Abstract

Various stimuli like thrombin induce endothelial cell shape change and stress fiber formation via Rho/Rho-kinase-mediated reorganization of the actin cytoskeleton. LIM-kinases regulate actin cytoskeletal reorganization through phosphorylation of cofilin at Ser3. The LIMK family kinases possess characteristic structural features, consisting of two LIM domains, a PDZ domain and a C-terminal kinase domain. In cell transfection studies it has been shown that LIMK2 is phosphorylated at Thr505 by Rho-kinase thereby activating the enzyme. Recently it has been reported that nuclear LIMKs suppress cyclin D1 expression in a manner independent of cofilin phosphorylation and actin polymerization. In this study, we found that endothelial cells express both LIMK1 and LIMK2. By using live cell imaging, we confirm previous findings that thrombin induces stress fiber formation, ruffle formation and cell contraction. Furthermore, the cell-cell contacts were disrupted and F-actin fibers connecting two cells were broken. Thrombin induced a rapid and sustained Rho-kinase activation and subsequent phosphorylation of LIM-kinase and cofilin. Pretreatment of endothelial cells with the specific Rho-kinase inhibitor Y27632 inhibited MYPT1 phosphorylation, LIM-kinase and cofilin phosphorylation and blocked stress fiber formation in thrombin-stimulated cells. Notably, thrombin induced actin stress fiber formation was abolished in cells transfected with dominant negative LIMK2. LIMK2 was mainly localized in the cytoplasm. By using Leptomycin B (a specific inhibitor of CRM-1 dependent nuclear export) and FRAP and FLIP analysis, we demonstrate that LIMK2 in resting endothelial cells shuttles between the nucleus and cytoplasm. The LIM domains of LIMK2, but not of LIMK1 inhibited its nuclear import thereby keeping LIMK2 mainly in the cytoplasm. Mutational analysis of the unique basic amino acid-rich motif (amino acids 480-503) indicated that this motif regulates the nuclear and nucleolar localization of LIMK2. Activation of PKC in PMA-stimulated endothelial cells stimulated the phosphorylation of LIMK2 at Ser283 and the translocation of LIMK2 and the PDZ-kinase construct of LIMK2 from the nucleus to the cytoplasm. Of the various PKC isoforms, PKC- and PKC- were found to be mainly responsible for Ser283 phosphorylation and the regulation of translocation of LIMK2. Mutational analysis indicated that LIMK2 phosphorylation at Ser283 and Thr494 play a role in the regulation of nucleocytoplasmic shuttling of LIMK2 by PKC. These results show that LIM-kinase activation is mediated by Rho-kinase in stimulated endothelial cells, and that LIM-kinase-mediated cofilin phosphorylation plays an essential role in thrombin-induced stress fiber formation. LIMK2 shuttles between nucleus and cytoplasm in resting endothelial cells. Phosphorylation of LIMK2 at Ser283 and Thr494 by PKC regulates nucleocytoplasmic shuttling and suggests that LIMK2 might also have a function in the nucleus such as the suppression of cyclin D1 expression.