Abstract
Amino acid hydroxylation is a post-translational modification that regulates intra- and inter-molecular protein-protein interactions. The modifications are regulated by a family of 2-oxoglutarate- (2OG) dependent enzymes and, although the biochemistry is well understood, until now only a few substrates have been described for these enzymes. Using quantitative interaction proteomics, we screened for substrates of the proline hydroxylase PHD3 and the asparagine hydroxylase FIH, which regulate the HIF-mediated hypoxic response. We were able to identify hundreds of potential substrates. Enrichment analysis revealed that the potential substrates of both hydroxylases cluster in the same pathways but frequently modify different nodes of signaling networks. We confirm that two proteins identified in our screen, MAPK6 (Erk3) and RIPK4, are indeed hydroxylated in a FIH- or PHD3-dependent mechanism. We further determined that FIH-dependent hydroxylation regulates RIPK4-dependent Wnt signaling, and that PHD3-dependent hydroxylation of MAPK6 protects the protein from proteasomal degradation.
Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Sequence
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Amino Acids, Dicarboxylic / chemistry
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Chromatography, High Pressure Liquid
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HEK293 Cells
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Humans
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Hypoxia-Inducible Factor 1, alpha Subunit / chemistry
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Hypoxia-Inducible Factor 1, alpha Subunit / metabolism
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Hypoxia-Inducible Factor-Proline Dioxygenases / antagonists & inhibitors
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Hypoxia-Inducible Factor-Proline Dioxygenases / genetics
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Hypoxia-Inducible Factor-Proline Dioxygenases / metabolism*
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Immunoblotting
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Immunoprecipitation
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Mitogen-Activated Protein Kinase 6 / antagonists & inhibitors
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Mitogen-Activated Protein Kinase 6 / metabolism
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Mixed Function Oxygenases / chemistry
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Mixed Function Oxygenases / genetics
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Mixed Function Oxygenases / metabolism*
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Peptides / analysis
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Peptides / chemistry
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Protein Interaction Maps
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Protein Serine-Threonine Kinases / chemistry
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Protein Serine-Threonine Kinases / metabolism
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RNA Interference
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RNA, Small Interfering / metabolism
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Repressor Proteins / chemistry
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Repressor Proteins / genetics
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Repressor Proteins / metabolism*
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Signal Transduction
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Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
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Substrate Specificity
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Ubiquitination
Substances
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Amino Acids, Dicarboxylic
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Hypoxia-Inducible Factor 1, alpha Subunit
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Peptides
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RNA, Small Interfering
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Repressor Proteins
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Mixed Function Oxygenases
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HIF1AN protein, human
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EGLN3 protein, human
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Hypoxia-Inducible Factor-Proline Dioxygenases
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RIPK4 protein, human
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Protein Serine-Threonine Kinases
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Mitogen-Activated Protein Kinase 6
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oxalylglycine