Cloning of a Human Type II Phosphatidylinositol 4-Kinase Reveals a Novel Lipid Kinase Family

Phosphoinositide lipids regulate numerous cellular processes in all eukaryotes. The versatility of this phospholipid is provided by combinations of phosphorylation on the 3′, 4′, and 5′ positions of the inositol head group. Two distinct structural families of phosphoinositide (PI) kinases have so far been identified and named after their prototypic members, the PI 3-kinase and phosphatidylinositol (PtdIns) phosphate kinase families, both of which have been found to contain structural homologues possessing PI 4-kinase activity. Nevertheless, the prevalent PtdIns 4-kinase activity in many mammalian cell types is conferred by the widespread type II PtdIns 4-kinase, which has so far resisted molecular characterization. We have partially purified the human type II isoform from plasma membrane rafts of human A431 epidermoid carcinoma cells and obtained peptide mass and sequence data. The results allowed the cDNA containing the full open reading frame to be cloned. The predicted amino acid sequence revealed that the type II enzyme is the prototypic member of a novel, third family of PI kinases. We have named the purified protein type IIα and a second human isoform, type IIβ. The type IIα mRNA appears to be expressed ubiquitously in human tissues, and homologues appear to be expressed in all eukaryotes. Phosphoinositide lipids regulate numerous cellular processes in all eukaryotes. The versatility of this phospholipid is provided by combinations of phosphorylation on the 3′, 4′, and 5′ positions of the inositol head group. Two distinct structural families of phosphoinositide (PI) kinases have so far been identified and named after their prototypic members, the PI 3-kinase and phosphatidylinositol (PtdIns) phosphate kinase families, both of which have been found to contain structural homologues possessing PI 4-kinase activity. Nevertheless, the prevalent PtdIns 4-kinase activity in many mammalian cell types is conferred by the widespread type II PtdIns 4-kinase, which has so far resisted molecular characterization. We have partially purified the human type II isoform from plasma membrane rafts of human A431 epidermoid carcinoma cells and obtained peptide mass and sequence data. The results allowed the cDNA containing the full open reading frame to be cloned. The predicted amino acid sequence revealed that the type II enzyme is the prototypic member of a novel, third family of PI kinases. We have named the purified protein type IIα and a second human isoform, type IIβ. The type IIα mRNA appears to be expressed ubiquitously in human tissues, and homologues appear to be expressed in all eukaryotes. phosphatidylinositol base pair electrospray ionization glutathione S-transferase matrix-assisted laser desorption mass spectrometry open reading frame polymerase chain reaction phosphoinositide phosphoinositide 3-kinase phospholipase C phosphatidylinositol 4-kinase phosphatidylinositol 4-phosphate PtdIns phosphate kinase 4-morpholineethanesulfonic acid dithiothreitol 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol polyacrylamide gel electrophoresis high pressure liquid chromatography Phosphoinositides have been implicated in a vast range of cellular functions, including receptor signaling, vesicle trafficking, endocytosis and cytoskeletal rearrangement (1de Camilli P. Emr S.D. McPherson P.S. Novick P. Science. 