Geminin escapes from ubiquitin-mediated degradation by Aurora-A-induced phosphorylation during mitosis

Objectives:DNA synthesis is restricted to one time per a cell cycle for proper cell division. This restriction is regulated by Geminin, which is known as a repressor of re-replication. Geminin expression is observed from S to M phase and its protein level is regulated by the ubiqutin-mediated proteolysis. Geminin is degraded by APC/CCdh1 ubiqutin ligase complex at early G1 phase. APC/C^Cdh1 has activity from late M phase to late G1 phase and is inactivated by APC/CCdh1 inhibitor, Emi1 during S phase to beginning of M phase. Although APC/C^Cdh1 is active at late M phase, degradation of Geminin occurs at G1 phase. Therefore, we thought that Geminin might be protected from APC/C^Cdh1-mediated proteolysis during late M phase to G1 phase. In this study, we examined the mechanism of Geminin stabilization during mitosis. Methods: We used HeLa, U2OS and 293T cells in this study. To know the role of phosphorylation of Geminin, we generated phospho-defect and -mimicking mutants by mutagaenesis. We also generated phospho-specific antibody against Thr25 of Geminin. BY using these materials, we examined the mechanism of Geminin stabilization. Results:We found that stabilization of Geminin and its band shift during mitosis by Western blotting. This band shift was caused by mitotic-specific phosphorylation at Thr25 within its D-box. Moreover, we found that Geminin co-localized with Aurora-A known as mitotic kinase. Interestingly, Thr25 of Geminin was phosphorylated by Aurora-A and phospho-mimicking mutant of Thr25 was not degraded by APC/C^Cdh1 at G1 phase. Additonally, we found that phospho-mimicking mutant showed the inhibition of ubiquitylation by APC/C^Cdh1 because of reduced interaction with Cdh1. Conclusions:Aurora-A phosphorylated Geminin at Thr25 and the phosphorylated Geminin was stabilized during mitosis. This novel mechanism may play an important role in the proper regulation of cell division and DNA replication.