Functional analysis of hypoxia-inducible factor-1 alpha-mediated transactivation. Identification of amino acid residues critical for transcriptional activation and/or interaction with CREB-binding protein.
The hypoxia-inducible factor-1 alpha (HIF-1 alpha) is a key regulator of adaptive responses to hypoxia. HIF-1 alpha has two independent transactivation domains (TADs). Whereas the N-terminal TAD (N-TAD) also constitutes a degradation box, the C-terminal TAD (C-TAD) functions in a strictly hypoxia-inducible fashion. Oxygen-dependent hydroxylation of an asparagine residue has recently been reported to regulate C-TAD function by disrupting the interaction with the CH1 domain of the p300/CBP coactivator at normoxia. Here we have performed alanine-scanning mutagenesis of a predicted alpha-helix within the C-TAD of mouse HIF-1 alpha to identify residues important for transactivation and interaction of the C-TAD with transcriptional coactivators. We observed that several hydrophobic residues, Ile(802), Leu(808), Leu(814), Leu(815), and Leu(818), were critical for transactivation and binding to the CH1 domain of CBP in hypoxic cells. Moreover, E812A/E813A and D819A mutations impaired hypoxia-dependent transactivation without disrupting binding to CH1. In the context of full-length HIF-1 alpha, mutation of the leucine residues conferred conformational changes to the protein and significantly reduced the transactivation function as well as functional interaction with the transcriptional coactivators CBP and SRC-1. These mutations also affected intranuclear redistribution of HIF-1 alpha in the presence of CBP, indicating that the integrity of the C-TAD is critical for intracellular localization of mouse HIF-1 alpha.
Department of Cell and Molecular Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden.
Jorge L
JL
Ruas
Lorenz
L
Poellinger
Teresa
T
Pereira
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D050882:CREB-Binding Protein; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D051548:Histone Acetyltransferases; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D007930:Leucine / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism*; D056920:Nuclear Receptor Coactivator 1; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015534:Trans-Activators / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000502:physiology*
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D050882:CREB-Binding Protein; C494363:CREBBP protein, human; C494364:Crebbp protein, mouse; D051548:Histone Acetyltransferases; C538859:NCOA1 protein, human; C538861:Ncoa1 protein, mouse; D056920:Nuclear Receptor Coactivator 1; D007930:Leucine