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PNAS 103 (40): 14767-14772

Copyright © 2006 by the National Academy of Sciences.

BIOLOGICAL SCIENCES / BIOCHEMISTRY

Posttranslational hydroxylation of ankyrin repeats in I{kappa}B proteins by the hypoxia-inducible factor (HIF) asparaginyl hydroxylase, factor inhibiting HIF (FIH)

Matthew E. Cockman{dagger}, David E. Lancaster{ddagger}, Ineke P. Stolze{dagger}, Kirsty S. Hewitson{ddagger}, Michael A. McDonough{ddagger}, Mathew L. Coleman{dagger}, Charlotte H. Coles{ddagger}, Xiaohong Yu§, Ronald T. Hay, Steven C. Ley||, Christopher W. Pugh{dagger}, Neil J. Oldham{ddagger}, Norma Masson{dagger}, Christopher J. Schofield{ddagger}, and Peter J. Ratcliffe{dagger},{dagger}{dagger}

{dagger}Henry Wellcome Building for Molecular Physiology, University of Oxford, Oxford OX3 7BN, United Kingdom; {ddagger}Chemistry Research Laboratory, Department of Chemistry, University of Oxford, Oxford OX1 3TA, United Kingdom; §Centre for Ecology and Hydrology, Oxford OX1 3SR, United Kingdom; Centre for Interdisciplinary Research, School of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom; and ||Division of Immune Cell Biology, National Institute for Medical Research, Mill Hill, London NW7 1AA, United Kingdom

Communicated by David Weatherall, University of Oxford, Oxford, United Kingdom, August 10, 2006

Received for publication June 1, 2006.

Abstract: Studies on hypoxia-sensitive pathways have revealed a series of Fe(II)-dependent dioxygenases that regulate hypoxia-inducible factor (HIF) by prolyl and asparaginyl hydroxylation. The recognition of these unprecedented signaling processes has led to a search for other substrates of the HIF hydroxylases. Here we show that the human HIF asparaginyl hydroxylase, factor inhibiting HIF (FIH), also efficiently hydroxylates specific asparaginyl (Asn)-residues within proteins of the I{kappa}B family. After the identification of a series of ankyrin repeat domain (ARD)-containing proteins in a screen for proteins interacting with FIH, the ARDs of p105 (NFKB1) and I{kappa}B{alpha} were shown to be efficiently hydroxylated by FIH at specific Asn residues in the hairpin loops linking particular ankyrin repeats. The target Asn residue is highly conserved as part of the ankyrin consensus, and peptides derived from a diverse range of ARD-containing proteins supported FIH enzyme activity. These findings demonstrate that this type of protein hydroxylation is not restricted to HIF and strongly suggest that FIH-dependent ARD hydroxylation is a common occurrence, potentially providing an oxygen-sensitive signal to a diverse range of processes.

Key Words: NF-{kappa}B • 2-oxoglutarate-dependent dioxygenase • protein hydroxylation

Freely available online through the PNAS open access option.

Author contributions: N.M., C.J.S., and P.J.R. contributed equally to this work; M.E.C., D.E.L., I.P.S., K.S.H., C.W.P., N.M., C.J.S., and P.J.R. designed research; M.E.C., D.E.L., I.P.S., K.S.H., M.L.C., C.H.C., N.J.O., and N.M. performed research; X.Y., R.T.H., S.C.L., and N.J.O. contributed new reagents/analytic tools; M.E.C., D.E.L., I.P.S., K.S.H., M.A.M., S.C.L., N.M., C.J.S., and P.J.R. analyzed data; and M.E.C., C.J.S., and P.J.R. wrote the paper.

{dagger}{dagger}To whom correspondence should be addressed. E-mail: pjr{at}well.ox.ac.uk

© 2006 by The National Academy of Sciences of the USA

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