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

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.

SCCRO3 (DCUN1D3) antagonizes the neddylation and oncogenic activity of SCCRO (DCUN1D1).

The activity of cullin-RING type ubiquitination E3 ligases is regulated by neddylation, a process analogous to ubiquitination that culminates in covalent attachment of the ubiquitin-like protein Nedd8 to cullins. As a component of the E3 for neddylation, SCCRO/DCUN1D1 plays a key regulatory role in neddylation and, consequently, cullin-RING ligase activity. The essential contribution of SCCRO to neddylation is to promote nuclear translocation of the cullin-ROC1 complex. The presence of a myristoyl sequence in SCCRO3, one of four SCCRO paralogues present in humans that localizes to the membrane, raises questions about its function in neddylation. We found that although SCCRO3 binds to CAND1, cullins, and ROC1, it does not efficiently bind to Ubc12, promote cullin neddylation, or conform to the reaction processivity paradigms, suggesting that SCCRO3 does not have E3 activity. Expression of SCCRO3 inhibits SCCRO-promoted neddylation by sequestering cullins to the membrane, thereby blocking its nuclear translocation. Moreover, SCCRO3 inhibits SCCRO transforming activity. The inhibitory effects of SCCRO3 on SCCRO-promoted neddylation and transformation require both an intact myristoyl sequence and PONY domain, confirming that membrane localization and binding to cullins are required for in vivo functions. Taken together, our findings suggest that SCCRO3 functions as a tumor suppressor by antagonizing the neddylation activity of SCCRO.

Oncogenic function of SCCRO5/DCUN1D5 requires its Neddylation E3 activity and nuclear localization.

PURPOSE: To determine mechanisms by which SCCRO5 (aka DCUN1D5) promotes oncogenesis. EXPERIMENTAL DESIGN: SCCRO5 mRNA and protein expression were assessed in 203 randomly selected primary cancer tissue samples, matched histologically normal tissues, and cell lines by use of real-time PCR and Western blot analysis. SCCRO5 overexpression was correlated with survival. The effect of SCCRO5 knockdown on viability was assessed in selected cancer cell lines. Structure-function studies were performed to determine the SCCRO5 residues required for binding to the neddylation components, for neddylation-promoting activity, and for transformation. RESULTS: In oral and lung squamous cell carcinomas, SCCRO5 mRNA levels corresponded with protein levels and overexpression correlated with decreased disease-specific survival. Knockdown of SCCRO5 by RNAi resulted in a selective decrease in the viability of cancer cells with high endogenous levels, suggesting the presence of oncogene addiction. SCCRO5 promoted cullin neddylation while maintaining conserved reaction processivity paradigms involved in ubiquitin and ubiquitin-like protein conjugation, establishing it as a component of the neddylation E3. Neddylation activities in vitro required the potentiating of neddylation (PONY) domain but not the nuclear localization sequence (NLS) domain. In contrast, both the NLS domain and the PONY domain were required for transformation of NIH-3T3 cells. CONCLUSIONS: Our data suggest that SCCRO5 has oncogenic potential that requires its function as a component of the neddylation E3. Neddylation activity and nuclear localization of SCCRO5 are important for its in vivo function.