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J. Biol. Chem., Vol. 281, Issue 44, 33095-33106, November 3, 2006

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Identification of MAPK Phosphorylation Sites and Their Role in the Localization and Activity of Hypoxia-inducible Factor-1^*

Ilias Mylonis, Georgia Chachami, Martina Samiotaki^¶, George Panayotou^¶, Efrosini Paraskeva, Alkmini Kalousi, Eleni Georgatsou, Sofia Bonanou, and George Simos¹

From the Laboratories of Biochemistry and Physiology, Department of Medicine, University of Thessaly, Larissa 41222, Greece and the ^¶Protein Chemistry Laboratory, Biomedical Sciences Research Center "Alexander Fleming," Vari 16672, Greece

Hypoxia-inducible factor 1 (HIF-1) controls the expression of most genes induced by hypoxic conditions. Regulation of expression and activity of its inducible subunit, HIF-1, involves several post-translational modifications. To study HIF-1 phosphorylation, we have used human full-length recombinant HIF-1 as a substrate in kinase assays. We show that at least two different nuclear protein kinases, one of them identified as p42/p44 MAPK, can modify HIF-1. Analysis of in vitro phosphorylated HIF-1 by mass spectroscopy revealed residues Ser-641 and Ser-643 as possible MAPK phosphorylation sites. Site-directed mutagenesis of these residues reduced significantly the phosphorylation of HIF-1. When these mutant forms of HIF-1 were expressed in HeLa cells, they exhibited much lower transcriptional activity than the wild-type form. However, expression of the same mutants in yeast revealed that their capacity to stimulate transcription was not significantly compromised. Localization of the green fluorescent protein-tagged HIF-1 mutants in HeLa cells showed their exclusion from the nucleus in contrast to wild-type HIF-1. Treatment of the cells with leptomycin B, an inhibitor of the major exportin CRM1, reversed this exclusion and led to nuclear accumulation and partial recovery of the activity of the HIF-1 mutants. Moreover, inhibition of the MAPK pathway by PD98059 impaired the phosphorylation, nuclear accumulation, and activity of wild-type GFP-HIF-1. Overall, these data suggest that phosphorylation of Ser-641/643 by MAPK promotes the nuclear accumulation and transcriptional activity of HIF-1 by blocking its CRM1-dependent nuclear export.

Received for publication, May 26, 2006 , and in revised form, August 31, 2006.

^* This work was supported in part by a grant co-funded by the European Social Fund & National Resources, EPEAEK II, PYTHAGORAS, from the Greek Ministry of National Education and Religious Affairs (to G. S.). 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.

¹ To whom correspondence should be addressed: 22 Papakyriazi Str., Larissa 41222, Greece. Fax: 30-2410-565054; E-mail: simos{at}med.uth.gr.

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