Cloning and Characterization of a Wortmannin-sensitive Human Phosphatidylinositol 4-Kinase
1997; Elsevier BV; Volume: 272; Issue: 7 Linguagem: Inglês
10.1074/jbc.272.7.4384
ISSN1083-351X
AutoresRachel Meyers, Lewis C. Cantley,
Tópico(s)Toxin Mechanisms and Immunotoxins
ResumoPhosphatidylinositol (PtdIns) 4-kinases catalyze the synthesis of PtdIns-4-P, the immediate precursor of PtdIns-4,5-P2. Here we report the cloning of a novel, ubiquitously expressed PtdIns 4-kinase (PI4Kβ). The 2.4-kilobase pair cDNA encodes a putative translation product of 801 amino acids which shows greatest homology to the yeast PIK1 gene. The recombinant protein exhibits lipid kinase activity when expressed in Escherichia coli, and specific antibodies recognize a 110-kDa PtdIns 4-kinase in cell lysates. The biochemical properties of PI4Kβ are characteristic of a type III enzyme. Interestingly, both recombinant PI4Kβ and the endogenous protein are inhibited by 150 nM wortmannin, suggesting that we have cloned the previously described PtdIns 4-kinase that is responsible for regulating the synthesis of agonist-sensitive pools of polyphosphoinositides (Nakanishi, S., Catt, J. K., and Balla, T. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5317-5321). Phosphatidylinositol (PtdIns) 4-kinases catalyze the synthesis of PtdIns-4-P, the immediate precursor of PtdIns-4,5-P2. Here we report the cloning of a novel, ubiquitously expressed PtdIns 4-kinase (PI4Kβ). The 2.4-kilobase pair cDNA encodes a putative translation product of 801 amino acids which shows greatest homology to the yeast PIK1 gene. The recombinant protein exhibits lipid kinase activity when expressed in Escherichia coli, and specific antibodies recognize a 110-kDa PtdIns 4-kinase in cell lysates. The biochemical properties of PI4Kβ are characteristic of a type III enzyme. Interestingly, both recombinant PI4Kβ and the endogenous protein are inhibited by 150 nM wortmannin, suggesting that we have cloned the previously described PtdIns 4-kinase that is responsible for regulating the synthesis of agonist-sensitive pools of polyphosphoinositides (Nakanishi, S., Catt, J. K., and Balla, T. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 5317-5321). INTRODUCTIONThe metabolism of phosphoinositides has long been acknowledged to play a central role in the transduction of signals triggered by a variety of growth factors and hormones. Both the enzymes and their product phosphoinositides are present in virtually all eukaryotic organisms and tissues that have been studied. Over the past several years the complexity of phosphoinositide metabolism has become better appreciated. In the classically defined phosphatidylinositol (PtdIns) 1The abbreviations used are:PtdInsphosphatidylinositolPI4KβPtdIns 4-kinase βkbkilobase pair(s)PCRpolymerase chain reactionRACErapid amplification of cDNA endsGSTglutathione S-transferaseHPLChigh performance liquid chromatographybpbase pair(s)aaamino acid(s). turnover pathway, sequential phosphorylation of the 4 and 5 positions yields PtdIns-4-P and PtdIns-4,5-P2, the latter of which acts as a substrate for phospholipase C producing inositol 1,4,5-trisphosphate, a stimulator of intracellular Ca2+ release (2Berridge M. Heslop J. Irvine R.F. Brown K.D. Biochem. J. 1984; 222: 195-201Crossref PubMed Scopus (315) Google Scholar), and diacylglycerol, a stimulator of certain protein kinase C isoforms (3Nishizuka Y. Science. 1986; 233: 305-312Crossref PubMed Scopus (4018) Google Scholar). More recently PtdIns-4-P and PtdIns-4,5-P2 have been shown to regulate cytoskeletal rearrangement through the association with a variety of actin binding proteins (4Janmey P.A. Stossel T.P. Nature. 