Ubiquitylation-dependent oligomerization regulates activity of Nedd4 ligases

Ilan Attali, William Sam Tobelaim, Avinash Persaud, Khatereh Motamedchaboki, Kobi J. Simpson-Lavy, Bayan Mashahreh, Olga Levin-Kravets, Tal Keren-Kaplan, Inbar Pilzer, Martin Kupiec, Reuven Wiener, Dieter A. Wolf, Daniela Rotin, Gali Prag*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

44 Scopus citations

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.

Original languageAmerican English
Pages (from-to)425-440
Number of pages16
JournalEMBO Journal
Volume36
Issue number4
DOIs
StatePublished - 15 Feb 2017

Bibliographical note

Funding Information:
We thank Simona Polo for the kind gift of the human Nedd4 plasmid. Robert Kass (Columbia University) and Bernard Attali (Tel Aviv University) are acknowledged, respectively, for the kind gift of the tandem construct KCNE1-KCNQ1 MK24 and the help in performing the TIRF and electrophysiology experiments. We thank Zvulun Elazar and Alik Demishtein (Weizmann Institute of Science) for kindly providing us with Nedd4 antibodies. We thank Peter Schuck (NIBIB-NIH) for help and constructive comments on AUC. We thank Moran Jerabek-Willemsen from NanoTemper Technologies for its guidance with the MST experiment. This research was supported by grants from the Israeli Science Foundation (grants numbers 1695/08 and 464/11), from the EC FP7 Marie Curie International Reintegration Grant (PIRG03-GA- 2008-231079), from the Israeli Ministry of Health (5108), and from the Marianna and Jorge Saia Fund for HIV and Parkinson Diseases to GP. The Constantiner Institute for Molecular Genetics and the Anat Krauskopf Travel Fund supported IA. Parts of this work were funded by grants from the US National Institutes of Health to DAW (GM105802-01A1 and P30 grants CA030199-32 and GM085764-03), and the Canadian Institute of Health Research to (MOP-130422) and Canadian Foundation for Innovation (CRC, Tier I) to DR, and grants from the Israel Science Foundation and the Israel Cancer Research Fund to MK.

Publisher Copyright:
© 2017 The Authors

Keywords

  • Nedd4
  • Rsp5
  • inactivation
  • oligomerization
  • ubiquitylation

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