The human Dcn1-like protein DCNL3 promotes Cul3 neddylation at membranes Nathalie Meyer-Schallera, Yang-Chieh Choub,c, Izabela Sumaraa, Dale D. O. Martind, Thimo Kurza, Nadja Kathedera, Kay Hofmanne, Luc G. Berthiaumed, Frank Sicherib,c, and Matthias Petera,1 aInstitute of Biochemistry, Eidgeno¨ssiche Technische Hochschule, 8093 Zurich, Switzerland; bCenter for Systems Biology, Samuel Lunenfeld Research Institute, Toronto, ON, Canada M5G 1X5; cDepartment of Molecular Genetics, University of Toronto, Toronto, ON, Canada M5S 1A8; dDepartment of Cell Biology, University of Alberta, Edmonton, AB, Canada T6G 2H7; and eBioinformatics Group, Miltenyi Biotec, 51429 Bergisch-Gladbach, Germany Edited by Michael Rape, University of California, Berkeley, CA, and accepted by the Editorial Board June 9, 2009 (received for review December 9, 2008) Cullin (Cul)-based E3 ubiquitin ligases are activated through the enzyme and promotes Nedd8 conjugation through formation of attachment of Nedd8 to the Cul protein. In yeast, Dcn1 (defective this complex (14, 15). Human cells harbor 5 Dcn1-like proteins in Cul neddylation 1 protein) functions as a scaffold-like Nedd8 termed DCNL1–DCNL5 (also named DCUN1D 1–5 for defec- E3-ligase by interacting with its Cul substrates and the Nedd8 E2 tive in Cul neddylation 1 domain-containing protein 1–5) (Fig. Ubc12. Human cells express 5 Dcn1-like (DCNL) proteins each S1). These DCNLs have distinct amino-terminal domains, but containing a C-terminal potentiating neddylation domain but dis- share a conserved C-terminal potentiating neddylation (PONY) tinct amino-terminal extensions. Although the UBA-containing domain, which in yeast Dcn1 is necessary and sufficient for Cul DCNL1 and DCNL2 are likely functional homologues of yeast Dcn1, neddylation in vivo and in vitro (14). The Cul interaction surface DCNL3 also interacts with human Culs and is able to complement at the C terminus of the PONY domain, the DAD patch (D226, the neddylation defect of yeast dcn1⌬ cells. DCNL3 down-regula- A253, D259 in scDcn1), is conserved in all human DCNLs. Like tion by RNAi decreases Cul neddylation, and overexpression of a yeast Dcn1, DCNL1 and DCNL2 harbor a predicted amino- Cul3 mutant deficient in DCNL3 binding interferes with Cul3 terminal UBA domain, which directly binds ubiquitin. DCNL1 is function in vivo. Interestingly, DCNL3 accumulates at the plasma highly amplified in various tumors including squamous cell membrane through a conserved, lipid-modified motif at the N carcinomas and based on this finding is also referred to as terminus. Membrane-bound DCNL3 is able to recruit Cul3 to mem- SCCRO (squamous cell carcinoma-related oncogene) (16). In CELL BIOLOGY branes and is functionally important for Cul3 neddylation in vivo. contrast, DCNL3 levels appear to decrease in liver, bladder, and We conclude that DCNL proteins function as nonredundant Cul renal tumors (17), suggesting that modulation of DCNLs may be Nedd8-E3 ligases. Moreover, the diversification of the N termini in important for cancer development. Although DCNL1 was shown mammalian Dcn1 homologues may contribute to substrate speci- to neddylate Culs (14, 18), the functional significance and targets ficity by regulating their subcellular localization. of other DCNLs remain to be investigated. In this study, we functionally characterized the human DCNL Cullin ͉ DCUN1D ͉ Nedd8 ͉ ubiquitin ͉ proteins with respect to Cul neddylation in vivo and in vitro. We squamous cell carcinoma-related oncogene found that DCNL1, DCNL2, and DCNL3 interact with Culs and modulate Cul neddylation in a nonredundant manner. Unlike biquitination is a major protein modification that targets DCNL1 and DCNL2, DCNL3 localizes to the plasma membrane Uspecific proteins for degradation by 26S proteasomes (1). through a conserved amino-terminal membrane motif. Our Substrate specificity is achieved mainly through the recognition results suggest that this membrane localization is essential for of target proteins by ubiquitin E3 ligases. Cullin (Cul)-Ring- DCNL3-mediated Cul3 neddylation in vivo, implying that Cul3 Ligases (CRLs) comprise a large class of E3 ligases (2), which are may be activated at least in part at membranes. assembled around a Cul scaffold that interacts through con- Results served regions with substrate adaptors and the ring-finger protein Rbx1. DCNL3 Promotes Cul Neddylation in Vivo in a Nonredundant Manner. The assembly and activity of Cul-based ligases is regulated Like yeast Dcn1, human DCNL1 and DCNL2 possess an amino- through reversible conjugation of Nedd8, a ubiquitin-like pro- terminal UBA domain and a conserved Cul interaction domain tein, which is covalently attached to a conserved lysine residue (DAD patch), which was required for its interaction with mul- in the Cul backbone (3). In mammals, this modification, termed tiple Culs [Figs. S1A and S2 A and B (14, 18)]. Moreover, both neddylation, is essential for CRL activity by at least two mech- DCNL1 