1987; 325: 362-364Crossref PubMed Scopus (490) Google Scholar, 5Lassing I. Lindberg U. Nature. 1985; 314: 472-474Crossref PubMed Scopus (634) Google Scholar). PtdIns-4,5-P2 has also been shown to stimulate both phospholipase D (6Liscovitch M. Chalifa V. Pertile P. Chen C.-S. Cantley L.C. J. Biol. Chem. 1994; 269: 21403-21406Abstract Full Text PDF PubMed Google Scholar, 7Pertile P. Liscovitch M. Chalifa V. Cantley L.C. J. Biol. Chem. 1995; 270: 5130-5135Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) and β-adrenergic receptor kinase (8Pitcher J.A. Touhara K. Payne E.S. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 11707-11710Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Finally, all of these lipids are substrates of PtdIns 3-kinase, yielding an array of 3-phosphorylated products (9Carpenter C.L. Duckworth B.C. Auger K.R. Cohen B. Schaffhausen B.S. Cantley L.C. J. Biol. Chem. 1990; 265: 19704-19711Abstract Full Text PDF PubMed Google Scholar). It is now clear that the synthesis of a variety of polyphosphoinositides from the starting substrate PtdIns is catalyzed by at least three types of PtdIns kinases (10Whitman M. Kaplan D. Roberts T. Cantley L. Biochem. J. 1987; 247: 165-174Crossref PubMed Scopus (188) Google Scholar, 11Endemann G. Dunn S.N. Cantley L.C. Biochemistry. 1987; 26: 6845-6852Crossref PubMed Scopus (91) Google Scholar).PtdIns 3-kinase (a type I enzyme) catalyzes the phosphorylation of PtdIns at the D3 position of the inositol ring. This enzyme was initially identified through its association with viral oncoproteins and a number of growth factor receptors (12Whitman M. Downes C.P. Keeler M. Keller T. Cantley L. Nature. 1988; 332: 644-646Crossref PubMed Scopus (731) Google Scholar). More recently several additional classes of PtdIns 3-kinases have been identified including a G protein-activated enzyme (13Stephens L. Smrcka A. Cooke F.T. Jackson T.R. Sternweis P.C. Hawkins P.T. Cell. 1994; 77: 83-93Abstract Full Text PDF PubMed Scopus (519) Google Scholar) and VPS 34p, a protein involved in protein trafficking in yeast (14Schu P.V. Takegawa K. Fry M.J. Stack J.H. Waterfield M.D. Emr S.D. Science. 1993; 260: 88-91Crossref PubMed Scopus (803) Google Scholar).PtdIns 4-kinases catalyze the phosphorylation of PtdIns at the D4 position of the inositol ring and have been divided into two types (II and III) based on their size and sensitivity to various compounds (11Endemann G. Dunn S.N. Cantley L.C. Biochemistry. 1987; 26: 6845-6852Crossref PubMed Scopus (91) Google Scholar). The type II enzymes were initially characterized as membrane-associated 55-kDa proteins whose lipid kinase activity is highly stimulated by detergent and inhibited by both adenosine and the monoclonal antibody 4C5G (11Endemann G. Dunn S.N. Cantley L.C. Biochemistry. 1987; 26: 6845-6852Crossref PubMed Scopus (91) Google Scholar, 15Endemann G.C. Graziani A. Cantley L.C. Biochem. J. 1991; 273: 63-66Crossref PubMed Scopus (42) Google Scholar). The type III enzymes are membrane-associated proteins predicted to be >200 kDa in size that are less stimulated by detergent and are not inhibited by adenosine or 4C5G antibodies. The PtdIns 4-kinases are highly abundant and have been identified in a large number of membrane structures (reviewed Ref. 16Pike L. Endocr. Rev. 1992; 13: 692-706Crossref PubMed Scopus (74) Google Scholar).Recently several PtdIns 4-kinases have been cloned and found to be homologous to PtdIns 3-kinases. They all contain both a lipid kinase unique domain and a C-terminal catalytic domain with distant homology to protein kinases. In yeast, the PIK1 gene encodes a 125-kDa protein that is indispensable for cell growth and plays a role in cytokinesis (17Flanagan C.A. Schnieders E.A. Emerick A.W. Kunisawa R. Admon A. Thorner J. Science. 