HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Free via Author's Choice: AC AC Full Text Full Text (PDF) Supplemental Data AC All Versions of this Article: M800340-MCP200v1 8/3/535    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Glossary Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Cockman, M. E. Articles by Ratcliffe, P. J. Search for Related Content PubMed PubMed Citation Articles by Cockman, M. E. Articles by Ratcliffe, P. J. Social Bookmarking                      What's this?

Molecular & Cellular Proteomics 8:535-546, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

---

Research

Proteomics-based Identification of Novel Factor Inhibiting Hypoxia-inducible Factor (FIH) Substrates Indicates Widespread Asparaginyl Hydroxylation of Ankyrin Repeat Domain-containing Proteins^*,S

Matthew E. Cockman, James D. Webb, Holger B. Kramer, Benedikt M. Kessler^,¶,|| and Peter J. Ratcliffe^,¶

From the Central Proteomics Facility, Henry Wellcome Building for Molecular Physiology, Nuffield Department of Clinical Medicine, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, United Kingdom

Post-translational hydroxylation has been considered an unusual modification on intracellular proteins. However, following the recognition that oxygen-sensitive prolyl and asparaginyl hydroxylation are central to the regulation of the transcription factor hypoxia-inducible factor (HIF), interest has centered on the possibility that these enzymes may have other substrates in the proteome. In support of this certain ankyrin repeat domain (ARD)-containing proteins, including members of the IB and Notch families, have been identified as alternative substrates of the HIF asparaginyl hydroxylase factor inhibiting HIF (FIH). Although these findings imply a potentially broad range of substrates for FIH, the precise extent of this range has been difficult to determine because of the difficulty of capturing transient enzyme-substrate interactions. Here we describe the use of pharmacological "substrate trapping" together with stable isotope labeling by amino acids in cell culture (SILAC) technology to stabilize and identify potential FIH-substrate interactions by mass spectrometry. To pursue these potential FIH substrates we used conventional data-directed tandem MS together with alternating low/high collision energy tandem MS to assign and quantitate hydroxylation at target asparaginyl residues. Overall the work has defined 13 new FIH-dependent hydroxylation sites with a degenerate consensus corresponding to that of the ankyrin repeat and a range of ARD-containing proteins as actual and potential substrates for FIH. Several ARD-containing proteins were multiply hydroxylated, and detailed studies of one, Tankyrase-2, revealed eight sites that were differentially sensitive to FIH-catalyzed hydroxylation. These findings indicate that asparaginyl hydroxylation is likely to be widespread among the 300 ARD-containing species in the human proteome.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				C. Loenarz, W. Ge, M. L. Coleman, N. R. Rose, C. D.O. Cooper, R. J. Klose, P. J. Ratcliffe, and C. J. Schofield PHF8, a gene associated with cleft lip/palate and mental retardation, encodes for an N{varepsilon}-dimethyl lysine demethylase Hum. Mol. Genet., January 15, 2010; 19(2): 217 - 222. [Abstract] [Full Text] [PDF]