Fig. 7: Aurora B and CREPT regulate the expression of Cyclin B1.

a CREPT is phosphorylated in eukaryotic cells. Purified GST-CREPT in prokaryotic (E. coli) and eukaryotic (Mammals) cells were examined using general anti-phosphorylation antibodies by western blots. A pan-phosphorylation antibody, marked as p-CREPT(S/T/Y), and a specific anti-serine phosphorylation antibody, marked as p-CREPT(S), were used. b The phosphorylation of CREPT is impaired after adding lambda protein phosphatase (λ-PPase). GST-CREPT purified from eukaryotic cells was incubated with λ-PPase to allow proteins of dephosphorylation. A pan-phosphorylation antibody was used for the western blot. c Potential phosphorylation sites in CREPT by Aurora B is predicted. The Consensus phosphorylation sequence by Aurora B is shown. The potential phosphorylation sites were predicted by Netphos 3.1. d S145 residue is conserved across different species. Sequences of CREPTs among different species are presented. Red box indicates S145, which is conserved. e Aurora B phosphorylates CREPT at S145. Flag-Aurora B was purified by an immunoprecipitation with an anti-Flag antibody from HEK293T cells transfected with an expression vector for Flag-Aurora B. The GST, GST-CREPT, and GST-CREPT(S145A) were purified by GST beads. Phosphorylation of GST or GST-tagged CREPT and its mutant catalyzed by Flag-Aurora B was detected by a general anti-phosphorylation antibody (top). f S145 in CREPT is responsible for its activity on the transcriptional regulation of CCNB1 gene. Wild-type (WT) and different mutants of CREPT were transfected with CCNB1 promoter-luciferase reporter and pRL-TK plasmids into MGC803 cells. Luciferase activities were measured from three independent experiments; **p < 0.01, ***p < 0.001; ns no significant. g Aurora B promotes expression of the Cyclin B1 through CREPT. Luciferase activities were examined for cells with overexpression of CREPT and Aurora B in both wild-type (WT) and CREPT deletion (CREPT–/–) MGC803 cells. Note that overexpression of CREPT rescued but Aurora B failed the activity in CREPT deletion cells. The experiments were performed in three independent repeats; **p < 0.01, ***p < 0.001; ns no significant. h–i Wild-type CREPT, but not CREPT(S145A), restores the colony formation impaired by endogenous CREPT deletion. Vector and HA-CREPTs were exogenously expressed in MGC803 cells where endogenous CREPT was deleted by a CRISPR/Cas9 system. For each well, 1000 cells were seeded and colonies were stained (h). A quantitative presentation of colonies formation is shown (i) from 3 independent experiments; *p < 0.05, ns no significant. j A model for the regulation of Cyclin B1 expression by CREPT and Aurora B at the G2/M transition. Aurora B interacts with CREPT and phosphorylates CREPT at S145. The phosphorylated CREPT promotes the transcription of Cyclin B1, which promotes the phosphorylation of Cdk1, leading to a quick cell transition from the G2 phase into the M phase to accelerate cell proliferation and metastasis