Cloning of cDNAs Encoding Two Isoforms of 68-kDa Type I Phosphatidylinositol4-phosphate 5-Kinase

Abstract

Accumulating evidence suggests that phosphatidylinositol metabolism is essential for membrane traffic in the cell. Of particular importance, phosphatidylinositol transfer protein and the type I phosphatidylinositol- 4-phosphate 5-kinase (PI4P5K) have been identified as cytosolic components required for ATP-dependent, Ca2+-activated secretion. In order to identify PI4P5K isoforms that may play important roles in regulated insulin secretion from pancreatic β-cells, we employed the polymerase chain reaction with degenerate primers and screening of a cDNA library of the murine pancreatic β-cell line MIN6. Two novel cDNAs, designated PI4P5K-Iα and PI4P5K-Iβ, were identified, which contained complete coding sequences encoding 539- or 546-amino acid proteins, respectively. These cDNAs were expressed in mammalian cells with an adenoviral expression vector. Proteins of both isoforms migrated at 68 kDa on SDS-polyacrylamide gel electrophoresis and exhibited phosphatidylinositol-4-phosphate 5-kinase activity, which was activated by phosphatidic acid, indicating that these proteins were type I isoforms. While these isoforms share a marked amino acid sequence homology in their central portion, the amino- and carboxyl-terminal regions differ significantly. Northern blot analysis depicted that tissue distributions differed between the two isoforms. Molecular identification of type I PI4P5K isoforms in insulin-secreting cells should provide insights into the role of phosphatidylinositol metabolism in regulated exocytosis of insulin-containing large dense core vesicles. Accumulating evidence suggests that phosphatidylinositol metabolism is essential for membrane traffic in the cell. Of particular importance, phosphatidylinositol transfer protein and the type I phosphatidylinositol- 4-phosphate 5-kinase (PI4P5K) have been identified as cytosolic components required for ATP-dependent, Ca2+-activated secretion. In order to identify PI4P5K isoforms that may play important roles in regulated insulin secretion from pancreatic β-cells, we employed the polymerase chain reaction with degenerate primers and screening of a cDNA library of the murine pancreatic β-cell line MIN6. Two novel cDNAs, designated PI4P5K-Iα and PI4P5K-Iβ, were identified, which contained complete coding sequences encoding 539- or 546-amino acid proteins, respectively. These cDNAs were expressed in mammalian cells with an adenoviral expression vector. Proteins of both isoforms migrated at 68 kDa on SDS-polyacrylamide gel electrophoresis and exhibited phosphatidylinositol-4-phosphate 5-kinase activity, which was activated by phosphatidic acid, indicating that these proteins were type I isoforms. While these isoforms share a marked amino acid sequence homology in their central portion, the amino- and carboxyl-terminal regions differ significantly. Northern blot analysis depicted that tissue distributions differed between the two isoforms. Molecular identification of type I PI4P5K isoforms in insulin-secreting cells should provide insights into the role of phosphatidylinositol metabolism in regulated exocytosis of insulin-containing large dense core vesicles. INTRODUCTIONExocytotic release of neurotransmitters and hormones is a highly complex process. In addition to Ca2+ and ATP, regulated fusion of secretory granules with the plasma membrane requires membrane proteins, v-SNARE 1The abbreviations used here are: v-SNARE and t-SNAREvesicle membrane- and target membrane-soluble N-ethylmaleimide-sensitive attachment protein receptorsPI4P5Kphosphatidylinositol-4-phosphate 5-kinasePtdIns(4,5)P2phosphatidylinositol 4,5-bisphosphatePtdIns(3,4)P2phosphatidylinositol 3,4-bisphosphatePtdIns(4Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-204Google Scholar)Pphosphatidylinositol 4-phosphatePAphosphatidic acidPAGEpolyacrylamide gel electrophoresisHAhemagglutinin and t-SNARE. Also required are cytosolic proteins, including N-ethylmaleimide-sensitive factor and soluble N-ethylmaleimide-sensitive factor attachment proteins (reviewed in 1Sudhof T.C. Nature. 