title: Identification of MAPK phosphorylation sites and their role in the localization and activity of hypoxia-inducible factor-1alpha.
abstract: 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-1alpha, involves several post-translational modifications. To study HIF-1alpha phosphorylation, we have used human full-length recombinant HIF-1alpha 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-1alpha. Analysis of in vitro phosphorylated HIF-1alpha 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-1alpha. When these mutant forms of HIF-1alpha 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-1alpha mutants in HeLa cells showed their exclusion from the nucleus in contrast to wild-type HIF-1alpha. 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-1alpha mutants. Moreover, inhibition of the MAPK pathway by PD98059 impaired the phosphorylation, nuclear accumulation, and activity of wild-type GFP-HIF-1alpha. Overall, these data suggest that phosphorylation of Ser-641/643 by MAPK promotes the nuclear accumulation and transcriptional activity of HIF-1alpha by blocking its CRM1-dependent nuclear export.
authors.affiliation: Laboratory of Biochemistry, Department of Medicine, University of Thessaly, Larissa 41222, Greece.
authors.forename: Ilias
authors.initials: I
authors.lastname: Mylonis
authors.forename: Georgia
authors.initials: G
authors.lastname: Chachami
authors.forename: Martina
authors.initials: M
authors.lastname: Samiotaki
authors.forename: George
authors.initials: G
authors.lastname: Panayotou
authors.forename: Efrosini
authors.initials: E
authors.lastname: Paraskeva
authors.forename: Alkmini
authors.initials: A
authors.lastname: Kalousi
authors.forename: Eleni
authors.initials: E
authors.lastname: Georgatsou
authors.forename: Sofia
authors.initials: S
authors.lastname: Bonanou
authors.forename: George
authors.initials: G
authors.lastname: Simos
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002467:Cell Nucleus / Q000201:enzymology; D006367:HeLa Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism; D016415:Sequence Alignment; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics
chemical_list: D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D010768:Phosphoserine; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3