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EMBO J. 2017 Feb 15;36(4):425-440. doi: 10.15252/embj.201694314. Epub 2017 Jan 9.

Ubiquitylation-dependent oligomerization regulates activity of Nedd4 ligases.

Author information

1
Department of Biochemistry and Molecular Biology, the George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
2
Department of Physiology & Pharmacology, Sackler Tel Aviv University, Tel Aviv, Israel.
3
Cell Biology Program, The Hospital for Sick Children and Biochemistry Department, University of Toronto, Toronto, ON, Canada.
4
Tumor Initiation & Maintenance Program and NCI Cancer Centre Proteomics Facility, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA.
5
Department of Molecular Microbiology and Biotechnology, the George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
6
Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel.
7
School of Pharmaceutical Sciences, Xiamen University, Xiamen, China.
8
Department of Biochemistry and Molecular Biology, the George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel prag@post.tau.ac.il.
9
Sagol School of Neuroscience, Tel Aviv University, Tel Aviv, Israel.

Abstract

Ubiquitylation controls protein function and degradation. Therefore, ubiquitin ligases need to be tightly controlled. We discovered an evolutionarily conserved allosteric restraint mechanism for Nedd4 ligases and demonstrated its function with diverse substrates: the yeast soluble proteins Rpn10 and Rvs167, and the human receptor tyrosine kinase FGFR1 and cardiac IKS potassium channel. We found that a potential trimerization interface is structurally blocked by the HECT domain α1-helix, which further undergoes ubiquitylation on a conserved lysine residue. Genetic, bioinformatics, biochemical and biophysical data show that attraction between this α1-conjugated ubiquitin and the HECT ubiquitin-binding patch pulls the α1-helix out of the interface, thereby promoting trimerization. Strikingly, trimerization renders the ligase inactive. Arginine substitution of the ubiquitylated lysine impairs this inactivation mechanism and results in unrestrained FGFR1 ubiquitylation in cells. Similarly, electrophysiological data and TIRF microscopy show that NEDD4 unrestrained mutant constitutively downregulates the IKS channel, thus confirming the functional importance of E3-ligase autoinhibition.

KEYWORDS:

Nedd4; Rsp5; inactivation; oligomerization; ubiquitylation

Comment in

PMID:
28069708
PMCID:
PMC5437815
DOI:
10.15252/embj.201694314
[Indexed for MEDLINE]
Free PMC Article

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