Ishihara H, Shibasaki Y, Kizuki N, Wada T, Yazaki Y, Asano T, Oka Y
Third Department of Internal Medicine, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan.
J Biol Chem. 1998 Apr 10;273(15):8741-8. doi: 10.1074/jbc.273.15.8741.
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 (PIP5Kgamma) and the characterization of members of the type I PIP5K family. Type I PIP5Kgamma 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 alpha and beta isoforms. Northern blot analysis revealed that the gamma isoform is highly expressed in the brain, lung, and kidneys. Among three isoforms, the beta isoform has the greatest Vmax value for the PtdIns(4)P kinase activity and the gamma 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 beta and gamma isoforms were found to confer the greatest Vmax value and the highest phosphatidic acid sensitivity, respectively. It was also discovered that lysine 138 in the putative ATP binding motif of the alpha isoform is essential for the PtdIns(4)P kinase activity. As was the case with the alpha 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 beta or the gamma 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.
I型磷脂酰肌醇4-磷酸（PtdIns(4)P）5-激酶（PIP5K）催化磷脂酰肌醇4,5-二磷酸的合成，这是一种在各种细胞过程中必不可少的脂质分子。在此，我们报告了I型PIP5K家族第三个成员（PIP5Kγ）的克隆及该家族成员的特性。I型PIP5Kγ有两种可变剪接形式，在SDS-聚丙烯酰胺凝胶电泳上迁移率分别为87 kDa和90 kDa。该同工型中部的氨基酸序列与α和β同工型的氨基酸序列显示出约80%的同一性。Northern印迹分析表明，γ同工型在脑、肺和肾中高度表达。在三种同工型中，β同工型的PtdIns(4)P激酶活性的Vmax值最大，γ同工型受磷脂酸的刺激最为明显。通过分析三种同工型的缺失突变体，确定这些同工型的最小激酶核心序列为一个约380个氨基酸的区域。此外，发现β和γ同工型的羧基末端区域分别赋予最大的Vmax值和最高的磷脂酸敏感性。还发现α同工型假定的ATP结合基序中的赖氨酸138对PtdIns(4)P激酶活性至关重要。与先前报道的α同工型情况一样（Shibasaki, Y., Ishihara, H., Kizuki, N., Asano, T., Oka, Y., Yazaki, Y. (1997) J. Biol. Chem. 272, 7578 - 7581），β或γ同工型的过表达导致COS7细胞中短肌动蛋白纤维增加，肌动蛋白应力纤维减少。令人惊讶的是，一种激酶缺陷型替代突变体也诱导了异常的肌动蛋白聚合，提示PIP5K通过与其他分子的结构相互作用发挥作用。
