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Published January 9, 2006 | Published
Journal Article Open

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.

Additional Information

© 2006 Cope and Deshaies, licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Submission date 23 Sep 2005; Acceptance date 9 Jan 2006; Publication date 9 Jan 2006 We thank W. Krek, W. Boelens, B. Clurman and S. Reed for generously providing antibodies and advice. We thank Nagesh Rao for performing FISH and kareotype analysis. We thank Rati Verma, Matt Petroski, Thibault Mayor and other members of the Deshaies lab for providing helpful insight and discussions during the course of this work. Authors Contributions: All experiments were performed by GC. Both GC and RJD conceived and designed the experiments. Both authors contributed to the writing of the manuscript and both authors read and approved the final manuscript.

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