1993; 262: 1444-1448Crossref PubMed Scopus (171) Google Scholar). It contains the lipid kinase unique domain at its far N terminus and the catalytic domain in the characteristic C-terminal position. Although it is intermediate in size, its biochemical properties suggest that it is more similar to the type III enzyme (18Flanagan C.A. Thorner J. J. Biol. Chem. 1992; 267: 24117-24125Abstract Full Text PDF PubMed Google Scholar). In Dictyostelium discoideum, a putative PtdIns 4-kinase has recently been cloned, whose domain structure is similar to PIK1, extending the identification of these proteins across several species (19Zhou K. Takegawa K. Emr S.D. Firtel R.A. Mol. Cell. Biol. 1995; 15: 5645-5656Crossref PubMed Scopus (111) Google Scholar). A second yeast gene, STT4, encodes a 200-kDa protein that is dispensable for growth in the presence of osmotic stabilizers and has been implicated in the protein kinase C pathway through its isolation in a screen for mutants sensitive to the protein kinase C inhibitor staurosporine (20Yoshida S. Ohya Y. Goebl M. Nakano A. Anraku Y. J. Biol. Chem. 1994; 269: 1166-1171Abstract Full Text PDF PubMed Google Scholar). Finally, the first PtdIns 4-kinase from higher eukaryotes, PI4Kα, was cloned and shown to encode a 100-kDa protein with significant homology to STT4 and biochemical properties of a type II enzyme (21Wong K. Cantley L.C. J. Biol. Chem. 1994; 269: 28878-28884Abstract Full Text PDF PubMed Google Scholar). This protein, as well as STT4, contains adjacent lipid kinase unique and catalytic domains at its C terminus. An alternative splice of the PI4Kα gene that generates a 230-kDa protein has also been recently reported (22Nakagawa T. Goto K. Kondo H. J. Biol. Chem. 1996; 271: 12088-12094Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar).These three types of PtdIns kinases all show homology to an ever expanding family of protein kinases whose substrates have not yet been identified. This family includes the TOR/FRAP proteins that are the cellular targets of the FK506-binding protein-rapamycin complex and are involved in cellular signaling and cell cycle control (23Brown E.J. Albers M.W. Shin T.B. Ichikawa K. Keith C.T. Lane W.S. Schreiber S.L. Nature. 1994; 369: 756-758Crossref PubMed Scopus (1640) Google Scholar, 24Sabatini D.M. Erdjument-Bromage H. Lui M. Tempst P. Snyder S.H. Cell. 1994; 78: 35-43Abstract Full Text PDF PubMed Scopus (1205) Google Scholar, 25Kunz J. Henriquez R. Schneider U. Deuter R.M. Movva N.R. Hall M.N. Cell. 1993; 73: 585-596Abstract Full Text PDF PubMed Scopus (721) Google Scholar, 26Helliwell S.B. Wagner P. Kunz J. Deuter R.M. Henriquez R. Hall M.N. Mol. Biol. Cell. 1994; 5: 105-118Crossref PubMed Scopus (312) Google Scholar, 27Zheng X.F. Fiorentino D. Chen J. Crabtree G.R. Schreiber S.L. Cell. 1995; 82: 121-130Abstract Full Text PDF PubMed Scopus (244) Google Scholar). It is interesting to note that although yeast TOR2 and mammalian FRAP/RAFT1 have associated PtdIns 4-kinase activities, these activities are probably not endogenous to the protein kinase catalytic site (27Zheng X.F. Fiorentino D. Chen J. Crabtree G.R. Schreiber S.L. Cell. 1995; 82: 121-130Abstract Full Text PDF PubMed Scopus (244) Google Scholar). Other members of this extended family include the ATM/MEC1/DNA-PK proteins that are involved in both cell cycle progression and checkpoint control and chromosomal maintenance and repair (28Weinert T.A. Kiser G.L. Hartwell L.H. Genes Dev. 1994; 8: 652-655Crossref PubMed Scopus (668) Google Scholar, 29Hartley K.O. Gell D. Smith G.C.M. Zhang H. Divecha N. Connelly M.A. Admon A. Lees-Miller