Testicular protein kinase 1 (TESK1) is a serine/threonine kinase with a structure composed of a kinase domain related to those of LIM-kinases and a unique C-terminal proline-rich domain. Like LIM-kinases, TESK1 phosphorylated cofilin specifically at Ser-3, both in vitro and in vivo. When expressed in HeLa cells, TESK1 stimulated the formation of actin stress fibers and focal adhesions. In contrast to LIM-kinases, the kinase activity of TESK1 was not enhanced by Rho-associated kinase (ROCK) or p21-activated kinase, indicating that TESK1 is not their downstream effector. Both the kinase activity of TESK1 and the level of cofilin phosphorylation increased by plating cells on fibronectin. Y-27632, a specific inhibitor of ROCK, inhibited LIM-kinase-induced cofilin phosphorylation but did not affect fibronectin-induced or TESK1-induced cofilin phosphorylation in HeLa cells. Expression of a kinase-negative TESK1 suppressed cofilin phosphorylation and formation of stress fibers and focal adhesions induced in cells plated on fibronectin. These results suggest that TESK1 functions downstream of integrins and plays a key role in integrin-mediated actin reorganization, presumably through phosphorylating and inactivating cofilin. We propose that TESK1 and LIM-kinases commonly phosphorylate cofilin but are regulated in different ways and play distinct roles in actin reorganization in living cells.

Actin filament dynamics play a critical role in mitosis and cytokinesis. LIM motif-containing protein kinase 1 (LIMK1) regulates actin reorganization by phosphorylating and inactivating cofilin, an actin-depolymerizing and -severing protein. To examine the role of LIMK1 and cofilin during the cell cycle, we measured cell cycleassociated changes in the kinase activity of LIMK1 and in the level of cofilin phosphorylation. Using synchronized HeLa cells, we found that LIMK1 became hyperphosphorylated and activated in prometaphase and metaphase, then gradually returned to the basal level as cells entered into telophase and cytokinesis. Although Rho-associated kinase and p21-activated protein kinase phosphorylate and activate LIMK1, they are not likely to be involved in mitosis-specific activation and phosphorylation of LIMK1. Immunoblot and immunofluorescence analyses using an anti-phosphocofilin-specific antibody revealed that the level of cofilin phosphorylation, similar to levels of LIMK1 activity, increased during prometaphase and metaphase then gradually declined in telophase and cytokinesis. Ectopic expression of LIMK1 increased the level of cofilin phosphorylation throughout the cell cycle and induced the formation of multinucleate cells. These results suggest that LIMK1 is involved principally in control of mitosis-specific cofilin phosphorylation and that dephosphorylation and reactivation of cofilin at later stages of mitosis play a critical role in cytokinesis of mammalian cells.

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.