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

Background: SCCRO/DCUN1D1 is commonly amplified in squamous cell carcinomas. Although the SCCRO PONY domain is required for its function, the contribution of its UBA domain is undefined. Results: Binding of polyubiquitin chains to UBA domain inhibits SCCRO-promoted neddylation and oncogenic activity. Conclusion: UBA domain serves as a sensor and regulator of SCCRO. Significance: Targeting the UBA domain may inhibit SCCRO function in human cancers. Amplification of squamous cell carcinoma-related oncogene (SCCRO) activates its function as an oncogene in a wide range of human cancers. The oncogenic activity of SCCRO requires its potentiating neddylation domain, which regulates its E3 activity for neddylation. The contribution of the N-terminal ubiquitin-associated (UBA) domain to SCCRO function remains to be defined. We found that the UBA domain of SCCRO preferentially binds to polyubiquitin chains in a linkage-independent manner. Binding of polyubiquitin chains to the UBA domain inhibits the neddylation activity of SCCRO in vivo by inhibiting SCCRO-promoted nuclear translocation of neddylation components and results in a corresponding decrease in cullin-RING-ligase-promoted ubiquitination. The results of colony formation and xenograft assays showed a mutation in the UBA domain of SCCRO that reduces binding to polyubiquitin chains, significantly enhancing its oncogenic activity. Analysis of 47 lung and head and neck squamous cell carcinomas identified a case with a frameshift mutation in SCCRO that putatively codes for a protein that lacks a UBA domain. Analysis of data from The Cancer Genome Atlas showed that recurrent mutations cluster in the UBA domains of SCCRO, lose the ability to bind to polyubiquitinated proteins, and have increased neddylation and transformation activities. Combined, these data suggest that the UBA domain functions as a negative regulator of SCCRO function. Mutations in the UBA domain lead to loss of inhibitory control, which results in increased biochemical and oncogenic activity. The clustering of mutations in the UBA domain of SCCRO suggests that mutations may be a mechanism of oncogene activation in human cancers.

Amplification of squamous cell carcinoma-related oncogene (SCCRO) activates its function as an oncogene in a wide range of human cancers. The oncogenic activity of SCCRO requires its potentiating neddylation domain, which regulates its E3 activity for neddylation. The contribution of the N-terminal ubiquitinassociated (UBA) domain to SCCRO function remains to be defined. We found that the UBA domain of SCCRO preferentially binds to polyubiquitin chains in a linkage-independent manner. Binding of polyubiquitin chains to the UBA domain inhibits the neddylation activity of SCCRO in vivo by inhibiting SCCRO-promoted nuclear translocation of neddylation components and results in a corresponding decrease in cullin-RINGligase-promoted ubiquitination. The results of colony formation and xenograft assays showed a mutation in the UBA domain of SCCRO that reduces binding to polyubiquitin chains, significantly enhancing its oncogenic activity. Analysis of 47 lung and head and neck squamous cell carcinomas identified a case with a frameshift mutation in SCCRO that putatively codes for a protein that lacks a UBA domain. Analysis of data from The Cancer Genome Atlas showed that recurrent mutations cluster in the UBA domains of SCCRO, lose the ability to bind to polyubiquitinated proteins, and have increased neddylation and transformation activities. Combined, these data suggest that the UBA domain functions as a negative regulator of SCCRO function. Mutations in the UBA domain lead to loss of inhibitory control, which results in increased biochemical and oncogenic activity. The clustering of mutations in the UBA domain of SCCRO suggests that mutations may be a mechanism of oncogene activation in human cancers.