S.P. Jackson S.P. Cell. 1995; 82: 849-856Abstract Full Text PDF PubMed Scopus (669) Google Scholar, 30Kastan M.B. Zhan Q. El-Diery W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace A.J. Cell. 1992; 71: 587-597Abstract Full Text PDF PubMed Scopus (2923) Google Scholar). All these proteins share a conserved C-terminal catalytic domain found in both lipid and protein kinases.Within this conserved domain are specific amino acid stretches that distinguish the subfamily of PtdIns 4-kinases from PtdIns 3-kinases and the other family members. We have taken advantage of this subfamily specificity to design degenerate PCR primers for the use in cloning novel PtdIns 4-kinases. We have identified and cloned one such gene and analyzed the biochemical properties of the encoded protein, which we call PI4Kβ. Interestingly, PI4Kβ is wortmannin-sensitive and shows great similarity to a recently described wortmannin-inhibitable PtdIns 4-kinase that was partially purified from bovine adrenal cortex (1Nakanishi S. Catt J.K. Balla T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5317-5321Crossref PubMed Scopus (308) Google Scholar). Nakanishi et al. (1Nakanishi S. Catt J.K. Balla T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5317-5321Crossref PubMed Scopus (308) Google Scholar) demonstrate that this enzyme is responsible for regulating the hormone-sensitive pools of inositol phospholipids. Recent studies in which the effects of 100 nM to 1 μM wortmannin have been used to implicate phosphatidylinositol 3-kinase in membrane trafficking, cytoskeletal rearrangement, and signal transduction must be reconsidered in view of the nearly ubiquitous expression of wortmannin-sensitive PI4Kβ.DISCUSSIONWe have identified and characterized PI4Kβ, a novel PtdIns 4-kinase that is widely expressed in a variety of tissues. The cDNA encodes an 801-aa protein that exhibits lipid kinase activity when expressed in E. coli Both the bacterially expressed and the endogenous proteins exhibit properties consistent with the characterization of PI4Kβ as a type III enzyme. Antibodies raised against PI4Kβ detect a ∼110-kDa protein in a number of cell types across several species. Interestingly, PI4Kβ is the first cloned PtdIns 4-kinase that is inhibitable by wortmannin, potentially implicating PI4Kβ in a variety of wortmannin-sensitive cellular pathways.Sequence analysis of PI4Kβ places it within the PtdIns 4-kinase family and more generally places it in the larger family of lipid/protein kinases. It contains a conserved C-terminal catalytic domain with distant homology to protein kinases as well as strong homology to the dual specificity kinases such as PtdIns 3-kinase. Within this conserved domain is lysine 549 which, based on homology to PtdIns 3-kinase, is the likely site of wortmannin reactivity (45Wymann M.P. Bulgarelli-Leva G. Zvelebil M.J. Pirola L. Vanhaesbroeck B. Waterfield M.D. Panayotou G. Mol. Cell. Biol. 1996; 16: 1722-1733Crossref PubMed Scopus (630) Google Scholar). All members of this lipid/protein kinase family contain this conserved lysine, yet many, including PIK1 and PI4Kα, are not inhibited by micromolar concentrations of the drug, suggesting that additional residues within the active site confer wortmannin sensitivity.Members of this extended family have diverse cellular functions. For example, the yeast protein MEC1 and its Drosophila homologue MEI41 are checkpoint control genes which appear to monitor the state of the genome at the G1/S and G2/M transitions (28Weinert T.A. Kiser G.L. Hartwell L.H. Genes Dev. 1994; 8: 652-655Crossref PubMed Scopus (668) Google Scholar, 46Paulovich A.G. Hartwell L.H. Cell. 1995; 82: 841-847Abstract Full Text PDF PubMed Scopus (525) Google Scholar, 47Hari K.L. Santerre A. Sekelsky J.J. McKim K.S. Boyd J.B. Hawley R.S. Cell. 1995; 82: 815-821Abstract Full Text PDF PubMed Scopus (247) Google Scholar). Another family member, DNA-PK, was originally identified as a DNA-dependent protein kinase (48Anderson C.W. Lees-Miller S.P. Crit. Rev. Eukaryotic Gene Expr. 1992; 2: 283-314PubMed Google Scholar) but was subsequently shown to function in immunoglobulin gene rearrangement and DNA repair (29Hartley K.O. Gell D. Smith G.C.M. Zhang H. Divecha N. Connelly M.A. Admon A. Lees-Miller S.P. Jackson S.P. Cell. 1995; 82: 849-856Abstract Full Text PDF PubMed Scopus (669) Google Scholar).PI4Kβ has properties similar to the wortmannin-sensitive PtdIns 4-kinase described by Nakanishi et al. (1Nakanishi S. Catt J.K. Balla T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5317-5321Crossref PubMed Scopus (308) Google Scholar). The partially purified enzyme was inhibited by wortmannin with an IC50 of ∼50 nM, not dissimilar to the 120-140 nM IC50 observed for PI4Kβ. The wortmannin-sensitive enzyme had an apparent molecular mass of 125 kDa, as judged by gel filtration, in agreement with the 110-kDa molecular mass observed for PI4Kβ. The reported enzymatic properties of this protein are also very similar to those of PI4Kβ. It is likely that we have cloned the PtdIns 4-kinase that regulates the formation of agonist-sensitive inositol phospholipids that are required for intracellular signaling in some cells.It should be noted that a PtdIns 4-kinase from the particulate fraction of Schizosaccharomyces pombe has been observed to be sensitive to the wortmannin analogue demethoxyviridin (49Woscholski R. Kodaki T. McKinnon M. Waterfield M.D. Parker P.J. FEBS Lett. 1994; 342: 109-114Crossref PubMed Scopus (96) Google Scholar). Curiously, this enzyme was not inhibited by wortmannin. Additionally, attempts to isolate a drug-sensitive PtdIns 4-kinase from rat brain particulate fractions were unsuccessful (49Woscholski R. Kodaki T. McKinnon M. Waterfield M.D. Parker P.J. FEBS Lett. 1994; 342: 109-114Crossref PubMed Scopus (96) Google Scholar). Although we have detected PI4Kβ in rat brain, both our experiments and those of Nakanishi et al. (1Nakanishi S. Catt J.K. Balla T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5317-5321Crossref PubMed Scopus (308) Google Scholar) suggest that it is only loosely associated with the membrane. Furthermore, PI4Kβ is not the major PtdIns 4-kinase present in membrane fractions, and therefore lipid kinase assays on these crude fractions would not be expected to show wortmannin sensitivity.Taken together, these data suggest that we need to reevaluate the interpretation of experiments employing wortmannin as an inhibitor in biological assays. For example, recent experiments have demonstrated that wortmannin inhibits the proper targeting of the lysosomal enzyme procathepsin D in a variety of cell types (50Davidson H.W. J. Cell Biol. 1995; 130: 797-805Crossref PubMed Scopus (184) Google Scholar, 51Brown W.J. DeWald D.B. Emr S.D. Plutner H. Balch W.E. J. Cell Biol. 1995; 130: 781-796Crossref PubMed Scopus (251) Google Scholar). The concentration of wortmannin used was as high as 1 μM with an estimated IC50 of ∼100 nM. Clearly, these elevated levels of wortmannin could be inhibiting PI4Kβ thereby implicating it in protein trafficking. Furthermore, both wortmannin and demethoxyviridin have been reported to inhibit phospholipase D, PtdIns-phospholipase C, and phospholipase A2 in vivo (41Arcaro A. Wymann M.P. Biochem. J. 1993; 296: 297-301Crossref PubMed Scopus (1047) Google Scholar, 52Cross M.J. Stewart A. Hodgkin M.N. Kerr D.J. Wakelam M.J.O. J. Biol. Chem. 1995; 270: 