title: Substrate-Trapped Interactors of PHD3 and FIH Cluster in Distinct Signaling Pathways.
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.
authors.affiliation: Systems Biology Ireland, University College Dublin, Dublin 4, Ireland; Edinburgh Cancer Research Centre, IGMM, University of Edinburgh, Edinburgh EH4 2XR, UK.
authors.forename: Javier
authors.initials: J
authors.lastname: Rodriguez
authors.affiliation: Systems Biology Ireland, University College Dublin, Dublin 4, Ireland.
authors.forename: Ruth
authors.initials: R
authors.lastname: Pilkington
authors.affiliation: Systems Biology Ireland, University College Dublin, Dublin 4, Ireland.
authors.forename: Amaya
authors.initials: A
authors.lastname: Garcia Munoz
authors.affiliation: Systems Biology Ireland, University College Dublin, Dublin 4, Ireland.
authors.forename: Lan K
authors.initials: LK
authors.lastname: Nguyen
authors.affiliation: Systems Biology Ireland, University College Dublin, Dublin 4, Ireland.
authors.forename: Nora
authors.initials: N
authors.lastname: Rauch
authors.affiliation: Systems Biology Ireland, University College Dublin, Dublin 4, Ireland.
authors.forename: Susan
authors.initials: S
authors.lastname: Kennedy
authors.affiliation: Systems Biology Ireland, University College Dublin, Dublin 4, Ireland.
authors.forename: Naser
authors.initials: N
authors.lastname: Monsefi
authors.affiliation: Systems Biology Ireland, University College Dublin, Dublin 4, Ireland.
authors.forename: Ana
authors.initials: A
authors.lastname: Herrero
authors.affiliation: Systems Biology Ireland, University College Dublin, Dublin 4, Ireland; Conway Institute, University College Dublin, Dublin 4, Ireland.
authors.forename: Cormac T
authors.initials: CT
authors.lastname: Taylor
authors.affiliation: Systems Biology Ireland, University College Dublin, Dublin 4, Ireland; Edinburgh Cancer Research Centre, IGMM, University of Edinburgh, Edinburgh EH4 2XR, UK. Electronic address: alex.vonkriegsheim@igmm.ed.ac.uk.
authors.forename: Alex
authors.initials: A
authors.lastname: von Kriegsheim
mesh_terms: D000595:Amino Acid Sequence; D000600:Amino Acids, Dicarboxylic / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D057809:HEK293 Cells; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000737:chemistry / Q000378:metabolism; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D047468:Immunoprecipitation; D048053:Mitogen-Activated Protein Kinase 6 / Q000037:antagonists & inhibitors / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000032:analysis / Q000737:chemistry; D060066:Protein Interaction Maps; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D054875:Ubiquitination
chemical_list: D000600:Amino Acids, Dicarboxylic; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D010455:Peptides; D034741:RNA, Small Interfering; D012097:Repressor Proteins; D006899:Mixed Function Oxygenases; C439790:HIF1AN protein, human; C488894:EGLN3 protein, human; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; C417187:RIPK4 protein, human; D017346:Protein Serine-Threonine Kinases; D048053:Mitogen-Activated Protein Kinase 6; C040947:oxalylglycine