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 IκBα. It is widely speculated that hydroxylation of IκBα has functional consequences, but the effects of hydroxylation have not been demonstrated. We prepared hydroxylated IκBα 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 NFκB showed no difference in the NFκB 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 IκBα by FIH-1 is inconsequential, at least for the functions we could assay in vitro.
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093-0378, USA.
Ingrid L
IL
Devries
Rachel J
RJ
Hampton-Smith
Melinda M
MM
Mulvihill
Vera
V
Alverdi
Daniel J
DJ
Peet
Elizabeth A
EA
Komives
D000595:Amino Acid Sequence; D002460:Cell Line; D006801:Humans; D006900:Hydroxylation; D051550:I-kappa B Kinase / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011487:Protein Conformation; D058767:Protein Unfolding; D012097:Repressor Proteins / Q000378:metabolism*; D020349:Surface Plasmon Resonance
D016328:NF-kappa B; D012097:Repressor Proteins; D051550:I-kappa B Kinase; D046988:Proteasome Endopeptidase Complex