Posttranslational modification by ubiquitin regulates the activity of proteins involved in a wide range of essential cellular processes. Although ubiquitin modifications primarily target proteins for degradation by the 26 S proteasome, they can also signal for nonproteolytic effects on protein function. The pleiotropic effects of the ubiquitin signal are encoded for by variations in length and linkage of the ubiquitin chain. In general, Lys 48 -linked polyubiquitin chains signal for protein degradation, whereas monoubiquitin and non-Lys 48 -linked polyubiquitin chains signal for nonproteolytic effects (Lys 6 , Lys 11 , Lys 27 , Lys 29 , Lys 33 , and Lys 63 ) (1)(2)(3)(4)(5). However, the same ubiquitin linkage can have varying consequences: for example, Lys 11linked chains can promote degradation of the targeted proteins by either the proteasome or the endoplasmic reticulum as well as promote endocytosis and nondegradative activities that lead to NF-B activation (6). The diversity of effects imparted by the ubiquitin signal are mediated by motifs that bind to ubiquitin and/or ubiquitin chains and transduce their effects, analogous to the role played by binding of phosphorylated proteins to phospho-binding domains (7)(8)(9)(10). Collectively known as ubiquitin-binding domains (UBDs), 2 these motifs comprise more than 20 different families. The ubiquitin-associated (UBA) domain, the prototypic and most common type of UBD, is composed of a motif of 40 residues that are arranged in three tightly packed ␣-helices that share structural homology with coupling of ubiquitin conjugation to endoplasmic reticulum degradation (CUE)-type UBDs (9,(11)(12)(13)(14). It is estimated that more than 79 proteins contain UBA domains. The majority of UBA domains recognize a canonical Ile 44 /Val 70 -binding patch on ubiquitin and preferentially bind to polyubiquitin chains (15,16). However, the affinities of individual UBA domains vary consider-ably, binding to ubiquitin chains of different lengths and linkages (10). UBA domains are thought to limit ubiquitin chain elongation and to help with localization of the bound ubiquitinated proteins to the 26 S proteasome for degradation. Recent studies show that UBA domains can also transduce their effects for a variety of other consequences, including modulation of protein interactions and subcellular localization as well as nonproteasomal turnover of the ubiquitinated proteins and/or the UBA domain-containing proteins (9,17,18).
The presence of a UBA domain in SCCRO (also known as DCUN1D1), a protein that functions as an E3 in neddylation (conjugation of the ubiquitin-like protein NEDD8), raises several intriguing possibilities about its function. In contrast to ubiquitination, only a limited number of proteins are subject to neddylation with the cullin protein family (CUL1, CUL2, CUL3, CUL4, CUL5, and CUL7) being the best characterized targets (19). Cullins serve as the scaffold for assembly of cullin-RINGligase (CRL)-type E3 ligases, the most common type of ubiquitination E3s (20,21). 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).
So what is the role of the UBA domain in SCCRO? The high degree of conservation and the existence of splice forms of the SCCRO orthologue DCN1 in lower organisms and SCCRO paralogues in higher organisms that lack a UBA domain suggest that, although it is not required, the UBA domain plays an important role in the function of SCCRO. In vitro studies confirm that the UBA domain is not required for the neddylation activity of SCCRO (26). Our finding that SCCRO regulates neddylation in vivo by promoting nuclear translocation of the neddylation components (27) combined with recent reports that the UBA domain promotes monoubiquitination of SCCRO leading to its nuclear export (28) suggests that the UBA domain may regulate the neddylation activity of SCCRO in vivo. In this study, we show that binding of polyubiquitin chains to the UBA domain inhibits the activity of SCCRO, a core protein in the ubiquitin-proteasome pathway. In this role, the UBA domain serves as a feedback regulator of the in vivo neddylation and oncogenic activity of SCCRO. Moreover, clustering of mutations in the UBA domain of SCCRO that mitigate binding to polyubiquitinated proteins suggests that it is a mechanism for oncogene activation. Furthermore, the presence of mutations in the UBA domain of many other proteins suggests that this may be an underappreciated mechanism of oncogene activation in human cancers. Given the conserved binding characteristics of the UBA domain, targeting it may have therapeutic benefit in the treatment of human cancer.

EXPERIMENTAL PROCEDURES
Alignment and Sequence Analyses-Database and BLAST searches were performed at the National Center for Biotechnology Information. Sequence alignments were performed using the ClustalW program.
Reagents-All mutants were generated by PCR mutagenesis and verified by sequencing. Proteins were expressed as glutathione S-transferase (GST) fusions in Escherichia coli using the pGEX4T3 vector. The proteins were purified using glutathione-Sepharose 4B beads (Amersham Biosciences) followed by thrombin cleavage where required. CUL1-ROC1 was expressed and purified from E. coli as described previously (29). APPBP1/UBA3, UBC12, NEDD8, and all ubiquitin chains were obtained from a commercial source (Boston Biochem, Cambridge, MA). The proteasome inhibitor Z-Leu-Leu-Leu-al (here referred to as MG132) was purchased from Sigma-Aldrich. The following antibodies were used in this study: anti-CUL1 (Zymed Laboratories Inc., South San Francisco, CA); anti-CUL3 (BD Biosciences); anti-UBC12 (Rockland Immunochemicals, Gilbertsville, PA); anti-NEDD8 (Invitrogen); anti-CAND1 (BD Biosciences); anti-␣-tubulin (Calbiochem); anti-HA, anti-His 6 , and anti-ROC1 (Abcam, Cambridge, MA); and anti-MYC and anti-ubiquitin (Santa Cruz Biotechnology, Dallas, TX). Anti-SCCRO (rabbit polyclonal) antibody was produced and used as described previously (30). Anti-SCCRO monoclonal antibody was raised against an N-terminal region of the protein in SCCRO Ϫ/Ϫ mice. This antibody was found to be highly sensitive and specific (data not shown). All cell lines used in this study were obtained from the American Type Culture Collection (Manassas, VA) and maintained as recommended.