1995; 375: 645-653Google Scholar). Recently, two other cytosolic protein components required for ATP-dependent priming for Ca2+ -activated secretion have been identified: phosphatidylinositol transfer protein (2Hay J.C. Martin T.F.J. Nature. 1993; 366: 572-575Google Scholar) and the type I phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) (3Hay J.C. Fisette P.L. Jenkins G.H. Fukami K. Takenawa T. Anderson R.A. Martin T.F.J. Nature. 1995; 374: 173-177Google Scholar). PI4P5K produces, from phosphatidylinositol 4-phosphate (PtdIns(4Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-204Google Scholar)P), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), which has been demonstrated to be important in various cellular processes. PtdIns(4,5)P2 acts as a substrate for phospholipase C, generating the major second messenger molecules, inositol 1,4,5-triphosphate and diacylglycerol (4Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-204Google Scholar). In addition, PtdIns(4,5)P2 itself has also been demonstrated to function as a regulator molecule in several cellular processes, including actin filament reorganization (5Janmey P.A. Stossel T.P. J. Biol. Chem. 1989; 264: 4825-4831Google Scholar, 6Fukami K. Furuhashi K. Inagaki M. Endo T. Hatano S. Takenawa T. Nature. 1992; 359: 150-152Google Scholar) and exocytosis (7Eberahard D.A. Cooper C.L. Low M.G. Holz R.W. Biochem. J. 1990; 268: 15-25Google Scholar). More recently, it has been postulated that PtdIns(4,5)P2 functions in the fusion of intracellular vesicles with target membranes (8Liscovitch M. Cantley L.C. Cell. 1995; 81: 659-662Google Scholar). Despite these important roles played by the type I PI4P5K, molecular identification of the enzyme has yet to be carried out.Insulin secretion from pancreatic β-cells is one example of regulated exocytosis. Extensive studies have been conducted, which suggest the importance of the polyphosphoinositide synthesis pathway and the breakdown of its products by phospholipase C (9Prentki M. Matschinsky F.M. Physiol. Rev. 1987; 67: 1185-1248Google Scholar, 10Farese F.V. DiMarco P.E. Barnes D.E. Sabir M.A. Larson R.E. Davis J.S. Morrison A.D. Endocrinology. 1986; 118: 1498-1503Google Scholar, 11Dunlop M.E. Larkins R.G. Biochem. Biophys. Res. Commun. 1985; 132: 467-473Google Scholar). We recently demonstrated that insulin secretion requires an ATP-dependent priming process (12Shibasaki Y. Ishihara H. Kikuchi M. Yazaki Y. Diabetes. 1996; 45: 1-243AGoogle Scholar), suggesting that the type I PI4P5K plays a significant role in insulin secretion from pancreatic β-cells. We have therefore made efforts to identify PI4P5K-like proteins in insulin-secreting MIN6 cells (13Ishihara H. Asano T. Tsukuda K. Katagiri H. Inukai K. Anai M. Kikuchi M. Yazaki Y. Miyazaki J.-I. Oka Y. Diabetologia. 1993; 36: 1139-1145Google Scholar). Here, we have identified two type I isoforms of PI4P5K from a cDNA library of the murine pancreatic β-cell line MIN6.DISCUSSIONWe have identified two type I isoforms of PI4P5K, designated PI4P5K-Iα and PI4P5K-Iβ, in this study. PI4P5K catalyzes the formation of PtdIns(4,5)P2 from PtdIns(4Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-204Google Scholar)P. PtdIns(4,5)P2 has been demonstrated to be an important molecule in various cellular processes, including inositol 1,4,5-trisphosphate mediated Ca2+ mobilization (4Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-204Google Scholar), actin filament reorganization (5Janmey P.A. Stossel T.P. J. Biol. Chem. 1989; 264: 4825-4831Google Scholar, 6Fukami K. Furuhashi K. Inagaki M. Endo T. Hatano S. Takenawa T. Nature. 