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, Ca 2؉ -activated secretion. In order to identify PI4P5K isoforms that may play important roles in regulated insulin secretion from pancreatic ␤-cells, we employed the polymerase chain reaction with degenerate primers and screening of a cDNA library of the murine pancreatic ␤-cell line MIN6. Two novel cDNAs, designated PI4P5K-I␣ and PI4P5K-I␤, 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.Exocytotic release of neurotransmitters and hormones is a highly complex process. In addition to Ca 2ϩ and ATP, regulated fusion of secretory granules with the plasma membrane requires membrane proteins, v-SNARE 1 and t-SNARE. Also required are cytosolic proteins, including N-ethylmaleimide-sensitive factor and soluble N-ethylmaleimide-sensitive factor attachment proteins (reviewed in Ref. 1). Recently, two other cytosolic protein components required for ATP-dependent priming for Ca 2ϩ -activated secretion have been identified: phosphatidylinositol transfer protein (2) and the type I phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) (3). PI4P5K produces, from phosphatidylinositol 4-phosphate (PtdIns(4)P), phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), which has been demonstrated to be important in various cellular processes. PtdIns(4,5)P 2 acts as a substrate for phospholipase C, generating the major second messenger molecules, inositol 1,4,5-triphosphate and diacylglycerol (4). In addition, PtdIns-(4,5)P 2 itself has also been demonstrated to function as a regulator molecule in several cellular processes, including actin filament reorganization (5, 6) and exocytosis (7). More recently, it has been postulated that PtdIns(4,5)P 2 functions in the fusion of intracellular vesicles with target membranes (8). Despite these important roles played by the type I PI4P5K, molecular identification of the enzyme has yet to be carried out.Insulin secretion from pancr...

Type I phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinases (PIP5K) catalyze the synthesis of phosphatidylinositol 4,5-bisphosphate, an essential lipid molecule in various cellular processes. Here, we report the cloning of the third member (PIP5K␥) and the characterization of members of the type I PIP5K family. Type I PIP5K␥ has two alternative splicing forms, migrating at 87 and 90 kDa on SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the central portion of this isoform shows approximately 80% identity with those of the ␣ and ␤ isoforms. Northern blot analysis revealed that the ␥ isoform is highly expressed in the brain, lung, and kidneys. Among three isoforms, the ␤ isoform has the greatest V max value for the PtdIns(4)P kinase activity and the ␥ isoform is most markedly stimulated by phosphatidic acid. By analyzing deletion mutants of the three isoforms, the minimal kinase core sequence of these isoforms were determined as an approximately 380-amino acid region. In addition, carboxyl-terminal regions of the ␤ and ␥ isoforms were found to confer the greatest V max value and the highest phosphatidic acid sensitivity, respectively. It was also discovered that lysine 138 in the putative ATP binding motif of the ␣ isoform is essential for the PtdIns(4)P kinase activity. As was the case with the ␣ isoform reported previously (Shibasaki, Y., Ishihara, H., Kizuki, N., Asano, T., Oka, Y., Yazaki, Y. (1997) J. Biol. Chem. 272, 7578 -7581), overexpression of either the ␤ or the ␥ isoform induced an increase in short actin fibers and a decrease in actin stress fibers in COS7 cells. Surprisingly, a kinase-deficient substitution mutant also induced an abnormal actin polymerization, suggesting a role of PIP5Ks via structural interactions with other molecules.Recent advances in cell biology have revealed that phosphoinositide metabolism plays an essential role in various cellular processes. Synthesis and breakdown of certain phosphoinositides at appropriate times and intracellular sites appear to be required for complex regulation of these cellular processes. One of the phosphoinositides, phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ), 1 is located at an important branchpoint in phosphoinositide metabolism. PtdIns(4,5)P 2 serves as a substrate for phosphoinositide-specific phospholipase C (EC 3

The Rho family GTP-binding proteins have been known to mediate extracellular signals to the actin cytoskeleton. Although several Rho interacting proteins have been found, downstream signals have yet to be determined. Many actin-binding proteins are known to be regulated by phosphatidylinositol 4,5-bisphosphate in vitro. Rho has been shown to enhance the activity of phosphatidylinositol-4-phosphate 5-kinase (PI4P5K), the phosphatidylinositol 4,5-bisphosphate synthesizing enzyme. Recently we isolated several isoforms of type I PI4P5K. Here we report that PI4P5K I␣ induces massive actin polymerization resembling "pine needles" in COS-7 cells in vivo. When truncated from the C terminus to amino acid 308 of PI4P5K I␣, both kinase activity and actin polymerizing activity were lost. Although the dominant negative form of Rho, RhoN19, alone decreased actin fibers, those induced by PI4P5K were not affected by the coexpression of RhoN19. These results suggest that PI4P5K is located downstream from Rho and mediates signals for actin polymerization through its phosphatidylinositol-4-phosphate 5-kinase activity.

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