cDNAs for mammalian transfection were cloned into the pBABE-puro vector (Addgene, Cambridge, MA) or pUSE-amp (Clontech). NIH 3T3 cells were doubly exposed to virus in the presence of 7 g/ml Polybrene (Sigma-Aldrich) for 24 h each. Infected cells were selected for by use of puromycin (Sigma-Aldrich). pUSE-amp transfection was performed using Lipofectamine 2000 reagent (Invitrogen) in accordance with the manufacturer's specifications.
GST Pulldown Assay and Immunoprecipitation-GSTtagged proteins were bound to glutathione-Sepharose beads by gentle rotation at 4°C for 45 min. The beads were washed five times with EBC buffer (50 mM Tris-HCl (pH 7.5), 2.5 mM MgCl 2 , 150 mM NaCl, and 0.5% Nonidet P-40) at 20ϫ bead volume. The beads were incubated with HeLa cell lysate or purified proteins as indicated at 4°C for 1 h followed by five washes with EBC buffer at 20ϫ bead volume. Bound proteins were eluted by the addition of 6ϫ Laemmli buffer, resolved by SDS-PAGE, and analyzed by Western blotting. GST-tagged proteins were visualized by use of Coomassie R-250 staining (Sigma-Aldrich). Immunoprecipitations were performed essentially as described previously (30). In brief, 1 mg of wholecell lysate was incubated with antibody bound to agarose beads by gentle rocking at 4°C for 2 h. The wash and detection were the same as described above for the GST pulldown assay.
Immunofluorescence-Rhodamine-conjugated anti-MYC and FITC-conjugated anti-HA antibodies were obtained from a commercial source (Jackson ImmunoResearch Laboratories, West Grove, PA). Cells transfected with plasmid(s) were seeded in 6-well plates with cover glass. Forty-eight hours after transfection, cells were washed with PBS and fixed in 4% formaldehyde for 10 min. The fixed cells were permeabilized with 0.5% Triton X-100 for 5 min, incubated in blocking buffer (PBS containing 10% FCS) for 30 min, and stained overnight at 4°C with fluorochrome-conjugated antibodies. The cells were washed three times with PBS, counterstained with DAPI, covered with ProLong Gold antifade reagent (Invitrogen), and examined with a Leica inverted confocal microscope fitted with appropriate fluorescence filters.
Colony Formation and Xenograft Assays-A soft agar assay was performed essentially as described previously (30). Xenograft assays were performed as described previously and in accordance with Institutional Animal Care and Use Committee guidelines. In brief, NIH 3T3 cells (5 ϫ 10 6 ) were implanted into the flanks of 8-week-old female BALB/c severe combined immunodeficiency (SCID) mice (Taconic, Hudson, NY). Tumor size was measured every 3 days by a single observer and analyzed using Microsoft Excel and GraphPad Prism 5 software (GraphPad Software, La Jolla, CA). According to institutional guidelines, mice were sacrificed when tumor burden impeded normal feeding and movement.
UBA Mutation Analysis-Sixty-four human UBA domaincontaining proteins from Simple Modular Architecture Research Tool (SMART) and 15 UBA domain-containing proteins from the literature were analyzed. Of these proteins, 28 were included in the TCGA data set. These 28 proteins were assessed for the presence and location of mutations against 29 cancer types by use of the cBioPortal for Cancer Genomics.
Statistical Analysis-Paired t tests were used to compare the means Ϯ S.D. of soft agar colony formation counts by use of SPSS 19 software (IBM, Armonk, NY). Survival curves were generated using the Kaplan-Meier method and compared using the log rank test by use of GraphPad Prism. Disease-free survival was defined as the time from primary treatment of cancer to development of recurrence. Survival outcomes were censored for patients who died of intercurrent disease or who survived to the end of the study. A two-tailed p value Յ0.05 was considered to indicate statistical significance.

