosphorylation at possible in their for PtdIns are to have over PtdIns 5-P species have been identified in J.C. J. Cell Biol. 1998; PubMed Scopus Google Scholar). of PI be by that the of insulin D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1310) Google Parker G.J. Boronenkov I.V. Anderson R.A. J. Biol. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). that PIKfyve has been identified by or to acute insulin to possible of PIKfyve enzymatic activity. acute insulin action in 3T3-L1 adipocytes stimulated monophosphoinositide by PIKfyve immune complexes insulin action the PtdIns 5-P levels but to the PtdIns 3-P of PIKfyve immune complexes a and of PtdIns 3-P by the PI the PtdIns 5-P remained that the two are products of A and and a of PI of PI with a diverse of or Jr., Annu. Rev. 1996; 36: PubMed Scopus Google Scholar). Although the role and of action are of that PI and their products are in insulin membrane to cell S.A. Birnbaum M.J. Biochem. Soc. Trans. 1997; 25: 981-988Crossref PubMed Scopus (59) Google Scholar, 7Shepherd P.R. Withers D.J. Siddle K. Biochem. J. 1998; 333: 471-490Crossref PubMed Scopus (837) Google Scholar, Jr., Annu. Rev. 1996; 36: PubMed Scopus Google Scholar, J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). remains to be identified PIKfyve with PI and a or by insulin, and other PI also the of in the of the with PI that predicted in In the an in vitro of PtdIns 5-P and PtdIns 3,5-P2 by the enzymatic action of by acute insulin action in cells. We for the of 5-P and PtdIns of and from and are We for the of the HPLC