Abstract
The hypoxia-inducible factors 1alpha (HIF-1alpha) and 2alpha (HIF-2alpha) are key regulators of the transcriptional response to low oxygen and are closely related in domain architecture, DNA binding, and activation mechanisms. Despite these similarities, targeted disruption of the HIF-alpha genes in mice results in distinctly different phenotypes demonstrating nonredundancy of function, although the underlying mechanisms remain unclear. Here we report on the novel and specific interaction of HIF-2alpha, but not HIF-1alpha, with the NF-kappaB essential modulator (NEMO) using immunoprecipitation, mammalian two-hybrid, and in vitro protein interaction assays. Reporter gene assays demonstrate that this interaction specifically enhances normoxic HIF-2alpha transcriptional activity, independently of the HIF-2alpha transactivation domain, consistent with a model by which NEMO aids CBP/p300 recruitment to HIF-2alpha. In contrast, HIF-2alpha overexpression does not alter NF-kappaB signaling, suggesting that the functional consequence of the HIF-2alpha/NEMO interaction is limited to the HIF pathway. The specificity of NEMO for HIF-2alpha represents one of the few known differential protein-protein interactions between the HIF-alpha proteins, which has important implications for the activity of HIF-2alpha and is also the first postulated NF-kappaB-independent role for NEMO.
Highlights
Aerobic organisms are critically dependent upon the supply of oxygen for fundamental processes such as energy production
We demonstrate that this physical interaction has a functional effect, increasing the normoxic activity of HIF-2␣, but not HIF-1␣, in a dose-dependent manner, consistent with a model by which NF-B essential modulator (NEMO) aids CBP/p300 recruitment to HIF-2␣
Identification of NEMO as a HIF-2␣-interacting Protein—To identify novel HIF-2␣-interacting proteins, amino acids 565– 820 of mouse HIF-2␣ fused to the Gal4 DNA-binding domain (Gal4DBD) were used as bait in a yeast two-hybrid screen of a mouse E10.5 cDNA library
Summary
Aerobic organisms are critically dependent upon the supply of oxygen for fundamental processes such as energy production. The activity of both proteins is controlled through hydroxylation of key proline and asparagine residues in the conserved oxygen-dependent degradation domain and transactivation domain (TAD), respectively, regulating protein stability and transactivation potential [2,3,4] Despite these similarities, HIF-1␣- and HIF-2␣-deficient mice manifest distinct phenotypes and have nonredundant functions. HIF-2␣-deficient mice have been reported to exhibit a multiple organ pathology, consistent with mitochondrial disease [11] Concomitant with these phenotypes, there is a growing body of evidence demonstrating different responses between the HIF-␣ proteins in relation to cellular localization, hypoxic responsiveness, and target gene expression [12,13,14]. We demonstrate that this physical interaction has a functional effect, increasing the normoxic activity of HIF-2␣, but not HIF-1␣, in a dose-dependent manner, consistent with a model by which NEMO aids CBP/p300 recruitment to HIF-2␣
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