Specific activation of LIM kinase 2 via phosphorylation of threonine 505 by ROCK, a Rho-dependent protein kinase.
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.
Sumi
T
T
Division of Biochemistry, Department of Oncology, Biomedical Research Center, Osaka University Graduate School of Medicine, Suita, Osaka 565-0871, Japan.
Matsumoto
K
K
Nakamura
T
T
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism; D000577:Amides / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D020134:Catalytic Domain / Q000187:drug effects; D003599:Cytoskeleton / Q000378:metabolism; D004268:DNA-Binding Proteins; D004789:Enzyme Activation / Q000187:drug effects; D006367:HeLa Cells; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D060850:LIM-Homeodomain Proteins; D054461:Lim Kinases; D008840:Microfilament Proteins / Q000378:metabolism; D009154:Mutation; D010766:Phosphorylation / Q000187:drug effects; D010769:Phosphothreonine / Q000378:metabolism*; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000187:drug effects; D011725:Pyridines / Q000494:pharmacology; D015398:Signal Transduction; D014157:Transcription Factors; D014162:Transfection; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D054460:rho-Associated Kinases
D051339:Actin Depolymerizing Factors; D000199:Actins; D000577:Amides; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D047908:Intracellular Signaling Peptides and Proteins; C085473:LHX1 protein, human; D060850:LIM-Homeodomain Proteins; D008840:Microfilament Proteins; D011725:Pyridines; D014157:Transcription Factors; D010769:Phosphothreonine; C108830:Y 27632; D011494:Protein Kinases; C102895:LIMK2 protein, human; D054461:Lim Kinases; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020830:rac1 GTP-Binding Protein