Abstract
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.
Original language | English (US) |
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Pages (from-to) | 260-268 |
Number of pages | 9 |
Journal | Journal of Proteome Research |
Volume | 19 |
Issue number | 1 |
DOIs | |
State | Published - Jan 3 2020 |
Bibliographical note
Funding Information:We would like to thank the members of the Chen lab for helpful suggestions and discussion. We are grateful to the University of Minnesota’s Center for Mass Spectrometry and Proteomics and the Masonic Cancer Center for LC–MS instrument access and support. We would also like to thank Do-Hyung Kim for providing access to the hypoxia chamber and helping us with immunofluorescence microscopy. This work was supported by the National Institute of Health (R35GM124896 to Y.C.).
Publisher Copyright:
Copyright © 2019 American Chemical Society.
Keywords
- Cul3
- KEAP1
- LCMS
- PHD2/EGLN1
- degradation
- hypoxia
- ubiquitination