DCNL1 Functions as a Substrate Sensor and Activator of Cullin 2-RING Ligase Pardeep Heir, Roxana I. Sufan, Samantha N. Greer, Betty P. Poon, Jeffrey E. Lee, Michael Ohh Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada Downloaded from Substrate engagement by F-box proteins promotes NEDD8 modification of cullins, which is necessary for the activation of cul- lin-RING E3 ubiquitin ligases (CRLs). However, the mechanism by which substrate recruitment triggers cullin neddylation re- mains 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-induc- ible factor 1␣ (HIF1␣) to the substrate receptor VHL increases DCNL1 binding to VHL as well as to CUL2. Notably, an engi- neered 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. http://mcb.asm.org/ ullin-RING ligases (CRLs), the largest family of E3 ubiquitin A235, and D241 in DCNL1), and it can also bind Ubc12 (12). It Cligases, are multiprotein complexes involved in protein deg- has been proposed that it is the concerted action of Dcn1 and Rbx1 radation (1, 2). They are assembled on a cullin scaffold (CUL1, that promotes cullin neddylation (14). However, the molecular CUL2, CUL3, CUL4A, CUL4B, and CUL5) and contain a RING events responsible for recruiting DCNL1 to cullin scaffolds for finger protein (RBX1 or RBX2) at the cullin C-terminal domain neddylation have remained unclear. (CTD) as well as a substrate receptor, which generally associates von Hippel-Lindau tumor suppressor protein (VHL) is the with the cullin N-terminal domain (NTD) via adaptor proteins substrate receptor of the CUL2-based CRL called ECV (elongins on December 14, 2020 at UNIV OF TORONTO (2). CUL3-based CRLs are the exception in which the Broad com- BC/CUL2/VHL) (15). VHL contains an ␣ domain, which binds plex, Tramtrack, and Bric-a-Brac (BTB)-domain containing pro- the elongin C adaptor protein that is critical for bridging VHL to tein mediates both substrate specificity and binding to CUL3 CUL2, and a ␤ domain, which recognizes substrates that have without the need of adaptor proteins (3). been posttranslationally modified for subsequent ubiquitylation The process of attaching ubiquitin onto a CRL substrate re- (16, 17). Hypoxia-inducible factor ␣ (HIF␣), the best-character- quires three enzymes. Using ATP, an E1 activating enzyme forms ized substrate of ECV, is targeted for polyubiquitylation upon a thioester bond between its catalytic cysteine residue and the hydroxylation of conserved proline residues within the oxygen- C-terminal carboxyl group of ubiquitin (4). The activated ubiqui- dependent degradation (ODD) domain, which is catalyzed by tin is then transferred onto the catalytic cysteine of an E2 conju- prolyl-hydroxylase enzymes in the presence of oxygen (18–21). gating enzyme (4). CRL through RBX1/2 helps transfer ubiquitin Under reduced oxygen tension or hypoxia, HIF␣ remains unhy- from E2 onto a lysine residue on the bound substrate (2). Neddy- droxylated, escapes ECV-dependent degradation, and het- lation, the process of conjugating the ubiquitin-like protein erodimerizes with HIF␤ to form an active transcription factor for NEDD8 onto a substrate, follows an analogous cascade (5, 6). the transactivation of numerous genes containing hypoxia-re- Cullins are modified by NEDD8 (7). Structural studies involving sponsive elements (HREs) to trigger various adaptive responses to CTD CTD the CTD of CUL5 (CUL5 )-RBX1 and NEDD8ϳCUL5 - hypoxia, including glycolysis, erythropoiesis, and angiogenesis RBX1 have demonstrated that in an unneddylated state, RBX1 has (22). Notably, mutations in VHL cause VHL disease, which is limited movement due to inhibitory cullin subdomains, thus re- commonly characterized by the stabilization of HIF␣, overexpres- straining its ability to recruit ubiquitin-charged E2 (8). Neddyla- sion of hypoxia-inducible genes, and the development of multiple tion causes a conformational change in cullin structure that re- tumors in several organ systems such as the central nervous system leases RBX1 from a confined configuration to enhance the binding and the kidney (23). of ubiquitin-charged E2 and thus CRL ubiquitylation capability Engagement of various substrates to their respective CRLs, in- (8, 9). The molecular events involved in the process of neddylation of cullins are not as straightforward as the resulting effects. For CUL1 Received 4 October 2012 Returned for modification 30 October 2012 neddylation to take place, rotation of the RING domain of RBX1 is Accepted 4 February 2013 needed in order to bring the catalytic Cys111 of UBC12 (NEDD8 Published ahead of print 11 February 2013 E2 enzyme) in close proximity to CUL1 Lys720, the NEDD8 ac- Address correspondence to Michael Ohh, [email protected]. ceptor site (10). Defective in cullin neddylation 1 (Dcn1) in Sac- Supplemental