Label-free Interactome Analysis Revealed an Essential Role of CUL3-KEAP1 Complex in Mediating the Ubiquitination and Degradation of PHD2.

Abstract

Prolyl Hydroxylase Domain-Containing Protein 2 (PHD2/EGLN1) is a key regulatory enzyme that plays a fundamental role in the cellular hypoxia response pathway, mediating proline hydroxylation-dependent protein degradation of selected target proteins. However, the regulation of PHD2 homeostasis at the protein level is not well understood. Here, we performed label-free quantitative interactome analysis through immunoprecipitation coupled with mass spectrometry analysis. We identified and validated Cullin 3 as a novel PHD2 interactor in vivo. Through candidate screening, we further identified CUL3-KEAP1 E3 ubiquitin ligase complex as the major enzyme that regulates PHD2 degradation. Overexpression of either CUL3, KEAP1 or both significantly increases PHD2 ubiquitination and reduces PHD2 protein abundance. Knockdown of CUL3 or KEAP1 decreased PHD2 ubiquitination and inhibited PHD2 degradation. Accordingly, loss of the CUL3-KEAP1 complex under hypoxia promoted PHD2 stabilization and led to significantly reduced abundance of the PHD2 target, hypoxia-inducible factor 1A (HIF1A). Thus, CUL3-KEAP1 is an essential pathway that regulates PHD2 ubiquitination and degradation in cells.