| The cell cycle is the basis of cell proliferation and individual development.Different phases of the cell cycle are regulated by Cyclin proteins and their cognate cyclin-dependent kinases(CDKs).The entry and exit of mitosis are mainly regulated by Cyclin B-CDK1.The main function of the mitosis is to divide S phase-duplicated genetic material equally into two progeny cells.Chromosome segregation errors during mitosis can lead to the formation of aneuploidy of daughter cells.Aneuploidy is closely related to the occurrence and development of tumors.About 90%of solid tumor cells are aneuploidy.In order to ensure the correct separation of chromosomes during mitosis,there is a strict quality monitoring and control mechanism--the spindle assembly checkpoint(SAC).In the presence of kinetochores that are not properly connected to microtubules,SAC is activated to prevent cells from entering into anaphase until all kinetochores are properly connected and the spindle forms the correct bipolarorientation.Mad2 is one of the core proteins underlying SAC machinery and plays a very important function in SAC.Mad2 has two conformations:C-Mad2 and O-Mad2.OMad2 binds to the heterotetrametric formed by Mad1 and C-Mad2 on kinetochore and is catalyzed to form C-Mad2,which then participates in SAC.Cyclin B-CDK1 also plays an important role in SAC.The Cyclin B family contains three members:Cyclin B1,B2 and B3.Among them,the domain organization and molecular weight of Cyclin B1 and B2 are similar.In addition,both Cyclin B1 and B2 are extensively expressed in somatic cells.At present,there are many studies on Cyclin B1-CDK1,but there are remaining gaps and even controversies on the localization and function of Cyclin B2CDK1 complex in mitosis.In this study,we systematically studied the localization and molecular mechanism of Cyclin B2 during interphase and mitosis.We found that Cyclin B2 localizes on the centrosome in interphase,and redistributes at the spindle pole during mitosis,nuclear envelope during prophase,and kinetochores with incorrectly connected microtubules during prometaphase.Surprisingly,Cyclin B2 specifically binds to SAC protein Mad2,but not Mad1,as judged by immunoprecipitation analysis and mass spectrometric analyses.Immunofluorescence studies showed that the kinetochore localization of Cyclin B2 depended on its direct binding to Mad2.Furthermore,we found that the carboxyl terminus of Cyclin B2 is very important for its kinetochore localization.Because its carboxyl terminus contains a conserved Mad2 interacting motif(MIM),and Cyclin B2 binds to Mad2 through the MIM.When endogenous Cyclin B2 was knocked down in HeLa cells or HCT116 cells,a higher proportion of anaphase with lagging chromosomes or chromosome bridges were found in the knockdown group compared with the control group,suggesting that Cyclin B2 knockdown resulted in impaired SAC function.RNA interference and rescue experiments showed that the cells expressing Cyclin B2 wild-type could achieve correct mitosis,while the cells expressing the MIM deletion Cyclin B2 mutant could not achieve correct mitosis.Moreover,when endogenous Cyclin B2 was knocked down,mitotic checkpoint complex assembly capacity and CDK1 activity decreased.These experiments indicated that Cyclin B2 is a novel kinetochore protein and involves in the function of SAC,and its correct kinetochore localization ensured the functional integrity of SAC.In summary,we have discovered a new kinetochore localization protein Cyclin B2.Cyclin B2 promotes robust SAC function and ensures correct chromosome separation in anaphase by Mad2-mediated localization to kinetochore incorrectly connected to microtubules.This study reveals the previously unidentified functions of Cyclin B2 in kinetochore and SAC and a new molecular mechanism by which Mad2 exerts SAC function.Taking together,this study sheds new light on our understanding of SAC regulation. |
