Vancurova I, Choi J H, Lin H, Kuret J, Vancura A
Department of Biological Sciences, St. John's University, Jamaica, New York 11439, USA.
J Biol Chem. 1999 Jan 8;274(2):1147-55. doi: 10.1074/jbc.274.2.1147.
Phosphatidylinositol ()P 5-kinase (PtdIns(4)P 5-kinase) catalyzes the last step in the synthesis of phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2). PtdIns(4,5)P2 is a precursor of diacylglycerol and inositol 1,4,5-trisphosphate and is also involved in regulation of actin cytoskeleton remodeling and membrane traffic. To satisfy such varied demands in several aspects of cell physiology, synthesis of PtdIns(4,5)P2 must be stringently regulated. In this paper we describe extraction, purification, and characterization of PtdIns(4)P 5-kinase from the plasma membranes of Schizosaccharomyces pombe. We also provide evidence that PtdIns(4)P 5-kinase is phosphorylated and inactivated by Cki1, the S. pombe homolog of casein kinase I. Phosphorylation by Cki1 in vitro decreases the activity of PtdIns(4)P 5-kinase. In addition, and most importantly, overexpression of Cki1 in S. pombe results in a reduced synthesis of PtdIns(4,5)P2 and in a lower activity of PtdIns(4)P 5-kinase associated with the plasma membrane. These results suggest that PtdIns(4)P 5-kinase is a target of Cki1 in S. pombe and that Cki1 is involved in regulation of PtdIns(4, 5)P2 synthesis by phosphorylating and inactivating PtdIns(4)P 5-kinase.
磷脂酰肌醇（）P5激酶（磷脂酰肌醇-4-磷酸5激酶）催化磷脂酰肌醇4,5-二磷酸（PtdIns(4,5)P2）合成的最后一步。PtdIns(4,5)P2是二酰甘油和肌醇1,4,5-三磷酸的前体，还参与肌动蛋白细胞骨架重塑和膜运输的调节。为了满足细胞生理学多个方面的这些不同需求，PtdIns(4,5)P2的合成必须受到严格调控。在本文中，我们描述了从粟酒裂殖酵母质膜中提取、纯化和鉴定磷脂酰肌醇-4-磷酸5激酶的过程。我们还提供证据表明，磷脂酰肌醇-4-磷酸5激酶被酪蛋白激酶I的粟酒裂殖酵母同源物Cki1磷酸化并失活。Cki1在体外的磷酸化降低了磷脂酰肌醇-4-磷酸5激酶的活性。此外，也是最重要的，在粟酒裂殖酵母中过表达Cki1会导致PtdIns(4,5)P2的合成减少，以及与质膜相关的磷脂酰肌醇-4-磷酸5激酶活性降低。这些结果表明，磷脂酰肌醇-4-磷酸5激酶是粟酒裂殖酵母中Cki1的作用靶点，并且Cki1通过磷酸化并使磷脂酰肌醇-4-磷酸5激酶失活参与PtdIns(4,5)P2合成的调控。
