Targeted silencing of Jab1/Csn5 in human cells downregulates SCF activity through reduction of F-box protein levels

BMC Biochem. 2006 Jan 9;7:1. doi: 10.1186/1471-2091-7-1.

Abstract

Background: SCF ubiquitin ligases target numerous proteins for ubiquitin dependent proteolysis, including p27 and cyclin E. SCF and other cullin-RING ligases (CRLs) are regulated by the ubiquitin-like protein Nedd8 that covalently modifies the cullin subunit. The removal of Nedd8 is catalyzed by the Jab1/MPN domain metalloenzyme (JAMM) motif within the Csn5 subunit of the Cop9 Signalosome.

Results: Here, we conditionally knock down Csn5 expression in HEK293 human cells using a doxycycline-inducible shRNA system. Cullin levels were not altered in CSN-deficient human cells, but the levels of multiple F-box proteins were decreased. Molecular analysis indicates that this decrease was due to increased Cul1- and proteasome-dependent turnover. Diminished F-box levels resulted in reduced SCF activity, as evidenced by accumulation of two substrates of the F-box protein Fbw7, cyclin E and c-myc, in Csn5-depleted cells.

Conclusion: We propose that deneddylation of Cul1 is required to sustain optimal activity of SCF ubiquitin ligases by repressing 'autoubiquitination' of F-box proteins within SCF complexes, thereby rescuing them from premature degradation.

Publication types

  • Comparative Study

MeSH terms

  • COP9 Signalosome Complex
  • Cell Line
  • Down-Regulation / genetics*
  • F-Box Proteins / antagonists & inhibitors*
  • F-Box Proteins / genetics
  • F-Box Proteins / metabolism*
  • Gene Silencing / physiology*
  • Gene Targeting / methods*
  • Humans
  • Intracellular Signaling Peptides and Proteins / deficiency*
  • Intracellular Signaling Peptides and Proteins / genetics*
  • Intracellular Signaling Peptides and Proteins / physiology
  • Peptide Hydrolases / deficiency*
  • Peptide Hydrolases / genetics*
  • Peptide Hydrolases / physiology
  • SKP Cullin F-Box Protein Ligases / genetics
  • SKP Cullin F-Box Protein Ligases / metabolism*

Substances

  • F-Box Proteins
  • Intracellular Signaling Peptides and Proteins
  • SKP Cullin F-Box Protein Ligases
  • Peptide Hydrolases
  • COPS5 protein, human
  • COP9 Signalosome Complex