Inhibitory phosphorylation site for Rho-associated kinase on smooth muscle myosin phosphatase

作     者：Feng, JH Ito, M Ichikawa, K Isaka, N Nishikawa, M Hartshorne, DJ Nakano, T 

作者机构：Mie Univ Sch Med Dept Internal Med 1 Tsu Mie 5148507 Japan Mie Univ Sch Med Dept Internal Med 2 Tsu Mie 5148507 Japan Univ Arizona Muscle Biol Grp Tucson AZ 85721 USA 

出 版 物：《JOURNAL OF BIOLOGICAL CHEMISTRY》 (生物化学杂志)

年 卷 期：1999年第274卷第52期

页      面：37385-37390页

核心收录：

学科分类：0710[理学-生物学] 071010[理学-生物化学与分子生物学] 07[理学] 

基　　金：NHLBI NIH HHS [HL23615] Funding Source: Medline 

主　　题：3T3细胞 酰胺类/药理学 抗体特异性 细胞内信号肽和蛋白质类 溶血磷脂素类/药理学 肌 平滑/酶学 诱变 定点 肌球蛋白轻链磷酸酶 磷蛋白磷酸酶类/化学 磷蛋白磷酸酶类/免疫学 磷蛋白磷酸酶类/代谢 磷酰化 蛋白质丝氨酸苏氨酸激酶/生理学 吡啶类/药理学 苏氨酸 rho相关激酶类 动物 小鼠 

摘      要：It is clear from several studies that myosin phosphatase (MP) can be inhibited via a pathway that involves RhoA. However, the mechanism of inhibition is not established;These studies were carried out to test the hypothesis that Rho-kinase (Rho-associated kinase) via phosphorylation of the myosin phosphatase target subunit 1 (MYPT1) inhibited MP activity and to identify relevant sites of phosphorylation. Phosphorylation by Rho-kinase inhibited MP activity and this reflected a decrease in V-max. Activity of MP with different substrates also was inhibited by phosphorylation, Two major;sites of phosphorylation on MYPT1 were Thr(695) and Thr(850). Various point mutations were designed for these phosphorylation sites. Following thiophosphorylation by Rho kinase and assays of phosphatase activity it was determined that Thr(695) was responsible for inhibition. A site- and phosphorylation-specific antibody was developed for the sequence flanking Thr(695) and this recognized only phosphorylated Thr(695) in both native and recombinant MYPT1. Using this antibody it was shown that stimulation of serum-starved Swiss 3T3 cells by lysophosphatidic acid, thought to activate RhoA pathways, induced an increase in Thr(695) phosphorylation on MYPT1 and this effect was blocked by a Rho-kinase inhibitor, Y-27632. In summary, these results offer strong support for a physiological role of Rho-kinase in regulation of MP activity.