DCNL1 Functions as a Substrate Sensor and Activator of Cullin 2-RING Ligase

Abstract

Substrate engagement by F-box proteins promotes NEDD8 modification of cullins, which is necessary for the activation of cullin-RING E3 ubiquitin ligases (CRLs). However, the mechanism by which substrate recruitment triggers cullin neddylation remains unclear. Here, we identify DCNL1 (defective in cullin neddylation 1-like 1) as a component of CRL2 called ECV (elongins BC/CUL2/VHL) and show that molecular suppression of DCNL1 attenuates CUL2 neddylation. DCNL1 via its DAD patch binds to CUL2 but is also able to bind VHL independent of CUL2 and the DAD patch. The engagement of the substrate hypoxia-inducible factor 1␣ (HIF1␣) to the substrate receptor VHL increases DCNL1 binding to VHL as well as to CUL2. Notably, an engineered mutant form of HIF1␣ that associates with CUL2, but not DCNL1, fails to trigger CUL2 neddylation and retains ECV in an inactive state. These findings support a model in which substrate engagement prompts DCNL1 recruitment that facilitates the initiation of CUL2 neddylation and define DCNL1 as a "substrate sensor switch" for ECV activation. Cullin-RING ligases (CRLs), the largest family of E3 ubiquitin ligases, are multiprotein complexes involved in protein degradation (1, 2). They are assembled on a cullin scaffold (CUL1, CUL2, CUL3, CUL4A, CUL4B, and CUL5) and contain a RING finger protein (RBX1 or RBX2) at the cullin C-terminal domain (CTD) as well as a substrate receptor, which generally associates with the cullin N-terminal domain (NTD) via adaptor proteins (2). CUL3-based CRLs are the exception in which the Broad complex, Tramtrack, and Bric-a-Brac (BTB)-domain containing protein mediates both substrate specificity and binding to CUL3 without the need of adaptor proteins (3).The process of attaching ubiquitin onto a CRL substrate requires three enzymes. Using ATP, an E1 activating enzyme forms a thioester bond between its catalytic cysteine residue and the C-terminal carboxyl group of ubiquitin (4). The activated ubiquitin is then transferred onto the catalytic cysteine of an E2 conjugating enzyme (4). CRL through RBX1/2 helps transfer ubiquitin from E2 onto a lysine residue on the bound substrate (2). Neddylation, the process of conjugating the ubiquitin-like protein NEDD8 onto a substrate, follows an analogous cascade (5, 6). Cullins are modified by NEDD8 (7). Structural studies involving the CTD of CUL5 (CUL5 CTD )-RBX1 and NEDD8ϳCUL5 CTD -RBX1 have demonstrated that in an unneddylated state, RBX1 has limited movement due to inhibitory cullin subdomains, thus restraining its ability to recruit ubiquitin-charged E2 (8). Neddylation causes a conformational change in cullin structure that releases RBX1 from a confined configuration to enhance the binding of ubiquitin-charged E2 and thus CRL ubiquitylation capability (8, 9).The molecular events involved in the process of neddylation of cullins are not as straightforward as the resulting effects. For CUL1 neddylation to take place, rotation of the RING domain of RBX1 is needed in order to bring the catalytic Cys111 of UBC12 (NEDD8 E2 enz...

Squamous cell carcinoma–related oncogene (SCCRO) neddylates Cul3 protein to selectively promote midbody localization and activity of Cul3KLHL21 protein complex during abscission

“…This suggests that binding of the substrate adaptor to the SCCRO-cullin-ROC1 complex is required to promote neddylation. Heir et al reported that binding of substrate (HIF1␣) to the substrate receptor von Hippel-Lindau (VHL) promotes binding of SCCRO to VHL and cullin (Cul2) (39). This led to the suggestion that SCCRO functions as a "substrate sensor switch," with binding of substrate to SCCRO via the substrate adaptor being required to trigger cullin neddylation.…”

The Ubiquitin-associated (UBA) Domain of SCCRO/DCUN1D1 Protein Serves as a Feedback Regulator of Biochemical and Oncogenic Activity

“…Neddylation of cullins promotes assembly of the CRL complex and optimizes its conformation to allow efficient transfer of ubiquitin from the E2 to the substrate protein. In its role in promoting neddylation, SCCRO activity regulates levels of ubiquitination activity in cells: an increase in SCCRO activity increases levels of ubiquitinated proteins by promoting CRL activity, whereas a decrease in SCCRO activity has the opposite effect (22)(23)(24)(25)(26)(27).…”

Regulation of glucose metabolism by p62/SQSTM1 through HIF1α

“…7 E), indicating that less HIF1α is recruited to VHL in the presence of p62. Because conjugation of Nedd8 to CUL2 (neddylation) is crucial for activation of CUL2, and the engagement of the substrate HIF1α with VHL prompts DCNL1 recruitment and then initiates CUL2 neddylation ( Heir et al, 2013 ), we studied whether p62 regulates CUL2 neddylation. Co-immunoprecipitation assays showed that p62 inhibited the DCNL1–CUL2 interaction in a dose-dependent manner ( Fig.…”

“…It is well known that CUL2 neddylation is important for its E3 ligase activity. DCNL1 mediates CUL2 neddylation upon engagement with HIF1α ( Heir et al, 2013 ). We provide the first evidence that p62 binds to and inhibits the activity of VHL ubiquitin ligase by inhibiting CUL2 neddylation.…”