Abstract
Squamous cell carcinoma-related oncogene (SCCRO)/DCUN1D1, a component of the neddylation E3 complex, regulates the activity of the cullin-RING-ligase type of ubiquitination E3s by promoting neddylation of cullin family members. Studies have shown that SCCRO regulates proliferation in vitro and in vivo Here we show that inactivation of SCCRO results in prolonged mitotic time because of delayed and/or failed abscission. The effects of SCCRO on abscission involve its role in neddylation and localization of Cul3 to the midbody. The Cul3 adaptor KLHL21 mediates the effects of SCCRO on abscission, as it fails to localize to the midbody in SCCRO-deficient cells during abscission, and its inactivation resulted in phenotypic changes identical to SCCRO inactivation. Ubiquitination-promoted turnover of Aurora B at the midbody was deficient in SCCRO- and KLHL21-deficient cells, suggesting that it is the target of Cul3KLHL21 at the midbody. Correction of abscission delays in SCCRO-deficient cells with addition of an Aurora B inhibitor at the midbody stage suggests that Aurora B is the target of SCCRO-promoted Cul3KLHL21 activity. The activity of other Cul3-anchored complexes, including Cul3KLHL9/KLHL13, was intact in SCCRO-deficient cells, suggesting that SCCRO selectively, rather than collectively, neddylates cullins in vivo Combined, these findings support a model in which the SCCRO, substrate, and substrate adaptors cooperatively provide tight control of neddylation and cullin-RING-ligase activity in vivo.
Highlights
Squamous cell carcinoma–related oncogene (SCCRO)/ DCUN1D1, a component of the neddylation E3 complex, regulates the activity of the cullin–RING–ligase type of ubiquitination E3s by promoting neddylation of cullin family members
We previously reported that SCCROϪ/Ϫ mice are runted, which was attributed to a decrease in cell proliferation rather than to alterations in cell size [9]
Proliferation defects were observed in SCCROϪ/Ϫ mouse embryonic fibroblasts (MEFs), which were rescued by re-expression of SCCRO by transfection
Summary
We previously reported that SCCROϪ/Ϫ mice are runted, which was attributed to a decrease in cell proliferation rather than to alterations in cell size [9]. Proliferation defects were observed in SCCROϪ/Ϫ mouse embryonic fibroblasts (MEFs), which were rescued by re-expression of SCCRO by transfection. Flow cytometry using propidium iodide staining for DNA content indicated an increased Ͼ4N fraction in SCCROϪ/Ϫ MEFs compared with wild-type MEFs (Fig. 1A). Immunostaining with DAPI and anti-pericentrin confirmed the increase in DNA content and disclosed an increase in polycentrosomy in SCCROϪ/Ϫ MEFs compared with wild-type MEFs (Fig. 1, B–E), suggesting the presence of a mitotic defect. Ploidy and centrosome defects were rescued in SCCROϪ/Ϫ MEFs by re-expression of SCCRO but not SCCROD241A, a mutant deficient in neddylation activity (Fig. 1, C–E), suggesting that neddylation activity is required for the effect of SCCRO on mitosis. Treatment of SCCROϩ/ϩ MEFs with MLN4924 (Active BioChem) resulted in mitotic delays and phenotypic changes similar to those seen in SCCROϪ/Ϫ MEFs, confirming the requirement for neddylation activity (Fig. 1, A–E)
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