1992; 359: 150-152Google Scholar), and exocytosis (7Eberahard D.A. Cooper C.L. Low M.G. Holz R.W. Biochem. J. 1990; 268: 15-25Google Scholar). Co-existence of at least three isoforms of the PI4P5K (one previously cloned isoform (type II or C) and two type I) in an insulin-secreting cell line, MIN6, suggests that these isoforms have distinct roles in various cellular processes. These isoforms may reside in different subcellular compartments and that spatially segregated synthesis or metabolism of PtdIns(4,5)P2 may be linked to different cellular functions. While these novel isoforms show approximately 75% identity in their central regions, the amino-terminal or carboxyl-terminal sequences are different. It is possible that the amino- and carboxyl-terminal regions have isoform-specific functions. Further studies of these isoforms are needed; of particular interest is their subcellular localization and possible association with other molecules, such as Rac1 (29Tolias K.F. Cantley L.C. Carpenter C.L. J. Biol. Chem. 1995; 270: 17656-17659Google Scholar) and Rho (30Ren X.-D. Bokoch G.M. Traynor-Kaplan A. Jenkins G.H. Anderson R.A. Schwartz M.A. Mol. Biol. Cell. 1996; 7: 435-442Google Scholar).Proteins of both the Iα and the Iβ isoform migrate at 68 kDa on SDS-PAGE, suggesting that these isoforms are identical or closely related to the type Ia isoform previously described (3Hay J.C. Fisette P.L. Jenkins G.H. Fukami K. Takenawa T. Anderson R.A. Martin T.F.J. Nature. 1995; 374: 173-177Google Scholar, 23Jenkins G.H. Fisette P.L. Anderson R.A. J. Biol. Chem. 1994; 269: 11547-11554Google Scholar). During the screening of the MIN6 cell cDNA library, we obtained another partial coding sequence closely homologous to the PI4P5K-Iα and Iβ isoforms. Further studies are needed to characterize the third sequence.Whether the type I PI4P5K plays an important roles in regulated exocytosis of insulin secretory granules remains to be determined. The type I but not the type II enzyme has been demonstrated to be activated by phosphatidic acid and appears to phosphorylate PtdIns(4Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-204Google Scholar)P on intact membrane (31Bazenet C.E. Ruano A.R. Brockman J.L. Anderson R.A. J. Biol. Chem. 1990; 265: 18012-18022Google Scholar). Recent studies on regulated exocytosis have postulated that a positive feedback loop catalyzed by phospholipase D and the type I PI4P5K is involved in exocytotic processes (8Liscovitch M. Cantley L.C. Cell. 1995; 81: 659-662Google Scholar), which may form a microdomain enriched in negatively charged phospholipids on the vesicle membrane. Our demonstration of several isoforms of the type I PI4P5K in insulin-secreting cells (MIN6), together with a previous demonstration of phospholipase D in pancreatic islets (32Metz S.A. Dunlop M. Arch. Biochem. Biophys. 1990; 283: 417-428Google Scholar), raises the possibility that such a positive feedback loop may operate in insulin-secreting cells. Furthermore, it was previously reported that glucose could cause activation of phosphatidylinositol metabolism, which was partly ascribed to increased de novo synthesis of PA and polyphosphoinositide in pancreatic islets (10Farese F.V. DiMarco P.E. Barnes D.E. Sabir M.A. Larson R.E. Davis J.S. Morrison A.D. Endocrinology. 1986; 118: 1498-1503Google Scholar, 11Dunlop M.E. Larkins R.G. Biochem. Biophys. Res. Commun. 