The UBA Domain of SCCRO Binds Polyubiquitin Chains in a
Linkage-independent Manner-Results of sequence and structural analyses show that the UBA domain of SCCRO is composed of a three-helical bundle architecture that shares homology with several other UBA and CUE domains (26,31). Hydrophobic residues that are critical for interactions with ubiquitin chains in structurally related domains are highly con-served in the UBA domain of SCCRO (Fig. 1A). Whereas previous studies have shown that the UBA domain of the yeast orthologue of SCCRO (DCN1) binds to monoubiquitin, structurally related domains preferentially bind to polyubiquitin chains and have a low affinity for monoubiquitin (16,22). To define the binding preferences, we used in vitro synthesized Lys 48 -linked ubiquitin chains (Ub 2 and Ub 4 ) and assessed their binding to SCCRO by size exclusion chromatography. The results showed a preferential 1:1 co-elution of the Ub 4 -SCCRO complex even in the presence of excess Ub or Ub 2 (Fig. 1B). To validate these binding preferences and precisely map the residues involved, GST-SCCRO and selected UBA and PONY domain mutants were used in a pulldown assay containing ubiquitin chains of varying lengths and linkages (Lys 48 or Lys 63 ). The results showed that SCCRO bound to both Lys 48and Lys 63 -linked Ub 4 or greater length chains with higher affinity than Ub or Ub 2 chains (Fig. 1C). Pulldown assays repeated using serial dilutions of equal concentrations of Lys 48 -and Lys 63 -linked polyubiquitin chains showed a higher affinity for binding to Lys 63 -linked chains (Fig. 1D). Mutations in conserved hydrophobic residues in the UBA domain of SCCRO (L30A and A40E) reduced binding, whereas mutations of nonconserved residues in the UBA domain or of residues in the PONY domain did not affect binding to polyubiquitin chains (Fig. 1C). Immunoprecipitation for MYC in lysates from U2OS cells expressing MYC-tagged SCCRO or SCCRO mutants followed by Western blot analysis for ubiquitin showed that SCCRO binds to polyubiquitinated proteins in vivo and that this interaction requires conserved hydrophobic residues (Phe 18 , Leu 30 , Ala 40 , and Phe 45 ) but not other residues in the UBA domain (Fig. 1E). Mutations in two (SCCRO F18K/L30A and SCCRO L30A/A40E ) or three conserved residues (SCCRO F18K/L30A/A40E ) in the UBA domain of SCCRO resulted in complete loss of binding to polyubiquitinated proteins (Fig. 1F). These findings confirm the requirement of the UBA domain in binding to polyubiquitinated proteins. Previous studies have suggested that UBA domain-containing proteins can be subclassified on the basis of the length and linkage of the ubiquitin chains to which they bind (32). Our results show that the UBA domain of SCCRO is best included in the class 4 subgroup, although it does not bind to monoubiquitin.
The UBA Domain of SCCRO Binds to Polyubiquitinated CRL Substrates-To determine whether SCCRO binds to proteins that are ubiquitinated by CRL complexes activated by neddylation, we performed tandem affinity purification to assess binding to known CUL1-and CUL3-anchored CRL substrates. GST-SCCRO pulldowns on whole-cell lysate from HeLa cells pretreated with MG132 were performed to enrich for SCCRObinding proteins. After thrombin cleavage to release these proteins from the glutathione beads, immunoprecipitation was performed using anti-ubiquitin antibody to enrich for ubiquitinated proteins. Immunoblotting showed that SCCRO bound to polyubiquitinated Aurora B and RHOA, established substrates of CUL3-anchored CRL complexes, but not to cyclin E or p27, substrates of CUL1-anchored CRL complexes ( Fig. 2A). However, as there is a limited amount of ubiquitinated Aurora B and RHOA in cellular lysates, extended exposure of the Western blot was required to detect binding. To verify these results, we repeated binding experiments on HeLa cells transfected with Aurora B or RHOA. Transfected cells were treated with MG132 and subjected to tandem affinity purification following the approach described above. As an additional control, we also included pulldowns with GST-SCCRO L30A on the same lysates. We found clear binding of polyubiquitinated Aurora B and RHOA to GST-SCCRO but not to GST or GST-SCCRO L30A (Fig. 2B). Combined, these findings suggest that SCCRO binds to CUL3-anchored CRL targets.
