摘要：

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 (PIP5Kγ) and the characterization of members of the type I PIP5K family. Type I PIP5Kγ 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 α and β isoforms. Northern blot analysis revealed that the γ isoform is highly expressed in the brain, lung, and kidneys. Among three isoforms, the β isoform has the greatest V max value for the PtdIns(4)P kinase activity and the γ 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 β and γ 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 α isoform is essential for the PtdIns(4)P kinase activity. As was the case with the α 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 β or the γ 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.

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