Modulation of p300 binding by posttranslational modifications of the C-terminal activation domain of hypoxia-inducible factor-1alpha.
Posttranslational modifications of hypoxia-inducible factor-1alpha (HIF-1alpha) influence HIF-mediated transcription, likely by affecting binding to p300/cAMP-response element-binding protein (CBP). To systematically analyze the HIF-1alpha-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-1alpha. After optimized for effectively capturing p300/CBP, the ... assay was utilized for evaluating direct effects of posttranslational modifications of the HIF-1alpha C-TAD on p300 binding. The results demonstrated that asparagine hydroxylation and S-nitrosylation of HIF-1alpha decrease p300 binding, while its phosphorylation does not affect p300 binding, which was reconfirmed by competitive inhibition analyses using mutant peptides.
Mesh Terms:
Amino Acid Motifs, Asparagine, Binding, Competitive, E1A-Associated p300 Protein, Fluorescence, Humans, Hydroxylation, Hypoxia-Inducible Factor 1, alpha Subunit, Nitrogen, Peptides, Phosphorylation, Protein Binding, Protein Processing, Post-Translational, Protein Structure, Tertiary, p300-CBP Transcription Factors
Amino Acid Motifs, Asparagine, Binding, Competitive, E1A-Associated p300 Protein, Fluorescence, Humans, Hydroxylation, Hypoxia-Inducible Factor 1, alpha Subunit, Nitrogen, Peptides, Phosphorylation, Protein Binding, Protein Processing, Post-Translational, Protein Structure, Tertiary, p300-CBP Transcription Factors
FEBS Lett.
Date: Apr. 17, 2007
PubMed ID: 17382325
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