The Function Research Of CUEDC2 In Mitosis

Mitosis is a very complicated and precise regulation procedure, which insures the hereditary information carried by chromosomes can be passed from one generation to next generation, though distributing the same number of chromosomes to daughter cells to maintain the genetic stability. In order to ensure segregation of the equal genetic material, the two kinetochores in each sister-chromatid pair must interact with microtubules that emanate from opposite spindle poles and establish the full tension, which could be monitored by spindle-assembly checkpoint (SAC). SAC can inhibit the APC/C activity, and then block the degradation of secruin and cyclin B1 to delay the chromosomes segregation and anaphase onset.In particular, the mitotic checkpoint is a mechanism that eukaryotic cells arrest at metaphase in mitosis until all sister kinetochores are attached to microtubules from opposite spindle poles. As cells proceed from prometaphase to metaphase, MCC (Mitotic checkpoint complex) containing Mad2, Cdc20, Bub3 and BubR1 assembles at unoccupied kinetochores, that cause spindle assembly checkpoint (SAC) activating. Though MCC inteact with the E3 ubiquitin ligase complex - anaphase promoting complex/cyclosome (APC/C), that blocking the degradation of its mitotic targets cyclin B1 and securin. In this state, exit from mitosis and the separation of sister chromatids are inhibited. As soon as kinetochore pairs are fully attached to the microtubules at opposite spindle poles, the MCC can depart from APC/C and the inhibitory signal is extinguished and the APC/C is fully activated. This leads to the ubiquitylation of cyclin B1 and securing, then allowing faithful segregation of chromosomes and anaphase onset.Although the mechanism of SAC activation is generally clear, how the SAC inactivation is still largely unknown. Here we show, phosphorylated CUEDC2 may interact with Cdc20 directly and then promote Mad2 disassociate from Cdc20. So CUEDC2 has a role in regulation metaphase to anaphase transition.In this study, we first found the band of CUEDC2 can shift obviously when cells were arrested in mitosis by nocodazole and taxol. And then we confirm this using other three cell lines. In order to validate the hypothesis CUEDC2 were phosphorylated, we found the band were totally reversed when addingλ-PPase into mitotic lysates. Further, we identify a phosphorylated site at Ser110 of CUEDC2 by using Mass spectrum, which was consistent with our hypothesis. Then we construct the phosphorylate-silenced mutant of CUEDC2, and found it only exists in the form of lower band, even if cells were synchronized in mitosis.Further in order to find the kinase of CUEDC2, we found the time course of band shift in CUEDC2 was the same of that in Cdc27, when cells were released into mitosis from double thymidine block. We also make use of bioinformatics method to predict its kinase is Cdk1. Further we found the band of CUEDC2 was reversed when cells were transfected with Cdk1 and cyclin B1. In addition, the band of CUEDC2 was also reversed when adding Cdk1 inhibitor RO3306 into mitotic lysates. All above results show that Cdk1 is the specific kinase of CUEDC2 in mitosis.We next investigated the function of CUEDC2 in mitosis. To do so, we monitored mitotic progression by time-lapse imaging of HeLa cells depleted CUEDC2 by siRNA. And we found most cells were arrested in metaphase, but the time period of prometaphase was unchanged. We also confirm this by using retinal pigment epithelium cell line (RPE). For exclusion the off-target function, we used another siRNA sequence, and get the same result. We also found the length of metaphase is consistent with the effect of siRNA. For further rule out the off-target function, we transfected the siRNA resistant contructs-CUEDC2 (WT) and CUEDC2 (S110A) into CUEDC2 siRNA cells ,and found the only CUEDC2 (WT) can reverse the effection of CUEDC2 siRNA induced metaphase arrest, but CUEDC2 (S110A) can not. The results show that phosphorylation of CUEDC2 is required for cells proceed from metaphase to anaphase.Further in order to find the mechanisim of CUEDC2 in mitosis, we found depleted CUEDC2 can not effect the formation and morphology of spindle. Inter-k tension was not effected by CUEDC2 siRNA by measure the distance of the different kinetochore pairs and the light intensity of BubR1 on metaphase kinetochores. And then we observe the dynamic movement of Mad2 by time lapse and measure the light intensity of Mad2 on prometaphase kinetochores, and exclude the function of CUEDC2 in microtubule and kinetochore attachment. By immunofluorescence we found CUEDC2 have a diffused localization in cytoplasm. All data shows CUEDC2 don`t take part in the activation of SAC.In order to see if CUEDC2 have a role in regulation of SAC inactivation, we found CUEDC2 is dependent of function of Mad2, and then only CUEDC2 (WT) can interact with Cdc20 in vivo and in vitro by immunoprecipitation, but CUEDC2 (S110A) can not. And Mad2 can also not interact with CUEDC2. This offers some molecular mechanisim for CUEDC2 function. Next, we found CUEDC2 (WT) overexpression caused drive cells exit from taxol induced mitosis block, generating interphase cells with multi-lobed nuclei, but CUEDC2 (S110A) can not. We also confirm this using the normal mammary epithelial cell line (MCF-10A). Overexpression of CUEDC2 (WT) in mitosis cells can increase the percentage of cells with lagging chromosomes, but CUEDC2 (S110A) can not also. All datas tell us CUEDC2 has an important role in SAC inactivation. Because overexpression CUEDC2 can make SAC inactivation in advance and precocious anaphase onset, with percentage of cells with lagging chromosomes obviously increased, that generating CIN. That demonstrated that CUEDC2 have an intimate relationship of tumorigensis.For research the role of CUEDC2 in tumorigensis, we quantify the amount of CUEDC2 by immunohistochemistry in many human tumors tissues. We found the amount of CUEDC2 is dramatically high in kidney cancer, ovarian cancer campared with normal tissue. That tell us high-expressed CUEDC2 can drive tumorigensis by abnormal regulation of SAC inactivation.Aneuploidy and chromosomal instability are major characteristics of human cancer. Most solid tumors are aneuploid, and many frequently mis-segregate whole chromosomes in a phenomenon called chromosomal instability (CIN). CIN positively correlates with poor patient prognosis. We found CUEDC2 overexpression can make abnormal regulation of SAC, so this can drive cells exit from taxol induced mitosis block, generating interphase cells with multi-lobed nuclei. During this time the percentage of cells with lagging chromosomes obviously increased, and then causing CIN.In conclusion, our findings demonstrate that phosphorylated CUEDC2 have an important role in metaphase to anaphase transition and SAC regulation, as well as the mechanism about chromosome stability regulation though CUEDC2. In additon, high expressed CUEDC2 might contribute to tumor development through causing chromosomal instability. So our study not only supplies the explanation the role of CUEDC2 in tumorigenesis, but offers the new clue for tumor therapy.