Abstract
BackgroundSCF 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.ResultsHere, 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.ConclusionWe 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.
Highlights
SCF ubiquitin ligases target numerous proteins for ubiquitin dependent proteolysis, including p27 and cyclin E
Depletion of CSN5 in HEK293 cells is not lethal To analyze the effect of loss of deneddylation in human cells, we utilized the doxycyline-inducible shRNA system developed by Clevers and colleagues [26] to conditionally down-regulate Csn5 protein levels
Eight days of doxycycline treatment of cells carrying the inducible Csn5-specific shRNA resulted in a drastic reduction in both Csn5 mRNA and protein levels when compared to induced cells expressing a scrambled shRNA (Figures 1A and 1B)
Summary
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 ubiquitinlike protein Nedd that covalently modifies the cullin subunit. Proteins are marked for degradation by the 26S proteasome via the covalent attachment of chains of the 76amino acid protein ubiquitin [reviewed in [1]]. One of the best-studied E3 ubiquitin ligase enzymes is the four subunit complex SCF [reviewed in [2]]. SCF consists of two activities: the first, contained within the Cul and RING domain Hrt1/Roc1/Rbx proteins, is the ability to recruit and activate the E2 to facilitate ubiquitin transfer from the E2 onto substrate; the second resides within the variable F-box proteins, which are linked to Cul via Skp and are thought to recruit substrates for ubiquitination by the Cul1/Hrt sub-complex. The large number of different F-box proteins gives SCF the opportunity to access a wide (page number not for citation purposes)
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