1996; 271: 1533-1539Crossref PubMed Scopus (661) Google Scholar, 2Hsuan J.J. Minogue S. dos Santos M. Adv. Cancer Res. 1998; 74: 167-216Crossref PubMed Google Scholar). Several distinct metabolic pathways of PtdIns1 phosphorylation exist in eukaryotic cells, producing important effectors such as PtdIns 4,5-bisphosphate, PtdIns 3,4-bisphosphate, and PtdIns 3,4,5-trisphosphate (3Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1319) Google Scholar). The first step in the phosphorylation of PtdIns in many receptor-dependent phospholipase C (PLC) and PI 3-kinase (PI3K) signaling pathways involves the synthesis of PtdIns 4-phosphate (PtdIns4P) by PtdIns 4-kinase (PtdIns4K) activity (4Pike L.J. Endocr. Rev. 1992; 13: 692-706Crossref PubMed Scopus (75) Google Scholar). The PI kinase sequences that have been determined so far fall into two families, the PI3K family (5Keith C.T. Schreiber S.L. Science. 1995; 270: 50-51Crossref PubMed Scopus (448) Google Scholar, 6Zvelebil M.J. MacDougall L. Leevers S. Volinia S. Vanhaesebroeck B. Gout I. Panayotou G. Domin J. Stein R. Pagès F. Waterfield M.D. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 1996; 351: 217-223Crossref PubMed Scopus (89) Google Scholar), which includes all the hitherto known PI3K and PtdIns4K sequences (3Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1319) Google Scholar), and the PtdIns phosphate kinase (PtdInsPK) family (3Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1319) Google Scholar, 7Loijens J.C. Boronenkov I.V. Parker G.J. Anderson R.A. Adv. Enzyme Regul. 1996; 36: 115-140Crossref PubMed Scopus (83) Google Scholar). All PI3Ks and PtdIns4Ks cloned so far display significant homology within their kinase domains. In contrast, members of the PtdInsPK family have quite distinct sequences. However, recent structural studies have shown that whereas the PtdInsPKs display little primary sequence homology with the PI3K family, they share a common protein fold that is also conserved in many protein kinases (8Rao V.D. Misra S. Boronenkov I.V. Anderson R.A. Hurley J.H. Cell. 1998; 94: 829-839Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, 9Walker E.H. Perisic O. Ried C. Stephens L. Williams R.L. Nature. 1999; 402: 313-320Crossref PubMed Scopus (417) Google Scholar). Early chromatographic purification of phosphoinositide kinase activities from bovine brain (4Pike L.J. Endocr. Rev. 1992; 13: 692-706Crossref PubMed Scopus (75) Google Scholar) and cultured rodent fibroblasts (10Whitman M. Kaplan D. Roberts T. Cantley L. Biochem. J. 1987; 247: 165-174Crossref PubMed Scopus (188) Google Scholar) identified three fractions containing PtdIns kinase activity, termed types I to III. The type II and III fractions contain different PtdIns4K activities, and the type I fraction was subsequently shown to contain PI3K activity (11Whitman M. Downes C.P. Keeler M. Keller T. Cantley L. Nature. 1988; 332: 644-646Crossref PubMed Scopus (740) Google Scholar). It is now known that the type I PI3K and type III PtdIns4K enzymes have related sequences and belong to the PI3K family (3Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1319) Google Scholar). However, the fraction containing the type II PtdIns4K (PtdIns4K II) has so far not been characterized at the molecular level despite the fact that in many mammalian cells, the predominant pathway of PtdIns phosphorylation is initiated by this strongly membrane-associated PtdIns4K (3Fruman D.A. Meyers R.E. Cantley L.C. Annu. Rev. Biochem. 1998; 67: 481-507Crossref PubMed Scopus (1319) Google Scholar). Perhaps surprisingly, although members of the PI3K and PtdInsPK families have been readily purified and cloned, numerous attempts to purify the PtdIns4K II enzyme (see Ref. 12Wetzker R. Klinger R. Hsuan J. Fry M.J. Kauffmann Z.A. Muller E. Frunder H. Waterfield M. Eur. J. Biochem. 