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J. Biol. Chem., Vol. 280, Issue 30, 27569-27577, July 29, 2005

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

Pankaj Goyal, Dharmendra Pandey, Antje Behring, and Wolfgang Siess

From the Institute for Prevention of Cardiovascular Diseases, University of Munich, 80336 München, Germany

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 G₁-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.

Received for publication, April 22, 2005

^* The work was supported by grants from the August-Lenz-Stiftung and the Deutsche Forschungsgemeinschaft (Graduate Program "Vascular Biology in Medicine" Grants GRK 438 (to P. G., D. H., and A. B.), SFB 413, and Si 274/9. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains two supplemental figures.

The results are part of the doctorate of Philosophy thesis of Pankaj Goyal at the University of Munich.

To whom correspondence should be addressed: Wolfgang Siess, Institut für Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Klinikum Innenstadt, Universität München, Pettenkoferstr. 9, D 80336 München, Germany. Tel.: 49-89-5160-4380; Fax: 49-89-5160-4382; E-mail: wsiess{at}med.uni-muenchen.de.

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This article has been cited by other articles:

				P. Goyal, D. Pandey, and W. Siess Phosphorylation-dependent Regulation of Unique Nuclear and Nucleolar Localization Signals of LIM Kinase 2 in Endothelial Cells J. Biol. Chem., September 1, 2006; 281(35): 25223 - 25230. [Abstract] [Full Text] [PDF]

				D. Pandey, P. Goyal, J. R. Bamburg, and W. Siess Regulation of LIM-kinase 1 and cofilin in thrombin-stimulated platelets Blood, January 15, 2006; 107(2): 575 - 583. [Abstract] [Full Text] [PDF]