Substrate-Trapped Interactors of PHD3 and FIH Cluster in Distinct Signaling Pathways

Javier Rodriguez; Ruth Pilkington; Amaya Garcia Munoz; Lan K Nguyen; Nora Rauch; Susan Kennedy; Naser Monsefi; Ana Herrero; Cormac T Taylor; Alex von Kriegsheim

doi:10.1016/j.celrep.2016.02.043

Substrate-Trapped Interactors of PHD3 and FIH Cluster in Distinct Signaling Pathways

Cell Rep. 2016 Mar 22;14(11):2745-60. doi: 10.1016/j.celrep.2016.02.043. Epub 2016 Mar 10.

Authors

Affiliations

¹ Systems Biology Ireland, University College Dublin, Dublin 4, Ireland; Edinburgh Cancer Research Centre, IGMM, University of Edinburgh, Edinburgh EH4 2XR, UK.
² Systems Biology Ireland, University College Dublin, Dublin 4, Ireland.
³ Systems Biology Ireland, University College Dublin, Dublin 4, Ireland; Conway Institute, University College Dublin, Dublin 4, Ireland.
⁴ 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.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Amino Acids, Dicarboxylic / chemistry
Chromatography, High Pressure Liquid
HEK293 Cells
Humans
Hypoxia-Inducible Factor 1, alpha Subunit / chemistry
Hypoxia-Inducible Factor 1, alpha Subunit / metabolism
Hypoxia-Inducible Factor-Proline Dioxygenases / antagonists & inhibitors
Hypoxia-Inducible Factor-Proline Dioxygenases / genetics
Hypoxia-Inducible Factor-Proline Dioxygenases / metabolism*
Immunoblotting
Immunoprecipitation
Mitogen-Activated Protein Kinase 6 / antagonists & inhibitors
Mitogen-Activated Protein Kinase 6 / metabolism
Mixed Function Oxygenases / chemistry
Mixed Function Oxygenases / genetics
Mixed Function Oxygenases / metabolism*
Peptides / analysis
Peptides / chemistry
Protein Interaction Maps
Protein Serine-Threonine Kinases / chemistry
Protein Serine-Threonine Kinases / metabolism
RNA Interference
RNA, Small Interfering / metabolism
Repressor Proteins / chemistry
Repressor Proteins / genetics
Repressor Proteins / metabolism*
Signal Transduction
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Substrate Specificity
Ubiquitination

Substances

Amino Acids, Dicarboxylic
Hypoxia-Inducible Factor 1, alpha Subunit
Peptides
RNA, Small Interfering
Repressor Proteins
Mixed Function Oxygenases
HIF1AN protein, human
EGLN3 protein, human
Hypoxia-Inducible Factor-Proline Dioxygenases
RIPK4 protein, human
Protein Serine-Threonine Kinases
Mitogen-Activated Protein Kinase 6
oxalylglycine

Grants and funding

C157/A18075/Cancer Research UK/United Kingdom