1991; 200: 179-185Crossref PubMed Scopus (28) Google Scholar and references therein) have failed to lead to the cDNA being identified. These preparations were probably often impure as PtdIns4K II is labile, difficult to solubilize, and tends to aggregate strongly with other proteins. Indeed, a published cDNA sequence was subsequently found to encode a long-chain fatty acid-CoA ligase (13Yamakawa A. Nishizawa M. Fujiwara K.T. Kawai S. Kawasaki H. Suzuki K. Takenawa T. J. Biol. Chem. 1991; 266: 17580-17583Abstract Full Text PDF PubMed Google Scholar). Despite the massive increase in genome data, approaches based on homology to known PtdIns4Ks, such as degenerate polymerase chain reaction (PCR) primers or data base trawling, have also failed to identify the PtdIns4K II sequence. Although three related PtdIns4Ks have so far been cloned (14Wong K. Cantley L.C. J. Biol. Chem. 1994; 269: 28878-28884Abstract Full Text PDF PubMed Google Scholar, 15Nakagawa T. Goto K. Kondo H. Biochem. J. 1996; 320: 643-649Crossref PubMed Scopus (47) Google Scholar, 16Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 4384-4390Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 17Balla T. Downing G.J. Jaffe H. Kim S. Zolyomi A. Catt K.J. J. Biol. Chem. 1997; 272: 18358-18366Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 18Nakagawa T. Goto K. Kondo H. J. Biol. Chem. 1996; 271: 12088-12094Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), the failure of such approaches with PtdIns4K II has raised the question of whether the PtdIns4K II enzyme might be a proteolytic fragment or splice variant of the known isozymes or belong to a different structural family altogether. Although there is indirect evidence for the regulation of the PtdIns4K II activity by serine and tyrosine residue phosphorylation (19Kauffmann Z.A. Klinger R. Endemann G. Waterfield M.D. Wetzker R. Hsuan J.J. J. Biol. Chem. 1994; 269: 31243-31251Abstract Full Text PDF PubMed Google Scholar, 20de Neef R.-S. Hardy-Dessources M.-D. Giraud F. Eur. J. Biochem. 1996; 235: 549-556Crossref PubMed Scopus (12) Google Scholar), receptor association (21Cochet C. Filhol O. Payrastre B. Hunter T. Gill G.N. J. Biol. Chem. 1991; 266: 637-644Abstract Full Text PDF PubMed Google Scholar, 22Kauffmann Z.A. Thomas G.M. Ball A. Prosser S. Cunningham E. Cockcroft S. Hsuan J.J. Science. 1995; 268: 1188-1190Crossref PubMed Scopus (161) Google Scholar), heterotrimeric G-proteins (23Pike L.J. Eakes A.T. J. Biol. Chem. 1987; 262: 1644-1651Abstract Full Text PDF PubMed Google Scholar), and substrate presentation by the PtdIns transfer protein (22Kauffmann Z.A. Thomas G.M. Ball A. Prosser S. Cunningham E. Cockcroft S. Hsuan J.J. Science. 1995; 268: 1188-1190Crossref PubMed Scopus (161) Google Scholar), the inability to purify or immunoprecipitate the enzyme has precluded definitive experiments to evaluate its function and regulation. PtdIns4K II has been identified in the plasma membrane, lysosomal, microsomal, transport vesicle, and nuclear compartments (4Pike L.J. Endocr. Rev. 1992; 13: 692-706Crossref PubMed Scopus (75) Google Scholar). More recently this isozyme has been shown to exist in subdomains of the plasma membrane termed non-caveolar membrane rafts (24Waugh M.G. Lawson D. Tan S.K. Hsuan J.J. J. Biol. Chem. 1998; 273: 17115-17121Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar), in which receptor-dependent PLC signaling also appears to be localized (25Pike L.J. Miller J.M. J. Biol. Chem. 1998; 273: 22298-22304Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). Rafts are typically small, cholesterol-rich membrane domains of low buoyant density, which are generally insoluble in 1% Triton X-100 at 4 °C (reviewed in Ref. 26Simons K. Ikonen E. Nature. 1997; 387: 569-572Crossref PubMed Scopus (8117) Google Scholar). To address questions regarding the pivotal role of this enzyme in many signaling pathways, we set out to purify sufficient human PtdIns4K II to allow the cDNA to be identified. Plasma membrane rafts containing PtdIns4K II were isolated from human A431 epidermoid carcinoma cells, employing similar methods to those described previously (24Waugh M.G. Lawson D. Tan S.K. Hsuan J.J. J. Biol. Chem. 1998; 273: 17115-17121Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). Two chromatographic purification steps were selected