p42/p44 mitogen-activated protein kinases phosphorylate hypoxia-inducible factor 1 alpha (HIF-1 alpha) and enhance the transcriptional activity of HIF-1

Hypoxia-inducible factor-1 (HIF-1) controls the expression of a number of g enes such as vascular endothelial growth factor and erythropoietin in low o xygen conditions. However, the molecular mechanisms that underlie the activ ation of the limiting subunit, HIF-1 alpha, are still poorly resolved, Resu lts showing that endogenous HIF-1 alpha migrated 12 kDa higher than in vitr o translated protein led us to evaluate the possible role of phosphorylatio n on this phenomenon. We report here that HIF-1 alpha is strongly phosphory lated in vivo and that phosphorylation is responsible for the marked differ ences in the migration pattern of HIF-1 alpha, In vitro, HIF-1 alpha: is ph osphorylated by p42 and p44 mitogen-activated protein kinases (MAPKs) and n ot by p38 MAPK or c-Jun N-terminal kinase, interestingly p42/p44 MAPK stoic hiometrically phosphorylate HIF-1 alpha in vitro, as judged by a complete u pper shift of HIF-1 alpha. More importantly, we demonstrate that activation of the p42/p44 MAPK pathway in quiescent cells induced the phosphorylation and shift of HIF-1 alpha, which was abrogated in presence of the MEK inhib itor, PD 98059, Finally, we found that in a vascular endothelial growth fac tor promoter mutated at sites previously shown to be MAPK-sensitive (SP1/AP 2-88-66 site), p42/p44 MAPK activation is sufficient to promote the transcr iptional activity of HIF-1, This interaction between HIF-1 alpha and p42/p4 4 MAPK suggests a cooperation between hypoxic and growth factor signals tha t ultimately leads to the increase in RIF-l-mediated gene expression.