Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis

Tobias Wauer, Kirby N. Swatek, Jane L. Wagstaff, Christina Gladkova, Jonathan N. Pruneda, Martin A. Michel, Malte Gersch, Christopher M. Johnson, Stefan M.V. Freund, David Komander

Research output: Contribution to journalArticle

141 Scopus citations

Abstract

The protein kinase PINK1 was recently shown to phosphorylate ubiquitin (Ub) on Ser65, and phosphoUb activates the E3 ligase Parkin allosterically. Here, we show that PINK1 can phosphorylate every Ub in Ub chains. Moreover, Ser65 phosphorylation alters Ub structure, generating two conformations in solution. A crystal structure of the major conformation resembles Ub but has altered surface properties. NMR reveals a second phosphoUb conformation in which β5-strand slippage retracts the C-terminal tail by two residues into the Ub core. We further show that phosphoUb has no effect on E1-mediated E2 charging but can affect discharging of E2 enzymes to form polyUb chains. Notably, UBE2R1- (CDC34), UBE2N/UBE2V1- (UBC13/UEV1A), TRAF6- and HOIP-mediated chain assembly is inhibited by phosphoUb. While Lys63-linked poly-phosphoUb is recognized by the TAB2 NZF Ub binding domain (UBD), 10 out of 12 deubiquitinases (DUBs), including USP8, USP15 and USP30, are impaired in hydrolyzing phosphoUb chains. Hence, Ub phosphorylation has repercussions for ubiquitination and deubiquitination cascades beyond Parkin activation and may provide an independent layer of regulation in the Ub system.

Original languageEnglish (US)
Pages (from-to)307-325
Number of pages19
JournalEMBO Journal
Volume34
Issue number3
DOIs
StatePublished - Feb 3 2015

Keywords

  • PINK1
  • Parkin
  • deubiquitinase
  • phosphorylation
  • ubiquitin

ASJC Scopus subject areas

  • Neuroscience(all)
  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

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    Wauer, T., Swatek, K. N., Wagstaff, J. L., Gladkova, C., Pruneda, J. N., Michel, M. A., Gersch, M., Johnson, C. M., Freund, S. M. V., & Komander, D. (2015). Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis. EMBO Journal, 34(3), 307-325. https://doi.org/10.15252/embj.201489847