Modulation of p300 binding by posttranslational modifications of the C-terminal activation domain of hypoxia-inducible factor-1α

Abstract 

Posttranslational modifications of hypoxia-inducible factor-1α (HIF-1α) influence HIF-mediated transcription, likely by affecting binding to p300/cAMP-response element-binding protein (CBP). To systematically analyze the HIF-1α–p300/CBP interaction, we developed a fluorescence polarization-based binding assay, employing fluorescein-labeled peptides derived from the C-terminal transactivation domain (C-TAD) of HIF-1α. After optimized for effectively capturing p300/CBP, the assay was utilized for evaluating direct effects of posttranslational modifications of the HIF-1α C-TAD on p300 binding. The results demonstrated that asparagine hydroxylation and S-nitrosylation of HIF-1α decrease p300 binding, while its phosphorylation does not affect p300 binding, which was reconfirmed by competitive inhibition analyses using mutant peptides.

Abbreviations: CBP, cAMP-response element-binding protein, CK2, casein kinase 2, C-TAD, C-terminal transactivation domain, FIH-1, factor inhibiting HIF-1α, FP, fluorescence polarization, HIF, hypoxia-inducible factor, VHL, von Hippel-Lindau protein

Keywords: Hypoxia-inducible factor-1α–p300/CBP, HIF-1α–p300/CBP interaction, Fluorescence polarization, Asparagine hydroxylation, S-Nitrosylation, Phosphorylation