The UBA Domain Does Not Affect Neddylation Activity of SCCRO in Vitro-To determine whether the UBA domain modulates the neddylation activity of SCCRO, we performed structure-function studies to investigate its effect in in vitro assays. In vitro reactions contained recombinant NEDD8, APPBP1/UBA3 (E1), UBC12 (E2), ATP, and whole-cell lysate from HeLa cells (as a source of CUL-ROC1 substrate) in the presence of varying concentrations of SCCRO or selected SCCRO mutants. As expected, the addition of SCCRO increased cullin neddylation in a dose-dependent manner. Deletion or mutation of the UBA domain (SCCRO ⌬N or SCCRO L30A ) did not alter the neddylation-promoting activity of SCCRO, whereas deletion or mutation of the PONY domain (SCCRO ⌬C or SCCRO D241N ) abrogated its activity (Fig. 3A) (23). Moreover, the addition of a 20-fold molar excess of Lys 48linked Ub 4 did not affect the efficiency with which SCCRO and SCCRO L30A increased levels of neddylated CUL1 or CUL3 (Fig.  3B). These findings are consistent with those reported previously for DCN1 in Caenorhabditis elegans and yeast (26), confirming that binding of ubiquitin chains to the UBA domain does not affect the neddylation activity of SCCRO in vitro.
The UBA Domain Affects Neddylation Activity of SCCRO in Vivo-Given our recent findings showing differential requirements for SCCRO in neddylation in vitro and in vivo (27), we next assessed the requirement of the UBA domain for the neddylation activity of SCCRO in vivo. To determine whether these effects were caused by binding of polyubiquitinated proteins to the UBA domain, HeLa cells were treated with sublethal doses of proteasome inhibitor (MG132), which resulted in accumulation of polyubiquitinated proteins relative to DMSO-treated controls. Whereas transfection of either SCCRO or SCCRO L30A increased levels of neddylated CUL1 and CUL3 in DMSOtreated cells, levels of neddylated CUL1 and CUL3 increased only in SCCRO L30A -transfected HeLa cells treated with MG132 (Fig. 4A).
The UBA Domain Affects Compartmentalization Activity of SCCRO-To determine the mechanisms by which the UBA domain modulates the neddylation activity of SCCRO, we first investigated whether binding of polyubiquitinated proteins to the UBA domain affects interactions between SCCRO and other neddylation components (27). Lysates from HeLa cells transfected with HA-UBC12, HA-CUL1, and MYC-SCCRO or MYC-SCCRO L30A were subjected to immunoprecipitation using anti-MYC antibody followed by Western blotting for neddylation components. We found no differences between the binding of MYC-SCCRO and MYC-SCCRO L30A to neddylation components (HA-CUL1 or HA-UBC12) (Fig. 5A). Similar results were obtained from GST pulldown assays with no detectable differences in binding to neddylation E3 components between GST-SCCRO and GST-SCCRO L30A in HeLa cell lysates treated with DMSO or MG132 (Fig. 5B).
Given the importance of nuclear localization to the function of SCCRO (27), we next investigated the effects of MG132 pretreatment on the subcellular localization of MYC-SCCRO or MYC-SCCRO L30A in U2OS cells. Treatment of U2OS cells with MG132 resulted in increased cytoplasmic localization of MYC-SCCRO compared with treatment with DMSO cells (Fig. 5C). In contrast, treatment with MG132 had no effect on localization of MYC-SCCRO L30A , which remained primarily nuclear. Next, we cotransfected U2OS cells with MYC-SCCRO or MYC-SCCRO L30A and HA-CUL1 ⌬610 -615 , a mutant that depends on SCCRO for nuclear translocation and neddylation in vivo (27). Both MYC-SCCRO and MYC-SCCRO L30A promoted nuclear translocation of HA-CUL1 ⌬610 -615 in U2OS cells treated with FIGURE 2. The UBA domain of SCCRO binds to polyubiquitinated CRL substrates. A, GST-SCCRO pulldown products from HeLa cell lysate pretreated with MG132 were released by thrombin cleavage. SCCRO and SCCRO-interacting proteins were further immunoprecipitated with antibody against polyubiquitinated proteins and analyzed by Western blotting with antibodies against Aurora B and RHOA (CUL3 substrates) and cyclin E and p27 (CUL1 substrates), which showed that SCCRO selectively bound to polyubiquitinated Aurora B and RHOA. B, experiments were performed as described for A except that HeLa cells were transfected with Aurora B or RHOA before MG132 treatment. Note that GST-SCCRO, but not GST or GST-SCCRO L30A , bound to polyubiquitinated Aurora B and RHOA. IP, immunoprecipitation; IB, immunoblotting; PD, pulldown.   1-3). In contrast, neither of the SCCRO mutants affected neddylation of CUL3 promoted by SCCRO L30A (lanes 4 -6).
