LIM-kinase 2 induces formation of stress fibres, focal adhesions and membrane blebs, dependent on its activation by Rho-associated kinase-catalysed phosphorylation at threonine-505.
LIM-kinase 1 and 2 (LIMK1 and LIMK2) phosphorylate cofilin and induce actin cytoskeletal reorganization. LIMK1 is activated by Rho-associated, coiled-coil-forming protein kinase (ROCK) and p21-activated kinase 1 (PAK1), but activation mechanisms and cellular functions of LIMK2 have remained to be determined. We report here that LIMK1 and LIMK2 phosphorylate both cofilin and actin-depolymerizing factor (ADF) specifically at Ser-3 and exhibit partially distinct substrate specificity when tested using site-directed cofilin mutants as substrates. We also show that LIMK2 is activated by ROCK by phosphorylation at Thr-505 within the activation loop. Wild-type LIMK2, but not its mutant (T505V) with replacement of Thr-505 by Val, was activated by ROCK in vitro and in vivo. LIMK2 mutants with replacement of Thr-505 by one or two Glu residues (T505E or T505EE) increased the kinase activity about 3.6-fold but were not further activated by ROCK. When expressed in HeLa cells, wild-type LIMK2, but not the T505V mutant, induced the formation of stress fibres, focal adhesions and membrane blebs. Furthermore, inhibitors of Rho and ROCK significantly suppressed LIMK2-induced stress fibres and membrane blebs. These results suggest that LIMK2 functions downstream of the Rho-ROCK signalling pathway and plays a role in reorganization of actin filaments and membrane structures, by phosphorylating cofilin/ADF proteins.
Amano
T
T
Biological Institute, Graduate School of Science, Tohoku University, Sendai 980-8578, and Department of Biology, Graduate School of Science, Kyushu University, Fukuoka 812-8581, Japan.
Tanabe
K
K
Eto
T
T
Narumiya
S
S
Mizuno
K
K
D051339:Actin Depolymerizing Factors; D000199:Actins / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D003599:Cytoskeleton / Q000648:ultrastructure*; D017931:DNA Primers; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D054461:Lim Kinases; D008840:Microfilament Proteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism*
D051339:Actin Depolymerizing Factors; D000199:Actins; D017931:DNA Primers; D008840:Microfilament Proteins; D013912:Threonine; D011494:Protein Kinases; C515892:LIMK1 protein, human; D054461:Lim Kinases