Effect Of Apc11 On Apoptosis And Apoptosis Of APC11L And NKD2 Proteins In HEK293T Cells

APC/C(Anaphase Promoting Complex/cyclosome) as a E3liganse,catalyses the ubiquitination of many key regulators in cell cycle, of which the dsyfuction may lead to disorders of cell proliferation, genomic instability and tumor.APC11is the only subunit with catalytic RING-finger domains in APC/C. In his thesis, we study the phenomenon of Ape11knockdown in the first part, which hints the function of APC11L in cell cycle; then in the second part, we screen for interactant of APC11L, and we verify that APC11L can interact with NKD2, which suggest NKD2functions in cell cycle control as well.In the first part, by aligning the7isoforms of Apc11, we find that there is only2kinds of proteins translated from them. According to their lengths, we name them APC11L and APC11S. We designed siRNA target sequences both at the CDS and3’UTR, which is shared by all the Apc11mRNAs. RT-PCR results showed that2of our targets sequences can reduce the mRNA amounts by50%and80%respectively. We drew growth curve in HEK293T, which are transitly transfected with siRNAs. And instead of mitotic arrest, targets siRNAs made cells grow faster than the control cells, which is similar to knockdown of CDH1not of CDC20. Then FACS assay back up the result, because siRNAs lead to more S phase cells. In rescue experiment, transfecting APC11L and APC11S can both rescue, which illuminates that APC11L may play a role in cell cycle control too.GST-pulldown assay showed that APC11L can bind to APC2by its CULLIN domain. And the localization of APC11L in Hela cells is similar to APC11S, which is diffusive in cytosol and accumulates around nuclear in granular structures. Also we got a picture of metaphase cell with APC11L on and around chromosomes, and diffusive in the whole cell. Overexpression of APC11L in HEK293T synchronized by Nocodazole,promotes CCNB1degradation, and inhibits the degradation of AURORA-A.Luciferase assay showed APC11L could influence different pathways, but the extent of the change is minor. All in all, it suppresses AP1, AP1-PMA, CRE, GRE, HRE, WNT, C-MYC and activate p53, E2F, NFAT, NF-kappaB, and RB pathway. And base on these results, we think APC11L may play a complex role in cell cycle, but the total influence could be minor.APC11L, sharing the same sequence at N-terminus with APC11S, can rescue the knockdown of Apc11, and affect the degradation of CCNB1and AURORA-A. APC11L deregulates pathways that promote proliferation in minor extent. Study on APC11L may give us new clues on cell cycle control.In the second part, we screened the adult liver cDNA-libraray for preys of APC11L, and we found NKD2. NKD is an inducinle negative factor in WNT pathway, and represses the pathway by binding to DVL-positive factor of WNT pathway. Human NKD2is a homologue of NKD, which also bind to DVL1, and related to protein trafficking as vesicles in cytosol as well. In soft tissue tumors, frequent amplication of Nkd2is detected, which illuminates the relevance between NKD2and cancer genesis. First, we verified the interaction by GST pulldown and Co-immunopreciation assay, and we also find the interaction dependent on presesnce of ZINC2+.Synchronization study indicates that the cell cycle is prolonged in the M/Gl transition in NKD2-GFP stable strains. So we performed experiments in synchronized HEK293T to see if NKD2contribute to the changes caused by APC11L. It turned out that NKD2could counteract the change caused by APC11L during metaphase to G1, but it didin’t work in prometaphase. We found cells of polyploidy and anueploidy too. We suppose that by changing APC11L’s localization, NKD2may affect cell cycle, which provides a new clue in cancer mechanism research.