DMSO. However, treatment with MG132 blocked MYC-SCCRO-but not MYC-SCCRO L30A -promoted nuclear translocation of HA-CUL1 ⌬610 -615 (Fig. 5C). These observations suggest that the subcellular localization of SCCRO is regulated by binding of polyubiquitinated proteins to the UBA domain. Combined with our previous work showing the importance of SCCRO-promoted nuclear translocation of CRL complexes for the neddylation of cullins, these results provide an explanation for why the UBA domain of SCCRO differentially affects cullin neddylation in vitro and in vivo.
Binding of Polyubiquitinated Proteins to the UBA Domain Promotes Monoubiquitination and Nuclear Export of SCCRO-Previous studies have shown that the UBA domain is required for monoubiquitination of SCCRO, which promotes its nuclear export (28). We therefore sought to determine whether cytoplasmic localization of SCCRO in MG132-treated cells is due to increased monoubiquitination of SCCRO. We expressed HA-SCCRO or HA-SCCRO L30A in U2OS cells. Whole-cell lysates from cells treated with either MG132 or DMSO were fractionated and immunoblotted for HA. HA-SCCRO, but not HA-SCCRO L30A , was monoubiquitinated and was present only in the cytoplasmic fraction of MG132-treated cells (Fig. 5D). Consistent with previous findings (28), SCCRO L30A was not monoubiquitinated under any conditions tested (Fig. 5D).
To determine whether monoubiquitination is required for nuclear export promoted by binding of polyubiquitinated proteins to the UBA domain of SCCRO, we cotransfected V5-SCCRO 3KR (a monoubiquitination-deficient mutant; Fig.  5E) and HA-CUL1 ⌬610 -615 into U2OS cells. In contrast to cells expressing SCCRO, treatment with MG132 had no effect on localization of V5-SCCRO 3KR -or V5-SCCRO 3KR -promoted nuclear translocation of HA-CUL1 ⌬610 -615 (Fig. 5C), suggesting that monoubiquitination is important for nuclear export. We next questioned whether the nuclear export of SCCRO is important for the inhibitory effects exerted by binding of polyubiquitinated proteins to the UBA of SCCRO. MYC-NLS-SCCRO and HA-CUL1 ⌬610 -615 when cotransfected into U2OS cells did not translocate to the cytoplasm upon treatment with MG132 (Fig. 5C). Combined with the observed changes in localization and neddylation activity of SCCRO that resulted from treatment with MG132, these data suggest that binding of polyubiquitinated proteins to the UBA domain promotes monoubiquitination and nuclear export of SCCRO, thereby inhibiting its neddylation activity.

Mutation of the UBA Domain Increases Oncogenic Activity of SCCRO-We reported previously that overexpression of SCCRO increases cullin neddylation, which promotes malignant transformation in vivo.
To determine whether the UBA domain affects the function of SCCRO in vivo, we assessed the effects of stable expression of HA-SCCRO, HA-SCCRO L30A , or HA-SCCRO F44Y on transformation of NIH 3T3 cells. Independent stable clones with relatively equal levels of transgene expression were selected for each construct. We first confirmed the functional effects of SCCRO and SCCRO UBA mutants. Levels of neddylated CUL1 or CUL3 were assessed by immunoblot analysis on lysates from at least two independent stable clones for each construct. We found that levels of neddylated CUL1 and CUL3 were higher in cells transfected with SCCRO L30A than in cells transfected with SCCRO or SCCRO F44Y (Fig. 6A, compare lanes 5, 6, and 7 with the other lanes). These observations suggest that the UBA domain modulate the neddylation activity of SCCRO in these cells. Next we assessed oncogenicity in stably transfected NIH 3T3 clones by soft agar assay, which showed that clones expressing SCCRO L30A formed significantly more colonies than NIH 3T3 clones expressing SCCRO (Fig. 6, B and C). In contrast, no differences in colony formation were seen between clones expressing SCCRO F44Y (which retains binding to polyubiquitinated proteins) and those expressing SCCRO. To confirm that the increase in anchorage-independent growth in UBA domain mutants depends on the neddylation activity of SCCRO, we investigated the transforming activity of the neddylation-dead mutant (SCCRO D241N ) and a double mutant in both the UBA domain and the PONY domain (SCCRO L30A/D241N ). Neither the SCCRO D241N mutant nor the SCCRO L30A/D241N double mutant was able to form colonies in soft agar, which suggests that the neddylation activity of SCCRO plays an essential role in its oncogenic activity (Fig. 6, B and C). To validate these findings, we performed in vivo xenograft assays in BALB/c nude mice using SCCRO-or SCCRO L30A -transfected NIH 3T3 clones. We found that there was shortened latency and significantly higher growth for SCCRO L30A -transfected than SCCROtransfected NIH 3T3 cells (Fig. 6, D-F). Necropsy of mice and histopathologic analyses showed that tumor xenografts resulting from injection of SCCRO L30A -expressing NIH 3T3 cells were poorly differentiated and metastasized to pelvic lymph nodes in all mice (data not shown). Conversely, tumor xenografts resulting from injection of SCCRO-expressing NIH 3T3 cells were more differentiated and rarely metastasized to lymph nodes. These results suggest that the oncogenic activity of SCCRO is modulated by its UBA domain. To determine the relevance of these findings to human tumors, we screened 47 lung and head and neck squamous cell carcinomas for mutations using the Sequenom method and identified one with a frameshift mutation that putatively produces a truncated SCCRO protein with an alternate start site downstream of the mutation and without a UBA domain (Fig. 7A). However, because of a lack of tumor tissue, we could not confirm the effect of this mutation on the production of SCCRO protein.
