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

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 alpha, involves several post-translational modifications. To study HIF-1 alpha phosphorylation, we have used human full-length recombinant HIF-1 alpha as a substrate in kinase assays. We show that at least two different nuclear protein kinases, one of them identified as p42/p44MAPK, can modify HIF-1 alpha. Analysis of in vitro phosphorylated HIF-1 alpha 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 alpha. When these mutant forms of HIF-1 alpha 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 alpha mutants in HeLa cells showed their exclusion from the nucleus in contrast to wild-type HIF-1 alpha. 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 alpha mutants. Moreover, inhibition of the MAPK pathway by PD98059 impaired the phosphorylation, nuclear accumulation, and activity of wild-type GFP-HIF-1 alpha. Overall, these data suggest that phosphorylation of Ser-641/643 by MAPK promotes the nuclear accumulation and transcriptional activity of HIF-1 alpha by blocking its CRM1-dependent nuclear export.