25352-25355Abstract Full Text Full Text PDF PubMed Scopus (251) Google Scholar). It is likely that this inhibition is a downstream effect of the inhibition of PtdIns 3-kinase and possibly PI4Kβ in these cells, as little direct inhibition of these enzymes was observed in vitro at μM concentrations of wortmannin. The assumption that PtdIns 3-kinase is a critical mediator of all the myriad pathways inhibited by wortmannin is likely to be an oversimplification. INTRODUCTIONThe metabolism of phosphoinositides has long been acknowledged to play a central role in the transduction of signals triggered by a variety of growth factors and hormones. Both the enzymes and their product phosphoinositides are present in virtually all eukaryotic organisms and tissues that have been studied. Over the past several years the complexity of phosphoinositide metabolism has become better appreciated. In the classically defined phosphatidylinositol (PtdIns) 1The abbreviations used are:PtdInsphosphatidylinositolPI4KβPtdIns 4-kinase βkbkilobase pair(s)PCRpolymerase chain reactionRACErapid amplification of cDNA endsGSTglutathione S-transferaseHPLChigh performance liquid chromatographybpbase pair(s)aaamino acid(s). turnover pathway, sequential phosphorylation of the 4 and 5 positions yields PtdIns-4-P and PtdIns-4,5-P2, the latter of which acts as a substrate for phospholipase C producing inositol 1,4,5-trisphosphate, a stimulator of intracellular Ca2+ release (2Berridge M. Heslop J. Irvine R.F. Brown K.D. Biochem. J. 1984; 222: 195-201Crossref PubMed Scopus (315) Google Scholar), and diacylglycerol, a stimulator of certain protein kinase C isoforms (3Nishizuka Y. Science. 1986; 233: 305-312Crossref PubMed Scopus (4018) Google Scholar). More recently PtdIns-4-P and PtdIns-4,5-P2 have been shown to regulate cytoskeletal rearrangement through the association with a variety of actin binding proteins (4Janmey P.A. Stossel T.P. Nature. 1987; 325: 362-364Crossref PubMed Scopus (490) Google Scholar, 5Lassing I. Lindberg U. Nature. 1985; 314: 472-474Crossref PubMed Scopus (634) Google Scholar). PtdIns-4,5-P2 has also been shown to stimulate both phospholipase D (6Liscovitch M. Chalifa V. Pertile P. Chen C.-S. Cantley L.C. J. Biol. Chem. 1994; 269: 21403-21406Abstract Full Text PDF PubMed Google Scholar, 7Pertile P. Liscovitch M. Chalifa V. Cantley L.C. J. Biol. Chem. 1995; 270: 5130-5135Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar) and β-adrenergic receptor kinase (8Pitcher J.A. Touhara K. Payne E.S. Lefkowitz R.J. J. Biol. Chem. 1995; 270: 11707-11710Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Finally, all of these lipids are substrates of PtdIns 3-kinase, yielding an array of 3-phosphorylated products (9Carpenter C.L. Duckworth B.C. Auger K.R. Cohen B. Schaffhausen B.S. Cantley L.C. J. Biol. Chem. 1990; 265: 19704-19711Abstract Full Text PDF PubMed Google Scholar). It is now clear that the synthesis of a variety of polyphosphoinositides from the starting substrate PtdIns is catalyzed by at least three types of PtdIns kinases (10Whitman M. Kaplan D. Roberts T. Cantley L. Biochem. J. 1987; 247: 165-174Crossref PubMed Scopus (188) Google Scholar, 11Endemann G. Dunn S.N. Cantley L.C. Biochemistry. 1987; 26: 6845-6852Crossref PubMed Scopus (91) Google Scholar).PtdIns 3-kinase (a type I enzyme) catalyzes the phosphorylation of PtdIns at the D3 position of the inositol ring. This enzyme was initially identified through its association with viral oncoproteins and a number of growth factor receptors (12Whitman M. Downes C.P. Keeler M. Keller T. Cantley L. Nature. 