New Substrates for Factor Inhibiting HIF

Abstract

Factor inhibiting hypoxia-inducible factor (FIH) is an asparaginyl hydroxylase, and hydroxylation of Asn of HIF (hypoxia-inducible factor) by FIH disrupts the interaction of HIF with the transcriptional coactivators CBP/p300. FIH activity is inhibited during hypoxia. Cockman et al . performed a yeast two-hybrid screen for proteins that interacted with FIH and found and confirmed the interaction with three ankryrin repeat domain (ARD)-containing proteins. Other ARD-containing peptides were also found to be substrates for FIH-dependent Asn hydroxylation. The interaction with the p105 precursor of the p50 component of the nuclear factor κB (NF-κB) transcription factor and with IκBα were characterized further. Interactions between the endogenous proteins were demonstrated by coimmunoprecipitation, and the interaction of p105 with FIH required Asn 678 . In vitro assays showed that in the presence of p105 or IκBα, FIH activity was stimulated, and liquid chromatography/mass spectrometry confirmed that p105 and IκBα were hydroxylated in vivo. RNA interference experiments showed that hydroxylation was dependent on FIH and hydroxylation of IκBα was decreased by hypoxic conditions. Despite using several different experimental approaches, the authors failed to show a clear consequence of ARD hydroxylation on NF-κB activity. Thus, it remains to be determined whether Asn hydroxylation is serving a structural role or is a regulatory posttranslational modification that alters the activity or interactions of these newly defined substrates. M. E. Cockman, D. E. Lancaster. I. P. Stolze, K. S. Hewitson, M. A. McDonough, M. L. Coleman, C. H. Coles, X. Yu, R. T. Hay, S. C. Ley, C. W. Pugh, N. J. Oldham, N. Masson, C. J. Schofield, P. J. Ratcliffe, Posttranslational hydroxylation of ankyrin repeats in IκB proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH). Proc. Natl. Acad. Sci. U.S.A. 103 , 14767-14772 (2006). [Abstract] [Full Text]