Abstract
Cullin-based E3 ligases are a large family of multi-subunit ubiquitin ligases with diverse cellular functions, including the regulation of the cell cycle, of the DNA damage response, and of various transcription factors. These ligases are composed of one of six mammalian cullin homologs (Cul1, Cul2, Cul3, Cul4a, Cul4b, and Cul5), the Ring finger containing protein Roc1/Rbx1, and cullin homolog-specific adaptor and substrate recognition subunits. To be active, cullin-based ligases require the covalent modification of a conserved lysine residue in the cullin protein with the ubiquitin-like protein Nedd8. We show in this study that in intact cells Cul1 neddylation is dependent on binding to adaptor proteins and substrate recognition subunits. Mutant Cul1 that is unable to recruit adaptor and substrate recognition subunits exhibits markedly reduced neddylation, and inhibiting binding of adaptor and substrate recognition subunits to wild type Cul1 reduces Nedd8 modification. This regulatory mechanism also extends to other cullin-based E3 ligases, including Cul2, Cul3, and Cul4a. The regulation of cullin neddylation by adaptor proteins and substrate recognition subunits in cells was found to be independent of both CAND1 and the COP9 signalosome, two negative regulators of cullin Nedd8 modification. Using hypoxia-inducible factor-1alpha (HIF-1alpha), a substrate of the Elongin B/C-Cul2-VHL ligase, we demonstrate the critical role of substrate binding to promote Cul2 neddylation in a manner that does not require substrate ubiquitination but may involve a conformational change. These findings suggest a mechanism through which availability of substrate recognition subunits and substrates can regulate the ubiquitin ligase activity.
Highlights
Substrate through a protein-protein interaction domain and the E2 enzyme through the RING motif, bringing substrate and E2 enzyme into close proximity and facilitating the transfer of ubiquitin molecules to lysine residues in the substrate
Using hypoxiainducible factor-1␣ (HIF-1␣), a substrate of the Elongin B/C-Cul2-VHL (ECV) E3 ligase, we demonstrate the critical role of substrate binding to promote Cul2 neddylation and provide evidence that this mechanism does not require substrate ubiquitination but is likely to involve a conformational change in the E3 ligase upon binding of the substrate
Mutant Cullin Proteins That Are Unable to Bind to Adaptor and Substrate Recognition Subunits Exhibit Markedly Reduced Nedd8 Modification—To characterize the role of substrate recognition subunits and substrates in regulating cullin neddylation, we initially generated Cul1 and Cul3 mutants that based on previous reports are unable to bind to Skp1 and F-box proteins or BTB domain proteins, respectively
Summary
Substrate through a protein-protein interaction domain and the E2 enzyme through the RING motif, bringing substrate and E2 enzyme into close proximity and facilitating the transfer of ubiquitin molecules to lysine residues in the substrate. We extend these studies and show that disrupting the interaction between cullin proteins and their adaptor and substrate recognition subunits in cells reduces neddylation This dependence on complete E3 ligase assembly for efficient cullin neddylation is a general mechanism that is observed in various cullin-based E3 ligases and appears to be independent of both CAND1 and CSN. Using hypoxiainducible factor-1␣ (HIF-1␣), a substrate of the ECV E3 ligase, we demonstrate the critical role of substrate binding to promote Cul neddylation and provide evidence that this mechanism does not require substrate ubiquitination but is likely to involve a conformational change in the E3 ligase upon binding of the substrate This is likely to constitute an important regulatory mechanism of cullin-based E3 ligase activity
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
