Visit our Expo - Redox and Inflammation signaling 2012

Session III : Protein kinase cascades as therapeutic targets Poster III, 38
Phosphorylation by p42/44 MAPK regulates the activity and localization of human
hypoxia inducible factor HIF-1alpha.
Ilias Mylonis1, Georgia Chachami1,2, Martina Samiotaki3, George Panayotou3,
Efrosyni Paraskeva2, Eleni Georgatsou1, Sophia Bonanou1 and George Simos1
1Laboratory of Biochemistry and 2Laboratory of Physiology, Department of Medicine,
University of Thessaly, Larissa, Greece.
3Protein Chemistry Laboratory, B.S.R.C. «Alexander Fleming», Vari, Greece
E-mail: simos@med.uth.gr
Hypoxia inducible factor 1 (HIF-1) is a heterodimeric transcriptional activator that controls
the expression of most of the genes induced by hypoxic conditions. The molecular
mechanisms that regulate the expression and activity of its inducible subunit HIF-1alpha,
involve several post-translational modifications including hydroxylation, acetylation and
phosphorylation. In order to study the role of HIF-1alpha phosphorylation we have used
human recombinant HIF-1alpha as a substrate in kinase assays with cell extracts. Our data
show that at least two different nuclear protein kinases can interact with and modify HIF-
1alpha and one of them was identified as the p42/p44 MAPK. Analysis of phosphorylated
HIF-1alpha by mass spectroscopy revealed two serine residues as possible MAPK
phosphorylation sites in its carboxy-terminal part. Site-directed mutagenesis of these two
residues significantly reduced the phosphorylation of HIF-1alpha by either cell extracts or
purified MAPK, showing that they represent major in vitro modification sites. When the
mutant forms of HIF-1alpha were introduced into HeLa cells, they exhibited much lower
transcriptional activity than the wild-type. Furthermore, the GFP-tagged HIF-1alpha mutants
were excluded from the nucleus in contrast to the predominant nuclear localization of the
wild-type form. These data suggest that phosphorylation of the identified sites by MAPK is
required for nuclear accumulation of HIF-1alpha and subsequent activation of the hypoxia
target genes.
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