Squamous Cell Carcinoma-related Oncogene (SCCRO) Family Members Regulate Cell Growth and Proliferation through Their Cooperative and Antagonistic Effects on Cullin Neddylation

Abstract

SCCRO (squamous cell carcinoma-related oncogene; also known as DCUN1D1) is a highly conserved gene that functions as an E3 in neddylation. Although inactivation of SCCRO in yeast results in lethality, SCCRO ؊/؊ mice are viable. The exclusive presence of highly conserved paralogues in higher organisms led us to assess whether compensation by SCCRO paralogues rescues lethality in SCCRO ؊/؊ mice. Using murine and Drosophila models, we assessed the in vivo activities of SCCRO and its paralogues in cullin neddylation. We found that SCCRO family members have overlapping and antagonistic activity that regulates neddylation and cell proliferation activities in vivo. In flies, both dSCCRO and dSCCRO3 promote neddylation and cell proliferation, whereas dSCCRO4 negatively regulates these processes. Analysis of somatic clones showed that the effects that these paralogues have on proliferation serve to promote cell competition, leading to apoptosis in clones with a net decrease in neddylation activity. We found that dSCCRO and, to a lesser extent, dSCCRO3 rescue the neddylation and proliferation defects promoted by expression of SCCRO4. dSCCRO and dSCCRO3 functioned cooperatively, with their coexpression resulting in an increase in both the neddylated cullin fraction and proliferation activity. In contrast, human SCCRO and SCCRO4 promote, and human SCCRO3 inhibits, neddylation and proliferation when expressed in flies. Our findings provide the first insights into the mechanisms through which SCCRO family members cooperatively regulate neddylation and cell proliferation.The highly conserved tripartite enzymatic cascade that results in posttranslational modification by ubiquitin regulates the activity of proteins involved in diverse cellular processes (1-3). Given its essential role, ubiquitination itself is subject to considerable regulatory control, primarily by modulation of E3 activity, the specificity-and rate-limiting step in the reaction (4). For cullin-RING ligase-type ubiquitination E3s, the largest class of E3s in mammals, assembly of the enzymatic complex serves as a key regulator of function. Neddylation, a process analogous to ubiquitination that results in conjugation of the ubiquitin-like protein NEDD8 to cullins, serves as a signal for assembly and activation of cullin-RING ligase complexes (5-15). We and others have shown that SCCRO 6 /DCUN1D1 is an essential component of the E3 ligase for neddylation (16 -21). Conservation of SCCRO from yeast to humans strongly suggests that it is required for fundamental processes in eukaryotic cells. Lending support to this idea, knocking out SCCRO orthologues DCN1 and Dcn1p in Caenorhabditis elegans and Saccharomyces cerevisiae results in lethality (19). Our finding that SCCRO knock-out mice are viable raises questions about the requirement of SCCRO for normal cellular function and development in higher organisms (17).The presence of SCCRO paralogues in higher organisms suggests that the viability of SCCRO Ϫ/Ϫ mice might be attributable to compensation by its paralogues (2...

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

“…We recently showed that SCCRO affects proliferation in vitro and in vivo (13). Consistent with the involvement of ned-dylation in proliferation, treatment with MLN2924, a neddylation E1 inhibitor, induces cell cycle arrest in normal and cancerous cells (14 -16).…”

“…Bioinformatics analysis shows that SCCRO has four paralogues in mammals, which can be classified into three subgroups on the basis of their phylogeny and N-terminal sequences: SCCRO and SCCRO2 (DCUN1D2) contain an ubiquitin-associated domain, SCCRO3 (DCUN1D3) contains a myristoyl sequence, and SCCRO4 (DCUN1D4) and SCCRO5 (DCUN1D5) contain a nuclear localization signal in the N terminus (12,45,46). We have shown that SCCRO paralogues have both independent and overlapping activities in regulating cullin neddylation in higher organisms (13). It is likely that SCCRO paralogues function to diversify the neddylation signal to provide tight control of CRL activity.…”

“…Finally, SCCRO is amplified and overexpressed in a variety of human cancers. The increased proliferation resulting from SCCRO overexpression in cellular and animal models (9,13), along with the high prevalence of polyploidy in tumors with SCCRO amplification, suggests the possibility that the cancerpromoting activity of SCCRO may result in premature removal of Aurora B, accelerated abscission, and aneuploidy and may contribute to genetic instability. Moreover, although proteasome and neddylation inhibitors have been shown to have therapeutic efficacy in humans, their broad-based activity induces severe side effects in a significant number of patients.…”

“…In contrast, SCCRO plays an essential role in neddylation in vivo by promoting nuclear translocation of cullin-ROC1 complexes, where neddylation is thought to occur (9). Although inactivation of other core components of neddylation in model organisms is lethal, inactivation of SCCRO is not lethal in mice or flies (9,13). Moreover, the effect of SCCRO on neddylation in vivo appears to be more selective than that of other core components, with the targeted inactivation of SCCRO variably and only partially reducing neddylation of individual cullins.…”

Coronin 3 negatively regulates G6PC3 in HepG2 cells, as identified by label-free mass-spectrometry

“…Coronin 3 inhibits glucose-6-phosphatase catalytic subunit 3 (G6PC3) expression in HepG2 cells. To verify the mass spectrometry results, 4 upregulated [human epithelial cell adhesion molecule (hepCAM), zinc binding alcohol dehydrogenase domain containing 2 (ZADH2), mitochondrial calcium uptake 1 (MICU1) and squamous cell carcinoma-related oncogene (DCUN1D1)] and 1 downregulated (G6PC3) proteins were selected out of these 249 proteins to perform qPCR verification (primers were listed in Table II), as they have been reported to serve important roles in tumorigenesis, growth and metastasis of cancer (15)(16)(17)(18)(19), and had a marked fold change in the MS analysis. Among these 5 selected proteins, only G6PC3 demonstrated alterations in mRNA expression levels when coronin 3 expression was dysregulated in the verification experiments.…”