Study Of Molecular Mechanisms. Phosphorylation Cuedc2 Adjustment Cell Mitosis

As an important feature of cell,mitosis has got lots of attention from biologists since it was found. Similar to the checkpoints of other phases, the checkpoint during mitosis—spindle assembly checkpoint (SAC) is always the core of mitosis related researches. And it is believed that the SAC is more important than others, because its defect often directly promote the generation of aneuploidy which has been observed in many tumor cells. So, the SAC related molecular mechanism and tumor therapy were hot.Spindle assembly checkpoint is a surveillance mechanism crucial for the accurate segregation of chromosomes during cell division which is essential to maintain genomic stability. It was found that lack of microtubules attachment to kinetochores or reduction in inter-kinetochore tension could cause prolonged metaphase through activating the SAC. Till the microtubule-kinetochore attachment and tension are fully established, the SAC signals are turned off, and the APC/C-Cdc20 is activated. And the activation of APC/C causes polyubiquitination and degradation of securin and cyclin B1, and is critical for the anaphase onset. Although it has known some about how the SAC been activated, the molecular mechanisms of SAC inactivation and the following activation of APC/C remain obscure.This paper focused on CUEDC2(CUE domain containing protein 2),which has been cloned and expressed first in our lab. It contains 287 residues, and its function has been not fully known. And in our lab, we found that CUEDC2 was phosphorylated specificly in mitosis. So we found that CUEDC2 had a role in the mitosis, besides the function of inhibition of transcriptional activity of progesterone receptor (PR) and the inhibition of activation of NF-κB pathway. Here, we found that, CUEDC2, is a novel cell cycle regulator that promotes spindle checkpoint inactivation and releases APC/C from checkpoint inhibition.To discover the function of CUEDC2 in mitosis, we employed the cells transfected with Control siRNA or CUEDC2 specific siRNA. And the related phonotype and molecular were detected after CUEDC2 was knocked-down. We found that knockdown CUEDC2 reduced the concentration of Nocodazole required to block cells in mitosis. And after release from Nocodazole block, cells exit mitosis slower. Through detecting the degradation of Cyclin B and securin, two critical molecular markers during mitotic exit, we found that compared to control siRNA cells, cyclin B and securin were dramatically stabilized in CUEDC2 knockdown cells. This result has been confirmed by employing the cell-free system, whose substrates were added in vitro. And then, the degradation delay of Cyclin B induced by CUEDC2 knockdown was reversed by expressing an siRNA-resistant wild type CUEDC2, but not the CUEDC2 S110A mutant. CUEDC2 was proved to function at downstream of the kinetochore pathway through triggering mitotic exit from a taxol arrest with the Aurora B inhibitor, ZM447439. After that, we found CUEDC2 depletion did not affect the degradation of cyclin A during mitosis, which was not regulated by the spindle checkpoint. Taker together, these results suggested that CUEDC2 was required for proper APC/C activation during SAC inactivation.Then the immunoprecipitation assays were used. We found that Cdc20, Mad2 and Cdc27 could be co-precipitated with CUEDC2, with an anti-CUEDC2 antibody. Further, only Cdc20 and Cdc27, but not Mad2, were still associated with CUEDC2 when cells were released from the nocodazole-mediated prometaphase arrest. And it was found that Cdc20 was able to bind to CUEDC2, whereas only slight association was found between CUEDC2 and Mad2.The interaction of Mad2 with Cdc20 following the release from nocodazole arrest was detected and greatly stabilized after CUEDC2 knocked-down. The ZM447439 and MG132 were added to taxol-treated cells, and the amount of Mad2 bound to Cdc20 was also higher in CUEDC2 knockdown cells. Therefore, these results indicated that CUEDC2 was critical for the disassociation of Mad2 from APC/C-Cdc20. And we use in vitro purified proteins to rule out the possibility that CUEDC2 directly compete with Mad2 for Cdc20 binding through mutually exclusive binding.We next investigated whether CUEDC2 overexpression caused chromosomal instability. Indeed, we found that expression of wild type CUEDC2, but not CUEDC2-S110A increased the incidences of aneuploidy. And the level of aneuploidy was further elevated after 20 additional generations in wild type CUEDC2 cells. These results indicated that overexpression of CUEDC2 induced chromosomal instability.Taken togher, we have shown that CUEDC2 is a novel mitotic regulator and promotes the onset of anaphase through releasing APC/C from checkpoint inhibition. CUEDC2 is phosphorylated by Cdk1 in early mitosis, and phosphorylation of CUEDC2 is essential for its binding to Cdc20. The interaction between CUEDC2 and Cdc20 is critical for the dissociation of Mad2 from the APC/C-Cdc20 complex. Furthermore, overexpression of CUEDC2 leads to chromosomal instability, a hallmark of cancer cells. Consistent with the possibility that CUEDC2 plays a role in tumorigenesis, the level of CUEDC2 is highly elevated in several human somatic tumors. Taken together, our data revealed a novel role of CUEDC2 in regulating mitotic checkpoint and promoting chromosome instability.