CLONING OF CDNAS ENCODING 2 ISOFORMS OF 68-KDA TYPE-I PHOSPHATIDYLINOSITOL-4-PHOSPHATE 5-KINASE

Accumulating evidence suggests that phosphatidylinositol metabolism is essential for membrane traffic in the cell, Of particular importance, phosphatidylinositol transfer protein and the type I phosphatidylinos itol-4-phosphate 5-kinase (PI4P5K) have been identified as cytosolic c omponents required for ATP-dependent, Ca2+-activated secretion. In ord er to identify PI4P5K isoforms that may play important roles in regula ted insulin secretion from pancreatic beta-cells, we employed the poly merase chain reaction with degenerate primers and screening of a cDNA library of the murine pancreatic beta-cell line MIN6, Two novel cDNAs, designated PI4P5K-I alpha and PI4P5K-I beta, were identified, which c ontained complete coding sequences encoding 539- or 546-amino acid pro teins, respectively. These cDNAs were expressed in mammalian cells wit h an adenoviral expression vector. Proteins of both isoforms migrated at 68 kDa on SDS-polyacrylamide gel electrophoresis and exhibited phos phatidylinositol-4-phosphate 5-kinase activity, which was activated by phosphatidic acid, indicating that these proteins were type I isoform s, While these isoforms share a marked amino acid sequence homology in their central portion, the amino- and carboxyl-terminal regions diffe r significantly. Northern blot analysis depicted that tissue distribut ions differed between the two isoforms. Molecular identification of ty pe I PI4P5K isoforms in insulin-secreting cells should provide insight s into the role of phosphatidylinositol metabolism in regulated exocyt osis of insulin-containing large dense core vesicles.