TI - Specific activation of LIM kinase 2 via phosphorylation of threonine 505 by ROCK , a Rho -dependent protein kinase . AB - LIM-kinase 1 ( LIMK1 ) and LIM-kinase 2 ( LIMK2 ) regulate actin cytoskeletal reorganization via cofilin phosphorylation downstream of distinct Rho family GTPases . We report our findings that ROCK , a downstream protein kinase of Rho , specifically activates LIMK2 but not LIMK1 downstream of RhoA . LIMK1 and LIMK2 activities toward cofilin phosphorylation were stimulated by co-expression with the active form of ROCK ( ROCK-Delta3 ) , whereas full-length ROCK selectively activates LIMK2 but not LIMK1 . Activation of LIMK2 by RhoA was inhibited by Y-27632 , a specific inhibitor of ROCK , but Rac1 -mediated activation of LIMK1 was not . ROCK directly phosphorylated the threonine 505 residue within the activation segment of LIMK2 and markedly stimulated LIMK2 activity . A LIMK2 mutant with replacement of threonine 505 by valine abolished LIMK2 activities for cofilin phosphorylation and actin cytoskeletal changes , whereas replacement by glutamate enhanced the protein kinase activity and stress fiber formation by LIMK2 . These results indicate that ROCK directly phosphorylates threonine 505 and activates LIMK2 downstream of RhoA and that this phosphorylation is essential for LIMK2 to induce actin cytoskeletal reorganization . Together with the finding that LIMK1 is regulated by Pak1 , LIMK1 and LIMK2 are regulated by different protein kinases downstream of distinct Rho family GTPases .