We further screened available whole-genome sequencing results from TCGA projects for different human cancers. This analysis showed that, although they are rare, mutations in SCCRO cluster in the UBA domain (Fig. 7B). To assess whether TCGA-identified UBA domain mutants bind to ubiquitin chains, we carried out an immunoprecipitation assay to assess their binding to ubiquitin chains of varying lengths and linkages (Lys 48 or Lys 63 ). In contrast to wild-type SCCRO, we found that none of the tumor-derived UBA mutants bound to ubiquitin chains (Fig. 7C). Moreover, immunoprecipitation for MYC in lysates from U2OS cells expressing MYC-tagged SCCRO or UBA mutants identified in TCGA followed by Western blot analysis for ubiquitin showed that all TCGA mutants lost the ability to bind to polyubiquitinated proteins in vivo (Fig. 7D).
To determine whether UBA mutants identified in the TCGA data set affect the biochemical and oncogenic functions of SCCRO, we assessed their effect on cullin neddylation and transformation. Whereas transfection of SCCRO, SCCRO R13H , or SCCRO Q20A increased levels of neddylated CUL1 and CUL3 in DMSO-treated HeLa cells, neddylated CUL1 and CUL3 only increased in SCCRO R13H -or SCCRO Q20A -transfected cells after  MG132 treatment (Fig. 8A). Next, to assess the transforming activities of tumor-derived SCCRO UBA mutants, we stably transfected NIH 3T3 cells and selected stable clones with similar transgene expression levels (Fig. 8B). These cells were subjected to soft agar assays, which showed significantly higher colony formation in NIH 3T3 cells expressing TCGA-identified SCCRO UBA mutants relative to those expressing wild-type SCCRO (Fig. 8, C and D). Combined, these findings suggest that the UBA domain of SCCRO modulates its neddylation activity and that this has functional relevance to tumorigenesis.

DISCUSSION
Through its role as an E3 in neddylation, SCCRO promotes CRL activity, which results in an increase in cellular levels of polyubiquitinated proteins (27). We found that binding of polyubiquitinated proteins to the N-terminal UBA domain of SCCRO inhibits its neddylation activity, suggesting the presence of a classical negative feedback loop that regulates CRL-promoted ubiquitination activity (Fig. 9). In this model, the N-terminal UBA domain of SCCRO serves as a sensor of polyubiquitinated proteins as well as an effector by promoting the autoubiquitination of SCCRO. The effects of the UBA domain do not appear to be allosteric as the addition of polyubiquitin chains or mutation of the UBA domain had no effect on the neddylation function of SCCRO in vitro, whereas accumulation of polyubiquitinated proteins showed classical negative feedback dynamics in vivo. Interestingly, the requirement for SCCRO in neddylation was also different in vitro and in vivo with its essential effects in vivo involving compartmentalization of neddylation components. As UBC12, the E2 for neddylation, is primarily located in the nucleus, SCCRO-promoted nuclear translocation of cullin-ROC1 complexes is required for neddylation. Consistent with this, we found that the effect that binding of polyubiquitinated proteins to the UBA domain had on the function of SCCRO was imparted by cytoplasmic translocation of SCCRO: an increase in levels of polyubiquitinated proteins increased cytoplasmic localization of SCCRO and inhibited nuclear localization of neddylation components, whereas mutation in the UBA domain abrogated these effects.
