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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • The 26S proteasome is vital for cellular protein homeostasis and implicated in numerous diseases.
  • Regulation of proteasome function is critical but not fully understood, particularly concerning posttranslational modifications like phosphorylation.
  • Many proteasome phosphorylation sites remain uncharacterized, hindering a complete understanding of proteasome control.

Purpose of the Study:

  • To investigate the functional significance of a highly prevalent proteasome phosphosite, Ser361 on the Rpn1 subunit of the 19S regulatory particle.
  • To identify kinases and phosphatases that regulate Rpn1-S361 phosphorylation.
  • To elucidate the mechanistic role of Rpn1-S361 phosphorylation in 26S proteasome assembly and function.

Main Methods:

  • CRISPR/Cas9-mediated gene editing to create phosphosite mutants.
  • Quantitative mass spectrometry to analyze phosphoproteome changes.
  • Human kinome screening to identify regulatory kinases.
  • Genetic code expansion system to study protein interactions during assembly.

Main Results:

  • Loss of Rpn1-S361 phosphorylation impairs 26S proteasome activity, reduces cell proliferation, and induces oxidative stress and mitochondrial dysfunction.
  • PIM1/2/3 kinases were identified as key enzymes catalyzing Rpn1-S361 phosphorylation.
  • UBLCP1 was identified as a phosphatase that reversibly controls Rpn1-S361 phosphorylation.
  • Rpn1-S361 phosphorylation is essential for 26S proteasome assembly, promoting Rpn1 interaction with Rpt2.

Conclusions:

  • Rpn1-S361 phosphorylation is a critical regulatory mechanism for 26S proteasome biogenesis and function.
  • This phosphosignaling pathway, involving PIM kinases and UBLCP1, influences cellular health by modulating proteasome activity.
  • The findings provide novel insights into the dynamic control of proteasome assembly and its implications in health and disease.