and DCNL2 coimmunoprecipitated hUbc12 (Fig. S2C), anisms. First, it promotes assembly of the Cul complex by and their down-regulation by RNAi decreased the neddylation dissociating the assembly inhibitor CAND1 (4). Second, Cul status of Cul1 and Cul3 (Fig. 1A and Fig. S3 A and C for neddylation enhances the recruitment of the activated E2 ubiq- statistics), implying that DCNL1 and to some extent DCNL2 uitin-conjugating enzyme to the Cul ligase (5) by inducing a promote Cul neddylation in vivo. conformational change in its carboxyl-terminal domain (6, 7). In addition to DCNL1 and DCNL2, human cells express other Cullins are not the only targets that are regulated by Nedd8 DCNL-members, which all share a conserved PONY domain but modification (3). For example, neddylation has been reported lack an amino-terminal UBA motif (Fig. S1A). Like DCNL1 and to inhibit the transcriptional activation of the tumor suppressor p53 and the breast cancer-associated protein 3 (BCA3) (8, 9). Author contributions: N.M.-S., Y.-C.C., I.S., D.D.O.M., T.K., L.G.B., F.S., and M.P. designed Like ubiquitination, neddylation of substrates is achieved by research; N.M.-S., Y.-C.C., I.S., D.D.O.M., T.K. and N.K. performed research; N.M.-S., Y.-C.C., an enzymatic cascade involving the Nedd8-activating enzyme N.K., and K.H. contributed new reagents/analytic tools; N.M.-S., Y.-C.C., I.S., D.D.O.M., T.K., APP-BP1(ULA1)/UBA3 and the Nedd8-conjugating enzyme K.H., L.G.B., F.S., and M.P. analyzed data; and N.M.-S. and M.P. wrote the paper. encoded by Ubc12 (3). Although the RING-finger protein Rbx1 The authors declare no conflict of interest. promotes Cul neddylation (10–12), recent data suggest that This article is a PNAS Direct Submission. M.R. is a guest editor invited by the Editorial Board. Dcn1 (defective in Cul neddylation 1) functions as an E3 ligase 1To whom correspondence should be addressed. E-mail: [email protected]. for Cul neddylation in yeast and Caenorhabditis elegans (13). This article contains supporting information online at www.pnas.org/cgi/content/full/ Indeed, yeast Dcn1 directly binds to the Cul and the Nedd8 E2 0812528106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812528106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 24, 2021 Fig. 1. Human DCNL proteins function in a nonredundant manner for Cul neddylation in vivo. (A) HeLa cells were transfected with control siRNA (scramble) or siRNA duplexes that specifically down-regulate hUbc12, DCNL1, or DCNL2. After 72 h, the neddylation state of Cul1 and Cul3 was analyzed by immunoblotting, and the ratio between neddylated and unneddylated Cul protein was quantified by LI-COR and normalized to scramble siRNA controls. The mean ratio from 4 independent experiments is indicated (see Fig. S3C for statistics). DCNL1 and DCNL2 comigrate and were detected with an antibody that recognizes both proteins (see Fig. S4C); mRNA levels were quantified by quantitative RT-PCR (Fig. S3A). (B) HeLa cells were transfected with scramble control siRNA or DCNL3 siRNA duplexes, and the neddylation state of Cul1–4 was analyzed after 72 h as described in A. The corresponding statistical analysis and mRNA quantifications are listed in Fig. S3 B and C.(C) HeLa cells transfected with scramble (Left) or DCNL3-specific siRNA duplexes (Right) were analyzed after 5 days by indirect immunofluorescence and DAPI staining. Multinucleated and butterfly nuclei were quantified from 3 independent experiments (bar graph). (Bars: 5 ␮m.) (D) Extracts from cells depleted for DCNL3 or treated with scramble RNAi controls (from B) were analyzed by immunoblotting for the Cul3 substrate Nrf2. (E) HA-tagged DCNL3 or the DCNL3 DAD-patch mutant were overexpressed in HeLa cells together with Flag-tagged Cul3. Total cell extracts were analyzed by immunoblotting with specific antibodies. DCNL2, ectopic expression of DCNL3 and DCNL5 restored activity (19), DCNL3 depletion resulted in the accumulation of neddylation of Cdc53 in dcn1⌬ cells, implying that these isoforms multinucleated cells and cells with butterfly nuclei (Fig. 1C) and also possess Cul neddylation activity in this heterologous assay increased levels of the Cul3-substrate Nrf2 (20) (Fig. 1D). (Fig. S3D). Indeed, RNAi depletion of DCNL3 strongly affected Conversely, overexpression of HA-tagged DCNL3 but not a the neddylation status of several Culs, including Cul3 (Fig. 1B DAD-patch mutant (D241A-A265R-D271A; Fig. S3E) signifi- and Fig. S3 B and C for statistics; and see Fig. S4 B and C for cantly increased Cul3 neddylation (Fig. 1E), whereas overex- specific antibodies). Furthermore, similar to cells lacking Cul3 pression of Rbx1 had no effect (Fig. S5A). Together, these data Fig. 2. DCNL3 directly binds Cul3 and hUbc12. (A) HeLa cell extracts were immunoprecipitated (IP) with specific DCNL3 antibodies or nonspecific control IgG and examined for associated Culs. An aliquot of total extract (input) and supernatants after immunoprecipitation were analyzed (Left).