1985; 132: 467-473Google Scholar). These observations suggest that activation of phosphatidylinositol metabolism, in concert with membrane depolarization, may serve as a link between glucose metabolism and exocytosis of insulin secretory granules. Molecular identification of the type I PI4P5K isoforms will provide essential information for the precise role of phosphatidylinositol metabolism in exocytosis of insulin-containing dense core vesicles. INTRODUCTIONExocytotic release of neurotransmitters and hormones is a highly complex process. In addition to Ca2+ and ATP, regulated fusion of secretory granules with the plasma membrane requires membrane proteins, v-SNARE 1The abbreviations used here are: v-SNARE and t-SNAREvesicle membrane- and target membrane-soluble N-ethylmaleimide-sensitive attachment protein receptorsPI4P5Kphosphatidylinositol-4-phosphate 5-kinasePtdIns(4,5)P2phosphatidylinositol 4,5-bisphosphatePtdIns(3,4)P2phosphatidylinositol 3,4-bisphosphatePtdIns(4Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-204Google Scholar)Pphosphatidylinositol 4-phosphatePAphosphatidic acidPAGEpolyacrylamide gel electrophoresisHAhemagglutinin and t-SNARE. Also required are cytosolic proteins, including N-ethylmaleimide-sensitive factor and soluble N-ethylmaleimide-sensitive factor attachment proteins (reviewed in 1Sudhof T.C. Nature. 1995; 375: 645-653Google Scholar). Recently, two other cytosolic protein components required for ATP-dependent priming for Ca2+ -activated secretion have been identified: phosphatidylinositol transfer protein (2Hay J.C. Martin T.F.J. Nature. 1993; 366: 572-575Google Scholar) and the type I phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) (3Hay J.C. Fisette P.L. Jenkins G.H. Fukami K. Takenawa T. Anderson R.A. Martin T.F.J. Nature. 1995; 374: 173-177Google Scholar). PI4P5K produces, from phosphatidylinositol 4-phosphate (PtdIns(4Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-204Google Scholar)P), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2), which has been demonstrated to be important in various cellular processes. PtdIns(4,5)P2 acts as a substrate for phospholipase C, generating the major second messenger molecules, inositol 1,4,5-triphosphate and diacylglycerol (4Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-204Google Scholar). In addition, PtdIns(4,5)P2 itself has also been demonstrated to function as a regulator molecule in several cellular processes, including actin filament reorganization (5Janmey P.A. Stossel T.P. J. Biol. Chem. 1989; 264: 4825-4831Google Scholar, 6Fukami K. Furuhashi K. Inagaki M. Endo T. Hatano S. Takenawa T. Nature. 1992; 359: 150-152Google Scholar) and exocytosis (7Eberahard D.A. Cooper C.L. Low M.G. Holz R.W. Biochem. J. 1990; 268: 15-25Google Scholar). More recently, it has been postulated that PtdIns(4,5)P2 functions in the fusion of intracellular vesicles with target membranes (8Liscovitch M. Cantley L.C. Cell. 1995; 81: 659-662Google Scholar). Despite these important roles played by the type I PI4P5K, molecular identification of the enzyme has yet to be carried out.Insulin secretion from pancreatic β-cells is one example of regulated exocytosis. Extensive studies have been conducted, which suggest the importance of the polyphosphoinositide synthesis pathway and the breakdown of its products by phospholipase C (9Prentki M. Matschinsky F.M. Physiol. Rev. 1987; 67: 1185-1248Google Scholar, 10Farese F.V. DiMarco P.E. Barnes D.E. Sabir M.A. Larson R.E. Davis J.S. Morrison A.D. Endocrinology. 1986; 118: 1498-1503Google Scholar, 11Dunlop M.E. Larkins R.G. Biochem. Biophys. Res. Commun. 1985; 132: 467-473Google Scholar). We recently demonstrated that insulin secretion requires an ATP-dependent priming process (12Shibasaki Y. Ishihara H. Kikuchi M. Yazaki Y. Diabetes. 1996; 45: 1-243AGoogle Scholar), suggesting that the type I PI4P5K plays a significant role in insulin secretion from pancreatic β-cells. We have therefore made efforts to identify PI4P5K-like proteins in insulin-secreting MIN6 cells (13Ishihara H. Asano T. Tsukuda K. Katagiri H. Inukai K. Anai M. Kikuchi M. Yazaki Y. Miyazaki J.-I. Oka Y. Diabetologia. 1993; 36: 1139-1145Google Scholar). Here, we have identified two type I isoforms of PI4P5K from a cDNA library of the murine pancreatic β-cell line MIN6.