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Nat Commun. 2017 Dec 20;8(1):2232. doi: 10.1038/s41467-017-01974-7.

Identifying the ubiquitination targets of E6AP by orthogonal ubiquitin transfer.

Author information

1
Department of Chemistry, Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, 30303, USA.
2
Department of Pharmacology, Northwestern University, Chicago, IL, 60611, USA.
3
Integrated Proteomics Core, Emory University, Atlanta, GA, 30322, USA.
4
Department of Chemistry, University of Chicago, Chicago, IL, 60637, USA.
5
Engineering Research Center of Cell & Therapeutic Antibody, Ministry of Education, and School of Pharmacy, Shanghai Jiao Tong University, Shanghai, 200240, China.
6
Department of Preventive Medicine, Northwestern University, Chicago, IL, 60611, USA.
7
Department of Pharmacology, Northwestern University, Chicago, IL, 60611, USA. kiyokawa@northwestern.edu.
8
Department of Chemistry, Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, 30303, USA. junyin@gsu.edu.

Abstract

E3 ubiquitin (UB) ligases are the ending modules of the E1-E2-E3 cascades that transfer UB to cellular proteins and regulate their biological functions. Identifying the substrates of an E3 holds the key to elucidate its role in cell regulation. Here, we construct an orthogonal UB transfer (OUT) cascade to identify the substrates of E6AP, a HECT E3 also known as Ube3a that is implicated in cancer and neurodevelopmental disorders. We use yeast cell surface display to engineer E6AP to exclusively transfer an affinity-tagged UB variant (xUB) to its substrate proteins. Proteomic identification of xUB-conjugated proteins in HEK293 cells affords 130 potential E6AP targets. Among them, we verify that MAPK1, CDK1, CDK4, PRMT5, β-catenin, and UbxD8 are directly ubiquitinated by E6AP in vitro and in the cell. Our work establishes OUT as an efficient platform to profile E3 substrates and reveal the cellular circuits mediated by the E3 enzymes.

PMID:
29263404
PMCID:
PMC5738348
DOI:
10.1038/s41467-017-01974-7
[Indexed for MEDLINE]
Free PMC Article

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