from the work of Deuel and co-workers (27Porter F.D. Li Y.S. Deuel T.F. J. Biol. Chem. 1988; 263: 8989-8995Abstract Full Text PDF PubMed Google Scholar), adapted to microscale purification and modified using detergents appropriate for cholesterol-rich rafts. A431 cells were cultured to confluence in six 24 × 24-cm dishes in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum. Monolayers were washed in phosphate-buffered saline and then scraped into 12 ml of buffer containing 10 mm Tris-HCl, pH 7.4, 10 mm EDTA, 10 mm EGTA, 0.25 msucrose, and protease inhibitors (Complete™ Mini, EDTA-free; Roche Molecular Biochemicals) prior to Dounce homogenization. The homogenate was centrifuged at 4000 × g for 5 min. Membranes were pelleted from the post-nuclear supernatant by centrifugation at 190,000 × g for 1 h at 4 °C. Low density membrane rafts were prepared using a modification of the method of Song et al. (28Song K.S. Li S. Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (920) Google Scholar). Briefly, the 190,000 ×g membrane pellet was resuspended in 2 ml of 100 mm Na2CO3, pH 11.0, 10 mm EGTA, 10 mm EDTA, 10 mmβ-octylglucoside, and 4 mm deoxycholate, containing Complete™ protease inhibitors. The membrane suspension was sonicated on ice (10 × 5-s bursts at 40% maximum power using a VC130PB sonicator (Sonics and Materials Inc.), and of in mm pH mm 10 mm EGTA, and 10 mm was was to a and with 4 ml of and 4 ml of both in mm pH mm 10 mm and 10 mm The was centrifuged at 4 °C for h at 190,000 × Rafts were then at the 5 and washed with ml of 10 mm Tris-HCl, pH 10 mm and by centrifugation at 4 °C for 1 h at 190,000 rafts were in ml of base buffer pH mm 1 mm EDTA, 1 mm EGTA, 1 mm containing Complete™ protease inhibitors and sonicated (10 × 5-s bursts at 40% maximum using a VC130PB The rafts were by the of and to 100 and with base and then to a in buffer pH mm 1 mm Triton on a chromatography The was washed in buffer and with a in buffer at mm at and mm at PtdIns4K activity was at with buffer mm pH mm 1 mm Triton and to a in buffer in buffer the was with a in buffer mm in 5 The of PtdIns4K activity was at min. and using were as described previously R. Klinger R. Hsuan J. Fry M.J. Kauffmann Z.A. Muller E. Frunder H. Waterfield M. Eur. J. Biochem. 1991; 200: 179-185Crossref PubMed Scopus (28) Google Scholar, Z.A. Klinger R. Endemann G. Waterfield M.D. Wetzker R. Hsuan J.J. J. Biol. Chem. 1994; 269: 31243-31251Abstract Full Text PDF PubMed Google Scholar). containing 5 of fractions or 10 of IIα were in containing mm Tris-HCl, pH 100 mm 10 mm Triton 1 mm mm EGTA, and were by the of and for at °C being by the of to the of phosphate to PtdIns was not The was R. Klinger R. Hsuan J. Fry M.J. Kauffmann Z.A. Muller E. Frunder H. Waterfield M. Eur. J. Biochem. 1991; 200: 179-185Crossref PubMed Scopus (28) Google Scholar), and the were by on Inc.) in acid containing 1% 5 acid by containing were by and in a liquid were by and with A. M. O. M. Chem. 1996; PubMed Scopus Google Scholar). The with activity was and to and using and acid by using modified laser desorption mass spectrometry was on a III of mass in the with and using a acid were using of and The of at was for peptide mass electrospray ionization liquid chromatography were on The a All were was on a × with acid in as and acid in as B. with a was to 40% in using a of The was to a mass set for liquid data using to 12 to the predicted of the PtdIns4K IIα open reading frame was to first cDNA synthesis from of isolated from A431 cells J. T. Molecular Scholar). was in using the (Life Technologies, of this reaction was subsequently to by using the the predicted and the the predicted was using polymerase were °C for °C for and °C for for to the predicted of the PtdIns4K IIα was gel purified and with polymerase into the as described by the The of the was by fragment containing the was from cloned into the and of Biochem. 