Targeted silencing of Jab1/Csn5 in human cells downregulates SCF activity through reduction of F-box protein levels
Gregory A Cope1,3 and Raymond J Deshaies1,2
1Department of Biology, California Institute of Technology Pasadena, CA 91125, USA
2Howard Hughes Medical Institute, 4000 Jones Bridge Road, Chevy Chase, MD, 20815-6789, USA
3Department of Biology, Stanford University, Palo Alto, CA 94305, USA
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.
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.
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.
BMC Biochemistry 2006, 7:1. This is an Open Access article distributed under the terms of the Creative Commons Attribution License.