Consequences of IkappaB alpha hydroxylation by the factor inhibiting HIF (FIH)

The factor inhibiting HIF-1 ( FIH-1) hydroxylates many ankyrin repeat-containing proteins including I kappa B alpha. It is widely speculated that hydroxylation of I kappa B alpha has functional consequences, but the effects of hydroxylation have not been demonstrated. We prepared hydroxylated I kappa B alpha and compared it to the unhydroxylated protein. Urea denaturation and amide H/D exchange experiments showed no change in the foldedness upon hydroxylation. Surface plasmon resonance measurements of binding to NF kappa B showed no difference in the NF kappa B binding kinetics or thermodynamics. Ubiquitin-independent proteasomal degradation experiments showed no difference in the half-life of the protein. Thus, it appears that hydroxylation of I kappa B alpha by FIH-1 is inconsequential, at least for the functions we could assay in vitro. Structured summary: MINT-8051494: NF-kappa-B p65 (uniprotkb:Q04207) physically interacts (MI: 0915) with NF-kappa-B p50 (uniprotkb:P25799) and I-kappa-B alpha (uniprotkb:O15111) by surface plasmon resonance (MI:0107) (c) 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.