Dual function of LIMK2 in endothelial cells

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.