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

Prolyl hydroxylase domain-containing protein 2 (PHD2/EGLN1)
is
a key regulatory enzyme that plays a fundamental role in the cellular
hypoxic 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 perform label-free quantitative interactome analysis through immunoprecipitation
coupled with mass spectrometry analysis. To minimize the side effects
caused by ectopic overexpression, in HeLa cells, we stably overexpressed
Flag-tagged PHD2 while suppressing the endogenous PHD2 by using an
shRNA targeting its 3′ UTR region. 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. The 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.