1988; 332: 644-646Crossref PubMed Scopus (731) Google Scholar). More recently several additional classes of PtdIns 3-kinases have been identified including a G protein-activated enzyme (13Stephens L. Smrcka A. Cooke F.T. Jackson T.R. Sternweis P.C. Hawkins P.T. Cell. 1994; 77: 83-93Abstract Full Text PDF PubMed Scopus (519) Google Scholar) and VPS 34p, a protein involved in protein trafficking in yeast (14Schu P.V. Takegawa K. Fry M.J. Stack J.H. Waterfield M.D. Emr S.D. Science. 1993; 260: 88-91Crossref PubMed Scopus (803) Google Scholar).PtdIns 4-kinases catalyze the phosphorylation of PtdIns at the D4 position of the inositol ring and have been divided into two types (II and III) based on their size and sensitivity to various compounds (11Endemann G. Dunn S.N. Cantley L.C. Biochemistry. 1987; 26: 6845-6852Crossref PubMed Scopus (91) Google Scholar). The type II enzymes were initially characterized as membrane-associated 55-kDa proteins whose lipid kinase activity is highly stimulated by detergent and inhibited by both adenosine and the monoclonal antibody 4C5G (11Endemann G. Dunn S.N. Cantley L.C. Biochemistry. 1987; 26: 6845-6852Crossref PubMed Scopus (91) Google Scholar, 15Endemann G.C. Graziani A. Cantley L.C. Biochem. J. 1991; 273: 63-66Crossref PubMed Scopus (42) Google Scholar). The type III enzymes are membrane-associated proteins predicted to be >200 kDa in size that are less stimulated by detergent and are not inhibited by adenosine or 4C5G antibodies. The PtdIns 4-kinases are highly abundant and have been identified in a large number of membrane structures (reviewed Ref. 16Pike L. Endocr. Rev. 1992; 13: 692-706Crossref PubMed Scopus (74) Google Scholar).Recently several PtdIns 4-kinases have been cloned and found to be homologous to PtdIns 3-kinases. They all contain both a lipid kinase unique domain and a C-terminal catalytic domain with distant homology to protein kinases. In yeast, the PIK1 gene encodes a 125-kDa protein that is indispensable for cell growth and plays a role in cytokinesis (17Flanagan C.A. Schnieders E.A. Emerick A.W. Kunisawa R. Admon A. Thorner J. Science. 1993; 262: 1444-1448Crossref PubMed Scopus (171) Google Scholar). It contains the lipid kinase unique domain at its far N terminus and the catalytic domain in the characteristic C-terminal position. Although it is intermediate in size, its biochemical properties suggest that it is more similar to the type III enzyme (18Flanagan C.A. Thorner J. J. Biol. Chem. 1992; 267: 24117-24125Abstract Full Text PDF PubMed Google Scholar). In Dictyostelium discoideum, a putative PtdIns 4-kinase has recently been cloned, whose domain structure is similar to PIK1, extending the identification of these proteins across several species (19Zhou K. Takegawa K. Emr S.D. Firtel R.A. Mol. Cell. Biol. 1995; 15: 5645-5656Crossref PubMed Scopus (111) Google Scholar). A second yeast gene, STT4, encodes a 200-kDa protein that is dispensable for growth in the presence of osmotic stabilizers and has been implicated in the protein kinase C pathway through its isolation in a screen for mutants sensitive to the protein kinase C inhibitor staurosporine (20Yoshida S. Ohya Y. Goebl M. Nakano A. Anraku Y. J. Biol. Chem. 1994; 269: 1166-1171Abstract Full Text PDF PubMed Google Scholar). Finally, the first PtdIns 4-kinase from higher eukaryotes, PI4Kα, was cloned and shown to encode a 100-kDa protein with significant homology to STT4 and biochemical properties of a type II enzyme (21Wong K. Cantley L.C. J. Biol. Chem. 1994; 269: 28878-28884Abstract Full Text PDF PubMed Google Scholar). This protein, as well as STT4, contains adjacent lipid kinase unique and catalytic domains at its C terminus. An alternative splice of the PI4Kα gene that generates a 230-kDa protein has also been recently reported (22Nakagawa T. Goto K. Kondo H. J. Biol. Chem. 1996; 271: 12088-12094Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar).These three types of PtdIns kinases all show homology to an ever expanding family of protein kinases whose substrates have not yet been identified. This family includes the TOR/FRAP proteins that are the cellular targets of the FK506-binding protein-rapamycin complex and are involved in cellular signaling and cell cycle control (23Brown E.J. Albers M.W. Shin T.B. Ichikawa K. Keith C.T. Lane W.S. Schreiber S.L. Nature. 