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J. Biol. Chem., Vol. 277, Issue 41, 38723-38730, October 11, 2002

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Functional Analysis of Hypoxia-inducible Factor-1-mediated Transactivation
IDENTIFICATION OF AMINO ACID RESIDUES CRITICAL FOR TRANSCRIPTIONAL ACTIVATION AND/OR INTERACTION WITH CREB-BINDING PROTEIN^*

Jorge L. Ruas, Lorenz Poellinger^§, and Teresa Pereira^¶

From the Department of Cell and Molecular Biology, Karolinska Institutet, S-171 77 Stockholm, Sweden

The hypoxia-inducible factor-1 (HIF-1) is a key regulator of adaptive responses to hypoxia. HIF-1 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 -helix within the C-TAD of mouse HIF-1 to identify residues important for transactivation and interaction of the C-TAD with transcriptional coactivators. We observed that several hydrophobic residues, Ile⁸⁰², Leu⁸⁰⁸, Leu⁸¹⁴, Leu⁸¹⁵, and Leu⁸¹⁸, 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, 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 in the presence of CBP, indicating that the integrity of the C-TAD is critical for intracellular localization of mouse HIF-1.

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