Identification of MAPK phosphorylation sites and their role in the localization and activity of hypoxia-inducible factor-1α

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-1α, involves several post-translational modifications. To study HIF-1α phosphorylation, we have used human full-length recombinant HIF-1α 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-1α. Analysis of in vitro phosphorylated HIF-1α 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-1α. When these mutant forms of HIF-1α 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-1α mutants in HeLa cells showed their exclusion from the nucleus in contrast to wild-type HIF-1α. Treatment of the cells with leptomycin B, an inhibitor of the major exportinCRM1,reversed this exclusion and led to nuclear accumulation and partial recovery of the activity of the HIF-1α mutants. Moreover, inhibition of the MAPK pathway by PD98059 impaired the phosphorylation, nuclear accumulation, and activity of wild-type GFP-HIF-1α. Overall, these data suggest that phosphorylation of Ser-641/643 by MAPK promotes the nuclear accumulation and transcriptional activity of HIF-1α by blocking its CRM1-dependent nuclear export. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.