Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop
Rho-associated kinase ROCK activates LIM-kinase 1 by phosphorylation at threonine 508 within the activation loop
复制标题
DOI：
10.1074/jbc.275.5.3577
复制
发表时间：
2000-02-04
影响因子：
4.8
通讯作者：
Mizuno, K
中科院分区：
文献类型：
--
作者：
Ohashi, K;Nagata, K;Mizuno, K
LIMK 1磷酸化肌动蛋白解聚因子cofilin，并调节肌动蛋白细胞骨架重组。LIMK 1被小G蛋白Rho及其下游蛋白激酶ROCK激活。我们现在报告由ROCK磷酸化LIMK 1的网站。体外激酶反应显示，活性形式的ROCK磷酸化LIMK 1的苏氨酸残基，并显着增加其cofilin磷酸化活性。一个LIMK 1突变体（T508 A）与替换的Thr-508内的激活环的激酶结构域的丙氨酸既不磷酸化，也不激活的ROCK。用丝氨酸替换Thr-508将ROCK催化的磷酸化残基从苏氨酸改变为丝氨酸。用两个谷氨酸取代Thr-508的LIMK 1突变体使激酶活性增加约a倍，但不被ROCK进一步激活。此外，野生型LIMK 1，而不是其T508 A突变体，通过与ROCK在培养的细胞中共表达而被激活。这些结果表明，ROCK在体外和体内通过Thr-508磷酸化激活LIMK 1。结合最近的发现，派（I），Rac的下游效应物，也通过Thr-508的磷酸化激活LIMK 1，这些结果表明LIMK 1的激活是Rho和Rac重组肌动蛋白细胞骨架的共同靶点之一。
LIM-kinase 1 (LIMK1) phosphorylates cofilin, an actin-depolymerizing factor, and regulates actin cytoskeletal reorganization. LIMK1 is activated by the small GTPase Rho and its downstream protein kinase ROCK. We now report the site of phosphorylation of LIMK1 by ROCK. In vitro kinase reaction revealed that the active forms of ROCK phosphorylated LIMK1 on the threonine residue and markedly increased its cofilin-phosphorylating activity. A LIMK1 mutant (T508A) with replacement of Thr-508 within the activation loop of the kinase domain by alanine was neither phosphorylated nor activated by ROCK. Replacement of Thr-508 by serine changed the ROCK-catalyzed phosphorylation residue from threonine to serine. A LIMK1 mutant with replacement of Thr-508 by two glutamates increased the kinase activity about a-fold but was not further activated by ROCK. In addition, wild-type LIMK1, but not its T508A mutant, was activated by co-expression with ROCK in cultured cells. These results suggest that ROCK activates LIMK1 in vitro and in vivo by phosphorylation at Thr-508. Together with the recent finding that PAI(I, a downstream effector of Rac, also activates LIMK1 by phosphorylation at Thr-508, these results suggest that activation of LIMK1 is one of the common targets for Rho and Rac to reorganize the actin cytoskeleton.