Inhibition of Nuclear Import of LIMK2 in Endothelial Cells by Protein Kinase C-dependent Phosphorylation at Ser-283

Abstract

LIM kinases (LIMKs) are mainly in the cytoplasm and regulate actin dynamics through cofilin phosphorylation. Recently, it has been reported that nuclear localization of LIMKs can mediate suppression of cyclin D1 expression. Using immunofluorescence monitoring of enhanced green fluorescent protein-tagged LIMK2 in combination with photobleaching techniques and leptomycin B treatment, we demonstrate that LIMK2 shuttles between the cytoplasm and the nucleus in endothelial cells. Sequence analysis predicted two PKC phosphorylation sites in LIMK2 but not in LIMK1. One site at Ser-283 is present between the PDZ and the kinase domain, and the other site at Thr-494 is within the kinase domain. Activation of PKC by phorbol ester treatment of endothelial cells stimulated LIMK2 phosphorylation at Ser-283 and inhibited nuclear import of LIMK2 and the PDZ kinase construct of LIMK2 (amino acids 142-638) but not of LIMK1. The PKC-␦ isoform phosphorylated LIMK2 at Ser-283 in vitro. Mutational analysis indicated that LIMK2 phosphorylation at Ser-283 but not Thr-494 was functional. Serum stimulation of endothelial cells also inhibited nuclear import of PDZK-LIMK2 by protein kinase C-dependent phosphorylation of Ser-283. Our study shows that phorbol ester and serum stimulation of endothelial cells inhibit nuclear import of LIMK2 but not LIMK1. This effect was dependent on PKC-␦-mediated phosphorylation of Ser-283. Since phorbol ester enhanced cyclin D1 expression and subsequent G 1 -to-S-phase transition of endothelial cells, we suggest that the PKC-mediated exclusion of LIMK2 from the nucleus might be a mechanism to relieve suppression of cyclin D1 expression by LIMK2.Contraction, migration, and proliferation of vascular endothelial cells are essential features of vascular permeability, endothelial repair after injury, and angiogenesis (1, 2) and are regulated by coordinated changes of actin dynamics (3, 4). The LIMK 1 family of proteins, a member of the class of serine/threonine protein kinases, consists of LIMK1 and LIMK2 that specifically phosphorylate and inactivate cofilin, an actin-depolymerizing protein, thereby regulating actin cytoskeleton rearrangement (5, 6). Other studies showed that cell cycle progression depends on the regulated activity of the LIMK-cofilin system (7,8). The kinase activity of LIMKs is regulated by members of the RhoGTPase family, Rho, Rac, and CdC42, via their downstream protein kinases Rho kinase and p21-activated kinases 1 and 4. These kinases phosphorylate LIMK1 at Thr-508 in the activation loop of the kinase domain (9 -11). Rho kinase activates LIMK2 by phosphorylation at 13).Several lines of evidence suggest that LIMKs also have a function in the nucleus. LIMK1 is predominantly localized in the cytoplasm but accumulates in the nucleus, when the cells are treated with the CRM1-dependent export inhibitor, leptomycin B (LMB) (14). In mouse tissues, various splice forms of LIMK2 have been reported that display an unique cellular localization (15, 16). LIMK2a (full-length) and LIMK2b containing only...

LIM kinase-2 induces programmed necrotic neuronal death via dysfunction of DRP1-mediated mitochondrial fission

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A Pathway for the Control of Anoikis Sensitivity by E-Cadherin and Epithelial-to-Mesenchymal Transition

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p53-Mediated transactivation of LIMK2b links actin dynamics to cell cycle checkpoint control

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