These findings raise obvious questions about how binding of polyubiquitinated proteins to the UBA domain affects the subcellular localization and function of SCCRO. Recent work by Wu et al. (28) shows that the UBA domain of SCCRO is required for its monoubiquitination and that monoubiquitination promotes nuclear export of SCCRO. Conversely, mutations in the UBA domain blocked monoubiquitination of SCCRO and consequently its nuclear export (Fig. 5, C and D) (28). These findings suggest that the binding of polyubiquitinated proteins to the UBA domain serves as a signal for the monoubiquitination of SCCRO. A limitation of this work is the reliance on SCCRO mutants to assess structure-function relationships. It remains possible that the generated mutants affect protein function in unanticipated ways. Mitigating this issue at least in part, we found that blocking the monoubiquitination of SCCRO by mutation of ubiquitination residues in SCCRO or restricting the nuclear export of SCCRO by NLS tag had identical effects on SCCRO localization and function as UBA domain mutants expressed in cells exposed to MG132.
UBD-coupled autoubiquitination has been reported for several other proteins, including STS1, STS2, EPS15, EPS15R, Vps27p/HRS, Vps9p, and RABEX-5 (9). Although the precise underlying mechanisms remain to be defined, a range of functional consequences are known to accompany UBD-coupled autoubiquitination, including effects on inter-and intraprotein interactions and subcellular localization. Various UBDs have been implicated in coupled autoubiquitination, including ubiquitin-interacting motif, CUE, motif interacting with ubiquitin, and A20 zinc finger domains; however, this has not been reported previously for UBA domain-containing proteins. In the case of SCCRO, coupled autoubiquitination leads to its cytoplasmic translocation and functional inhibition in vivo. What remains to be defined is how monoubiquitinated SCCRO is translocated and sequestered in the cytoplasm. Given the relatively low affinity of the UBA domain in SCCRO for monoubiquitin, it is unlikely that intraprotein interaction is the cause of changes in the localization of SCCRO. These effects are also unlikely to be caused by changes in interactions with neddylation components as the presence of polyubiquitin chains and/or mutations in the UBA domain did not affect binding between SCCRO and CUL1 or UBC12. Given that SCCRO and other neddylation E3 components lack a canonical nuclear localization sequence, one possibility for nuclear export is that monoubiquitination of SCCRO inhibits interactions with NLScontaining protein partners involved in nuclear import.
In the broader context, the principal presence of the UBA domain in E2s and E3s (33) raises the question of whether UBA domains serve as general feedback regulators of the ubiquitination pathway. In a related model, autoubiquitination and ubiquitin binding of the UBD in RABEX-5 affect its ubiquitin ligase

The UBA Domain of SCCRO/DCUN1D1
activity (34). It remains to be determined whether UBA domains play similar roles in other proteins.
Although specific mechanisms require definition, it is quite clear that mutations in the UBA domain that inhibit interactions with ubiquitinated proteins increase the neddylation activity of SCCRO in vivo. We have shown previously that increased neddylation activity resulting from overexpression of SCCRO promotes oncogenesis in vitro and in vivo (30). In primary tumors, the oncogenic activity of SCCRO is promoted by amplification that is present in many types of cancers, including lung, ovarian, head and neck, and esophageal cancers (35)(36)(37). Interestingly, we found that transgenic expression of UBA domain mutants of SCCRO was more oncogenic than that of wild-type SCCRO. Moreover, we found that, although they are rare, somatic mutations cluster in the UBA domain of SCCRO. As the spontaneously occurring somatic UBA domain mutations lose the ability to bind to polyubiquitinated proteins and have increased neddylation and transforming activity, they represent an alternate mechanism for activation of the oncogenic activity of SCCRO in human cancers.
The role of the UBA domain in cancer pathogenesis has been well established. For example, germ line mutations in the UBA domain of SQSTM1 are associated with the development of Paget disease of bone. These mutations impair the ability of SQSTM1 to bind to ubiquitin chains, altering proteasomeand/or autophagy-based turnover of the bound ubiquitinated proteins (38 -42). Although the specific polyubiquitinated proteins involved remain to be identified, the carcinogenic effects that result from loss of binding due to mutation in the UBA domain of SQSTM1 are achieved through activation of NF-B signaling (39,40). Moreover, the identification of mutation clusters in the UBA domains of other proteins suggests that these domains may play a larger and more direct role in oncogenesis than appreciated previously (supplemental Table 1). In the broader context, functional domains typically have similar activities even when present in different proteins. As such, functionally related mutations in cancers may cluster in domains rather than in individual genes or gene families. Domain-based analysis of genome-wide sequencing data may identify novel targets for anticancer therapies. For example, the activity of an oncogene may be mitigated by agents that bind to or mimic the UBA domain of the oncoprotein. In one such instance, overexpression of the UBA domain of hFAF1 significantly promotes cell death by increasing the degradation of polyubiquitinated proteins (43).