1991; PubMed Scopus Google Scholar), and expressed in the The as a of protein was by the of to and cells were after h of at °C. were sonicated in buffer containing 1 mm EDTA, 1 mm and Complete™ protease inhibitors. were by centrifugation at × g for after which the protein was to as described by the cDNA the amino of PtdIns4K IIα was by using the and the The pair was and with by as described previously J. T. Molecular Scholar). was then to a human The was high to the and then washed in × for 1 h at °C. the was and with a human as by the The vast of the PtdIns4K II in A431 cells is localized within non-caveolar membrane rafts (24Waugh M.G. Lawson D. Tan S.K. Hsuan J.J. J. Biol. Chem. 1998; 273: 17115-17121Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). purified rafts containing PtdIns4K II were in detergents and using and of activity was obtained at not fractions from the were by using 1 and PtdIns4K 1 of fraction that a of molecular mass and with the of the activity 1 of activity was at molecular mass as previously R. Klinger R. Hsuan J. Fry M.J. Kauffmann Z.A. Muller E. Frunder H. Waterfield M. Eur. J. Biochem. 1991; 200: 179-185Crossref PubMed Scopus (28) Google Scholar). other protein to with the as the protein or the kinase activity, the of a mass P. Biol. Full Text PDF PubMed Scopus Google Scholar) of the protein peptide the mass data were to the data base using the they were found to a cDNA a fragment of a human protein the of the human expressed sequence data base were using the 5′ of this cDNA to the revealed and using revealed two and of and a sequence M. J. Biol. 1991; PubMed Scopus Google Scholar) was identified from sequences and and were identified the data were using the sequence from the and The for all peptide for of the amino acid sequence To this the protein was prepared as the gel was washed with at and the peptide was with a to the of the of this by 24 the predicted with sequence and a mass of 100 acid sequences of human and type II PtdIns4K the predicted amino acid sequence of PtdIns4K IIα identified by and The sequence is kinase for the human PtdIns4K human PtdIns4K and the S. type II conserved The of and at predicted In this PtdIns4K and S. kinase domains and residue with the PtdIns4K IIα kinase a of we liquid to the of the first of the sequence for all of which the sequence in 2 the of the peptide in which the of fragment is The sequence obtained by data base was to These primers were to the cDNA by with a of cloned the of the The sequence a containing amino acid and a molecular mass of was found using a and which or Although the mammalian enzyme is strongly to sequence or for was The to the kinase homology with other known Although the enzyme is to by low of R. Klinger R. Hsuan J. Fry M.J. Kauffmann Z.A. Muller E. Frunder H. Waterfield M. Eur. J. Biochem. 1991; 200: 179-185Crossref PubMed Scopus (28) Google Scholar), was of data for sequences revealed a set of sequences from different including a second human sequence and a in on of 10 different PtdIns4K II homologues dos Santos and J. J. revealed kinase S.K. 1991; 200: PubMed Scopus Google Scholar), including a I sequence II or and homology was the to kinase and the of the PtdIns4K IIα The of sequence with the PI3K or PtdInsPK family that PtdIns4K IIα and its sequence homologues a novel, third PI kinase The PtdIns4K IIα mRNA has of and appears to be ubiquitously expressed in human which is with the purification of this enzyme from numerous different primary (see Ref. 12Wetzker R. Klinger R. Hsuan J. Fry M.J. Kauffmann Z.A. Muller E. Frunder H. Waterfield M. Eur. J. Biochem. 