Ohashi K, Nagata K, Maekawa M, Ishizaki T, Narumiya S, Mizuno K
Biological Institute, Graduate School of Science, Tohoku University, Sendai 980-8578, Japan.
J Biol Chem. 2000 Feb 4;275(5):3577-82. doi: 10.1074/jbc.275.5.3577.
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 2-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 PAK1, 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.
LIM激酶1（LIMK1）使肌动蛋白解聚因子cofilin磷酸化，从而调节肌动蛋白细胞骨架的重组。LIMK1由小GTP酶Rho及其下游蛋白激酶ROCK激活。我们现在报告ROCK对LIMK1的磷酸化位点。体外激酶反应显示，ROCK的活性形式使LIMK1的苏氨酸残基磷酸化，并显著增加其对cofilin的磷酸化活性。激酶结构域激活环内的苏氨酸508（Thr-508）被丙氨酸取代的LIMK1突变体（T508A）既不被ROCK磷酸化也不被其激活。将Thr-508替换为丝氨酸会使ROCK催化的磷酸化残基从苏氨酸变为丝氨酸。将Thr-508替换为两个谷氨酸的LIMK1突变体的激酶活性增加了约2倍，但不再被ROCK进一步激活。此外，在培养细胞中，野生型LIMK1与ROCK共表达时被激活，但其T508A突变体则不然。这些结果表明，ROCK在体外和体内通过对Thr-508的磷酸化激活LIMK1。连同最近发现的Rac的下游效应器PAK1也通过对Thr-508的磷酸化激活LIMK1，这些结果表明LIMK1的激活是Rho和Rac重组肌动蛋白细胞骨架的共同靶点之一。