material for this article may be found at http://dx.doi.org/10.1128 charomyces cerevisiae (DCNL1 is the human homolog; also known /MCB.01342-12. as SCCRO or DCUN1D1) has been reported to act as an E3 Copyright © 2013, American Society for Microbiology. All Rights Reserved. NEDD8 ligase of cullins (11–13). Dcn1 is able to bind Cdc53 doi:10.1128/MCB.01342-12 (CUL1 homolog) through its DAD patch (amino acids D211, April 2013 Volume 33 Number 8 Molecular and Cellular Biology p. 1621–1631 mcb.asm.org 1621 Heir et al. cluding HIF␣ to ECV, has been shown to induce the neddylation by GenScript and inserted into pcDNA3 using HindIII and BamHI. Con- of cullins, a phenomenon referred to as “substrate-mediated ned- structs that were cloned are described in Table S1 in the supplemental dylation” (24–30). Although the precise molecular mechanism by material. which substrate engagement triggers cullin neddylation and Immunoprecipitation and immunoblotting. Cells were harvested in thereby E3 enzymatic activity remains largely unknown, substrate EBC lysis buffer (50 mM Tris, pH 8, 120 mM NaCl, 0.5% NP-40) and receptors would be predicted to play an important, perhaps criti- supplemented with protease inhibitors (Roche) and MG132 (Peptides International) where indicated in the figure legends. Lysates were immu- cal, role in the substrate-induced CRL activation event, consider- noprecipitated using the indicated antibodies along with protein A-Sep- ing that the substrate receptors serve as the bridge connecting harose (Repligen). Bound proteins were washed five times in NETN buf- substrates to cullins. In this regard, components of the ECV com- fer (20 mM Tris, pH 8, 100 mM NaCl, 1 mM EDTA, 0.5% NP-40), eluted Downloaded from plex, namely, the VHL/elongins BC (VBC) subcomplex, have by boiling in sample buffer, and resolved by SDS-PAGE. Proteins were been crystallized in the presence and absence of a prolyl-hydroxy- electrotransferred onto polyvinylidene difluoride (PVDF) membrane lated HIF1␣ peptide (17, 31, 32). However, the addition of prolyl- (Bio-Rad), blocked, and probed with the antibodies indicated on the fig- hydroxylated HIF1␣ peptide did not cause an appreciable change ures. to the structure of VBC, leaving the mechanism of substrate-me- Luciferase assay. Cells were transfected in triplicate with pGL3- diated neddylation a mystery. 5ϫHRE-Luc (where Luc is luciferase) (kind gift from Richard P. Hill) and Here, we show that DCNL1 is a “substrate sensor switch” pRL-SV40 (where SV40 is simian virus 40) (Promega) Renilla luciferase ␮ whose incorporation into the ECV complex increases upon the control. Cells were treated with 10 M MG132 for4htohelp stabilize ␣ endogenous HIF1␣ in normoxia and lysed at 48 h posttransfection. A recognition of HIF1 by VHL and activates the E3 activity via http://mcb.asm.org/ dual-luciferase reporter assay system (Promega) was used, and lumines- promoting CUL2 neddylation. cence was measured using a Mithras LB940 plate reader (Berthold Tech- MATERIALS AND METHODS nologies). Relative light units (RLU) from firefly luciferase were normal- ized against Renilla luciferase values. Values were standardized against the Cells. 786-O, HEK293A, HEK293T, PC3, and U2OS cells (American Type value for the pGIPZ control cell line, which was arbitrarily set to 100. Culture Collection) were maintained in Dulbecco’s modified Eagle’s me- Lentiviral production and generation of stable shRNA knockdown dium supplemented with 10% heat-inactivated fetal bovine serum cell lines. The following shRNA constructs were obtained from Thermo (Wisent) at 37°C in a humidified atmosphere with 5% CO . TF-1 cells 2 Scientific: pGIPZ (catalog number RHS4346), shRNA targeting DCNL1 (American Type Culture Collection) were maintained in RPMI 1640 me- clone 1 (shDCNL1-1; V3LHS_371768), shDCNL1 clone 2 (shDCNL1-2; dium supplemented with 10% heat-inactivated fetal bovine serum V3LHS_371770), shDCNL3 (V3LHS_360875), and shVHL (V2LHS_ (Wisent) and 2 ng/ml granulocyte-macrophage colony-stimulating factor on December 14, 2020 at UNIV OF TORONTO 202399). HEK293T cells were transfected with psPAX2, pMDG1.vsvg (GM-CSF; Invitrogen). HEK293A and U2OS cells stably expressing short (kind gifts from Linda Z. Penn), and shRNA-containing plasmid. Lenti- hairpin RNA (shRNA) were maintained in 2 ␮g/ml puromycin. virus containing supernatant was collected at 48 and 72 h posttransfection Antibodies and reagents. The following antibodies were obtained and was used for infection after being filtered. HEK293A and U2OS cells from Cell Signaling Technology: glutathione S-transferase (GST; catalog number 2622), hemagglutinin (HA) (mouse, 2367; rabbit, 3724), VHL were selected in puromycin 24 h after infection. Protein purification. pGEX-4T-1-DCNL1 was expressed in (2738), and NEDD8 (2745).