Fig. 6

PPAT on COASY regulates TPX2 protein stability by interacting with CBP. a Schematic illustration of domains on COASY. COASY is composed of N terminus regulatory domain and two catalytic domain (PPAT and DPCK). R499C mutant inactivates DPCK. b Both wild and R499C of COASY cDNAs rescued multinucleation. MDA-MB-231 cells stably expressing control vector (pLKO.1), wild type or R499C COASY were transfected with COASY siRNA for 72 h. The multinucleation induced by COASY siRNA can be abolished by both wild type and R499C COASY. c Overexpression of PPAT rescued the multinucleation induced by COASY knockdown. *p < 0.05, **p < 0.01, two-tailed Student’s t-test, n = 3 independent repeats. d PPAT and CBP showed strong interaction. The cDNA of N-terminus domain, PPAT or DPCK were cotransfected with CBP cDNA to HEK-293T cells. The cells were then enriched in mitosis by nocodazole treatment and harvested for co-immunoprecipitation. e–g Both COASY and PPAT, but not DPCK, promote the degradation of TPX2 protein. COASY (e), PPAT domain (f), DPCK domain (g) was cotransfected with CBP and V5-tagged TPX2 into unsynchronized HEK-293T cells. After 24 h, the protein synthesis of the transfected cells was halted with 25 mg/ml cycloheximide and collected at the indicated times for western blots. The TPX2 protein level at indicated time points was quantified by Image J software and normalized to b-tubulin protein level. b, c N.S. not significant ***p < 0.001, **p < 0.01, two-tailed Student’s t-test, n = 3 independent repeats. e–g Two-way ANOVA: p < 0.0001 (e), p < 0.0001 (f), N.S. (g). Bonferroni post hoc tests, *p < 0.05, ***p < 0.001, n = 3 independent repeats. Bars show standard error of the mean