Identifying the ubiquitination targets of E6AP by orthogonal ubiquitin transfer

Yiyang Wang, Xianpeng Liu, Li Zhou, Duc Duong, Karan Bhuripanyo, Bo Zhao, Han Zhou, Ruochuan Liu, Yingtao Bi, Hiroaki Kiyokawa () and Jun Yin ()
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Yiyang Wang: Center for Diagnostics & Therapeutics, Georgia State University
Xianpeng Liu: Northwestern University
Li Zhou: Center for Diagnostics & Therapeutics, Georgia State University
Duc Duong: Emory University
Karan Bhuripanyo: Center for Diagnostics & Therapeutics, Georgia State University
Bo Zhao: Shanghai Jiao Tong University
Han Zhou: Center for Diagnostics & Therapeutics, Georgia State University
Ruochuan Liu: Center for Diagnostics & Therapeutics, Georgia State University
Yingtao Bi: Northwestern University
Hiroaki Kiyokawa: Northwestern University
Jun Yin: Center for Diagnostics & Therapeutics, Georgia State University

Nature Communications, 2017, vol. 8, issue 1, 1-14

Abstract: 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.

Date: 2017
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DOI: 10.1038/s41467-017-01974-7

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