The Cullin3 Ubiquitin Ligase Functions as a Nedd8-bound Heterodimer

Abstract

Cullins are members of a family of scaffold proteins that assemble multisubunit ubiquitin ligase complexes to confer substrate specificity for the ubiquitination pathway. Cullin3 (Cul3) forms a catalytically inactive BTB-Cul3-Rbx1 (BCR) ubiquitin ligase, which becomes functional upon covalent attachment of the ubiquitin homologue neural-precursor-cellexpressed and developmentally down regulated 8 (Nedd8) near the C terminus of Cul3. Current models suggest that Nedd8 activates cullin complexes by providing a recognition site for a ubiquitin-conjugating enzyme. Based on the following evidence, we propose that Nedd8 activates the BCR ubiquitin ligase by mediating the dimerization of Cul3. First, Cul3 is found as a neddylated heterodimer bound to a BTB domain-containing protein in vivo. Second, the formation of a Cul3 heterodimer is mediated by a Nedd8 molecule, which covalently attaches itself to one Cul3 molecule and binds to the winged-helix B domain at the C terminus of the second Cul3 molecule. Third, complementation experiments revealed that coexpression of two distinct nonfunctional Cul3 mutants can rescue the ubiquitin ligase function of the BCR complex. Likewise, a substrate of the BCR complex binds heterodimeric Cul3, suggesting that the Cul3 complex is active as a dimer. These findings not only provide insight into the architecture of the active BCR complex but also suggest assembly as a regulatory mechanism for activation of all cullin-based ubiquitin ligases. INTRODUCTIONThe conjugation of ubiquitin to a lysine residue of a target protein serves as a signaling mechanism in many biological processes, such as protein trafficking, DNA repair, protein-protein interactions, and proteolysis (Hershko and Ciechanover, 1998;Pines and Lindon, 2005). These ubiquitin linkages are formed by a series of enzymes designated as ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) (Ciechanover et al., 1980). A catalytically active ubiquitin ligase directly transfers ubiquitin molecules to the selected protein substrate, where a noncatalytic ubiquitin ligase shuttles the recruited substrate to a ubiquitin-conjugating enzyme (Glickman and Cienchanover, 2002). This latter group of E3 ligases includes cullin-based ubiquitin ligases, in which the cullin acts as a scaffold for the assembly of a multisubunit ubiquitin ligase complex that contains a RINGbox protein and a cullin-specific substrate adaptor protein (Ohta et al., 1999;Petroski and Deshaies, 2005). Cullin3 (Cul3) forms a complex called the BTB-Cul3-Rbx1 (BCR) ubiquitin ligase, which controls the degradation of several proteins, including cyclin E, the meiotic spindle-formation factor Mei-1, the transcription factor Nrf2, the Ci/Gli transcription factor, and the Dishevelled protein in the Wnt-␤-catenin pathway (Singer et al., 1999;Pintard et al., 2003b;Zhang et al., 2004Zhang et al., , 2006Angers et al., 2006). The BCR complex acquires its substrate specificity through a BTB domaincontaining protein bound at the N terminus ...

Hypertension-causing Mutations in Cullin3 Protein Impair RhoA Protein Ubiquitination and Augment the Association with Substrate Adaptors

“…Although the mechanism of dimer formation remains controversial, it is thought to occur through direct binding of substrate adaptors (16,32) or through the Nedd8 interface (35). To test whether Cul3⌬9 exists in heterodimers with Cul3WT, we first determined whether Cul3WT and Cul3⌬9 can form a complex.…”

Autophagy Suppresses Interleukin-1β (IL-1β) Signaling by Activation of p62 Degradation via Lysosomal and Proteasomal Pathways

“…S4A), we studied whether and how ATG16L1 regulates Cul-3 activity. Conjugation of Nedd8 (neddylation) to Cul-3 is critical for its dimerization and activity (11,12). To determine the neddylation of Cul-3, we overexpressed V5-Cul-3 and myc-Nedd8, and analyzed the neddylation by immunoblotting.…”

“…Cullin-3 (Cul-3) is a E3 ubiquitin ligase for various substrates and conjugation of Nedd8 to Cul-3 (neddylation) causes conformational changes in Cul-3 (10), which is critical for its dimerization and activation (11,12). Deletion of Cul-3 causes developmental defects in Drosophila, such as external sensory organ development, pattern formation and cell growth and survival (13).…”

PP2ACdc55regulates G1cyclin stability

“…57,58 Evidence points to SCF Fbw8 and SCF Fbx4-α/B-crystallin regulating cyclin D1 stability, 59,60 while cyclin E stability depends on SCF Fbw7 and the BCR (BTB-cul3-Rbx1) ubiquitin ligase complexes. 58,61 Cyclins D1 and E are highly stable in a number of cancers, a phenotype that is thought to aid in cell cycle proliferation and manifestation of the cancer phenotype. 1 In some cases, increased stability is associated with F-box gene mutations and deregulation of ubiquitin ligase activity.…”