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

Abstract

SummaryAmino 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.

New Insights into Protein Hydroxylation and Its Important Role in Human Diseases

“…Therefore, mass spectrometry fragmentation data need to be at high resolution and high coverage to confirm the assignment for the modification as previously shown [80]. Many hydroxylation events detected by mass spectrometry may not be mediated by prolyl hydroxylases.…”

“…[80] were able to identify some novel FIH and PHD substrates. As a proof of principle, they identified some of characterized PHD substrates from this proteomic approach, including CEP192 and FOXO3a [23, 80, 87].…”

“…[80] were able to identify some novel FIH and PHD substrates. As a proof of principle, they identified some of characterized PHD substrates from this proteomic approach, including CEP192 and FOXO3a [23, 80, 87]. Among potential PHD3/EglN3 binding partners/substrates, mitogen-activated protein kinase 6 (MAPK6) has been shown to be hydroxylated by PHD3/EglN3 on proline 25 (Pro25), which potentially leads to its dissociation from HECT, UBA and WWE domain containing 1, E3 ubiquitin protein ligase (HUWE1), thus protecting it from proteasomal degradation (Fig.…”

Hypoxia-dependent regulation of inflammatory pathways in immune cells

“…Another study identified FIH as a second, oxygen-dependent hydroxylase that targets HIF (77,78). Subsequent studies demonstrated that while HIF is clearly the major pathway regulated by hydroxylation, signaling components of other pathways such as NF-κB are also subject to enzymatic hydroxylation, although the functional consequences of hydroxylation for non-HIF targets remains an area under investigation (62)(63)(64)79).…”

Plant Genomics

“…Upon reconstitution, the EGFP has been observed to evidence a restored fluorescence upon protein-protein interactions in yeast, allowing for the characterization of its interactor. With standard Y2H assays, detection of an interaction requires that two proteins localize to the nucleus [32,33]. In 2007, a yeast-based genetic technology for the in vivo detection of membrane protein interactors was developed and is referred to as the split-ubiquitin membrane yeast two-hybrid system.…”