TY - JOUR
T1 - Cloning of cDNAs encoding two isoforms of 68-kDa type I phosphatidylinositol-4-phosphate 5-kinase
AU - Ishihara, Hisamitsu
AU - Shibasaki, Yoshikazu
AU - Kizuki, Nobuaki
AU - Katagiri, Hideki
AU - Yazaki, Yoshio
AU - Asano, Tomoichiro
AU - Oka, Yoshitomo
PY - 1996
Y1 - 1996
N2 - 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.
AB - 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.
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U2 - 10.1074/jbc.271.39.23611
DO - 10.1074/jbc.271.39.23611
M3 - Article
C2 - 8798574
AN - SCOPUS:0029795811
VL - 271
SP - 23611
EP - 23614
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 39
ER -