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


CONCLUSIONS
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.
Cope
Gregory A
GA
Department of Biology, California Institute of Technology Pasadena, CA 91125, USA. cope@caltech.edu
Deshaies
Raymond J
RJ
D000075686:COP9 Signalosome Complex; D002460:Cell Line; D015536:Down-Regulation / Q000235:genetics*; D044783:F-Box Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism*; D020868:Gene Silencing / Q000502:physiology*; D018390:Gene Targeting / Q000379:methods*; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000172:deficiency* / Q000235:genetics* / Q000502:physiology; D010447:Peptide Hydrolases / Q000172:deficiency* / Q000235:genetics* / Q000502:physiology; D044843:SKP Cullin F-Box Protein Ligases / Q000235:genetics / Q000378:metabolism*
D044783:F-Box Proteins; D047908:Intracellular Signaling Peptides and Proteins; D044843:SKP Cullin F-Box Protein Ligases; D010447:Peptide Hydrolases; C501488:COPS5 protein, human; D000075686:COP9 Signalosome Complex