Reversible phosphorylation of Rpn1 regulates 26S proteasome assembly and function

Proc Natl Acad Sci U S A. 2020 Jan 7;117(1):328-336. doi: 10.1073/pnas.1912531117. Epub 2019 Dec 16.

Abstract

The fundamental importance of the 26S proteasome in health and disease suggests that its function must be finely controlled, and yet our knowledge about proteasome regulation remains limited. Posttranslational modifications, especially phosphorylation, of proteasome subunits have been shown to impact proteasome function through different mechanisms, although the vast majority of proteasome phosphorylation events have not been studied. Here, we have characterized 1 of the most frequently detected proteasome phosphosites, namely Ser361 of Rpn1, a base subunit of the 19S regulatory particle. Using a variety of approaches including CRISPR/Cas9-mediated gene editing and quantitative mass spectrometry, we found that loss of Rpn1-S361 phosphorylation reduces proteasome activity, impairs cell proliferation, and causes oxidative stress as well as mitochondrial dysfunction. A screen of the human kinome identified several kinases including PIM1/2/3 that catalyze S361 phosphorylation, while its level is reversibly controlled by the proteasome-resident phosphatase, UBLCP1. Mechanistically, Rpn1-S361 phosphorylation is required for proper assembly of the 26S proteasome, and we have utilized a genetic code expansion system to directly demonstrate that S361-phosphorylated Rpn1 more readily forms a precursor complex with Rpt2, 1 of the first steps of 19S base assembly. These findings have revealed a prevalent and biologically important mechanism governing proteasome formation and function.

Keywords: PIM; UBLCP1; genetic code expansion; phosphorylation; proteasome.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics
  • Cell Line
  • Enzyme Assays
  • Gene Knock-In Techniques
  • Humans
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Mice
  • Mice, Knockout
  • Mice, Transgenic
  • Mitochondria / metabolism
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Oxidative Stress
  • Phosphoprotein Phosphatases / genetics
  • Phosphoprotein Phosphatases / metabolism*
  • Phosphorylation / physiology
  • Proteasome Endopeptidase Complex / genetics
  • Proteasome Endopeptidase Complex / metabolism*
  • Protein Serine-Threonine Kinases / genetics
  • Protein Serine-Threonine Kinases / metabolism*
  • Protein Subunits / genetics
  • Protein Subunits / metabolism*
  • RNA, Small Interfering / metabolism
  • Serine / metabolism
  • Trans-Activators / genetics
  • Trans-Activators / metabolism

Substances

  • Membrane Proteins
  • Nuclear Proteins
  • PSMD14 protein, mouse
  • Protein Subunits
  • RNA, Small Interfering
  • RPN1 protein, human
  • Trans-Activators
  • ribophorin
  • Serine
  • Protein Serine-Threonine Kinases
  • Phosphoprotein Phosphatases
  • UBLCP1 protein, human
  • Proteasome Endopeptidase Complex
  • ATP dependent 26S protease