Ubiquitin Ser65 phosphorylation affects ubiquitin structure, chain assembly and hydrolysis

EMBO J. 2015 Feb 3;34(3):307-25. doi: 10.15252/embj.201489847. Epub 2014 Dec 19.

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.

Keywords: PINK1; Parkin; deubiquitinase; phosphorylation; ubiquitin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism
  • Allosteric Regulation / physiology
  • Endopeptidases / genetics
  • Endopeptidases / metabolism
  • Endosomal Sorting Complexes Required for Transport / genetics
  • Endosomal Sorting Complexes Required for Transport / metabolism
  • Humans
  • Hydrolysis
  • Mitochondrial Proteins / genetics
  • Mitochondrial Proteins / metabolism
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Phosphorylation / physiology
  • Polyubiquitin / genetics
  • Polyubiquitin / metabolism*
  • Protein Multimerization / physiology*
  • Protein Structure, Tertiary
  • Serine / genetics
  • Serine / metabolism
  • TNF Receptor-Associated Factor 6 / genetics
  • TNF Receptor-Associated Factor 6 / metabolism
  • Thiolester Hydrolases / genetics
  • Thiolester Hydrolases / metabolism
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • Ubiquitin Thiolesterase / genetics
  • Ubiquitin Thiolesterase / metabolism
  • Ubiquitin-Conjugating Enzymes / genetics
  • Ubiquitin-Conjugating Enzymes / metabolism
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitin-Specific Proteases / genetics
  • Ubiquitin-Specific Proteases / metabolism
  • Ubiquitination / physiology*

Substances

  • Adaptor Proteins, Signal Transducing
  • Endosomal Sorting Complexes Required for Transport
  • Mitochondrial Proteins
  • Phosphoproteins
  • TAB2 protein, human
  • TNF Receptor-Associated Factor 6
  • Transcription Factors
  • Usp30 protein, human
  • Polyubiquitin
  • Serine
  • CDC34 protein, human
  • UBE2N protein, human
  • UBE2V1 protein, human
  • Ubiquitin-Conjugating Enzymes
  • RNF31 protein, human
  • Ubiquitin-Protein Ligases
  • parkin protein
  • Thiolester Hydrolases
  • Endopeptidases
  • USP15 protein, human
  • USP8 protein, human
  • Ubiquitin Thiolesterase
  • Ubiquitin-Specific Proteases

Associated data

  • PDB/4WZP