1991; 200: 179-185Crossref PubMed Scopus (28) Google Scholar and references in and and in and The protein expressed in molecular mass of and of the using the mass to The protein PtdIns4K activity, which was in preparations containing and the with a 5 the protein was phosphoinositide was to PtdIns not or not of the protein using the method of and for and which fall within the range of previously published (see Ref. 12Wetzker R. Klinger R. Hsuan J. Fry M.J. Kauffmann Z.A. Muller E. Frunder H. Waterfield M. Eur. J. Biochem. 1991; 200: 179-185Crossref PubMed Scopus (28) Google Scholar and references The activity of the protein was this is low with the enzyme purified from primary (see Ref. 12Wetzker R. Klinger R. Hsuan J. Fry M.J. Kauffmann Z.A. Muller E. Frunder H. Waterfield M. Eur. J. Biochem. 1991; 200: 179-185Crossref PubMed Scopus (28) Google Scholar and references therein) and in modification or The enzyme was by Triton X-100 5 and by with of 5 by Triton X-100 and by in the range are of PtdIns4K II purified from mammalian (4Pike L.J. Endocr. Rev. 1992; 13: 692-706Crossref PubMed Scopus (75) Google Scholar). The protein was also in a by the 5 which is of PtdIns4K II that from the type III PtdIns4K G. A. L. C. Scholar). The purification of PtdIns4K II in sufficient for on of the membrane of this enzyme (24Waugh M.G. Lawson D. Tan S.K. Hsuan J.J. J. Biol. Chem. 1998; 273: 17115-17121Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). The purification of rafts and their allowed the of a purification of the PtdIns4K II enzyme from cultured The of PtdIns4K II and PI appears to be for receptor-dependent (25Pike L.J. Miller J.M. J. Biol. Chem. 1998; 273: 22298-22304Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). of a of PtdIns4K II activity is typically characterized by its by detergents and by and the (4Pike L.J. Endocr. Rev. 1992; 13: 692-706Crossref PubMed Scopus (75) Google Scholar, M. Kaplan D. Roberts T. Cantley L. Biochem. J. 1987; 247: 165-174Crossref PubMed Scopus (188) Google R. Klinger R. Hsuan J. Fry M.J. Kauffmann Z.A. Muller E. Frunder H. Waterfield M. Eur. J. Biochem. 1991; 200: 179-185Crossref PubMed Scopus (28) Google Scholar, G. A. L. C. Scholar). The enzyme to those for the purified IIα was by Triton X-100 and by low of and by the PtdIns4K IIα enzyme a sequence family, be from the previously cloned PtdIns4K (14Wong K. Cantley L.C. J. Biol. Chem. 1994; 269: 28878-28884Abstract Full Text PDF PubMed Google Scholar), PtdIns4K T. Goto K. Kondo H. Biochem. J. 1996; 320: 643-649Crossref PubMed Scopus (47) Google Scholar, 16Meyers R. Cantley L.C. J. Biol. Chem. 1997; 272: 4384-4390Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 17Balla T. Downing G.J. Jaffe H. Kim S. Zolyomi A. Catt K.J. J. Biol. Chem. 1997; 272: 18358-18366Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar), and type III T. Goto K. Kondo H. J. Biol. Chem. 1996; 271: 12088-12094Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) as as the PI3K and PtdInsPK PtdIns4K PtdIns4K and the type III PtdIns4K belong in the PI3K We to the type II for PtdIns4K at the structural and range of enzymes is and to the human isoform purified in this and the second isoform identified by data base PtdIns4K IIα and PtdIns4K The of sequence in the of the PtdIns4K IIα and PtdIns4K this for by or regulation The of a PtdInsPK II in S. is as the type III and have been to for all of the PtdIns4K activity in cells A. M. Emr S.D. Biol. Cell. PubMed Scopus Google Scholar). Although a type PtdIns4K previously been partially purified from S. J.J. G.M. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar), the was that this was or of the type III It to be shown whether the and other PtdIns4K II homologues PtdIns4K activity. In the of the PtdIns4K IIα cDNA sequence has a family of PI kinases. It is now to approaches to to address numerous questions regarding the function of PtdIns4K including the the different PtdIns4K important step has been a molecular of the and function of this enzyme in and We The for S. M. and J. J. Wetzker for this Cancer for for the of the and for of