1994; 369: 756-758Crossref PubMed Scopus (1640) Google Scholar, 24Sabatini D.M. Erdjument-Bromage H. Lui M. Tempst P. Snyder S.H. Cell. 1994; 78: 35-43Abstract Full Text PDF PubMed Scopus (1205) Google Scholar, 25Kunz J. Henriquez R. Schneider U. Deuter R.M. Movva N.R. Hall M.N. Cell. 1993; 73: 585-596Abstract Full Text PDF PubMed Scopus (721) Google Scholar, 26Helliwell S.B. Wagner P. Kunz J. Deuter R.M. Henriquez R. Hall M.N. Mol. Biol. Cell. 1994; 5: 105-118Crossref PubMed Scopus (312) Google Scholar, 27Zheng X.F. Fiorentino D. Chen J. Crabtree G.R. Schreiber S.L. Cell. 1995; 82: 121-130Abstract Full Text PDF PubMed Scopus (244) Google Scholar). It is interesting to note that although yeast TOR2 and mammalian FRAP/RAFT1 have associated PtdIns 4-kinase activities, these activities are probably not endogenous to the protein kinase catalytic site (27Zheng X.F. Fiorentino D. Chen J. Crabtree G.R. Schreiber S.L. Cell. 1995; 82: 121-130Abstract Full Text PDF PubMed Scopus (244) Google Scholar). Other members of this extended family include the ATM/MEC1/DNA-PK proteins that are involved in both cell cycle progression and checkpoint control and chromosomal maintenance and repair (28Weinert T.A. Kiser G.L. Hartwell L.H. Genes Dev. 1994; 8: 652-655Crossref PubMed Scopus (668) Google Scholar, 29Hartley K.O. Gell D. Smith G.C.M. Zhang H. Divecha N. Connelly M.A. Admon A. Lees-Miller S.P. Jackson S.P. Cell. 1995; 82: 849-856Abstract Full Text PDF PubMed Scopus (669) Google Scholar, 30Kastan M.B. Zhan Q. El-Diery W.S. Carrier F. Jacks T. Walsh W.V. Plunkett B.S. Vogelstein B. Fornace A.J. Cell. 1992; 71: 587-597Abstract Full Text PDF PubMed Scopus (2923) Google Scholar). All these proteins share a conserved C-terminal catalytic domain found in both lipid and protein kinases.Within this conserved domain are specific amino acid stretches that distinguish the subfamily of PtdIns 4-kinases from PtdIns 3-kinases and the other family members. We have taken advantage of this subfamily specificity to design degenerate PCR primers for the use in cloning novel PtdIns 4-kinases. We have identified and cloned one such gene and analyzed the biochemical properties of the encoded protein, which we call PI4Kβ. Interestingly, PI4Kβ is wortmannin-sensitive and shows great similarity to a recently described wortmannin-inhibitable PtdIns 4-kinase that was partially purified from bovine adrenal cortex (1Nakanishi S. Catt J.K. Balla T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5317-5321Crossref PubMed Scopus (308) Google Scholar). Nakanishi et al. (1Nakanishi S. Catt J.K. Balla T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5317-5321Crossref PubMed Scopus (308) Google Scholar) demonstrate that this enzyme is responsible for regulating the hormone-sensitive pools of inositol phospholipids. Recent studies in which the effects of 100 nM to 1 μM wortmannin have been used to implicate phosphatidylinositol 3-kinase in membrane trafficking, cytoskeletal rearrangement, and signal transduction must be reconsidered in view of the nearly ubiquitous expression of wortmannin-sensitive PI4Kβ.
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