Abstract 4793
Background
ATM is an important cell cycle checkpoint kinase. This protein is an controller of cell cycle checkpoint that is required for cell response to DNA damage and for genome stability. The spindle assembly checkpoint (SAC) controls accurate chromosome segregation to maintain genome stability. All kinetochores correctly attach to the spindle, which allow mitosis to proceed. Bub3 is the mainly component of the SAC, in which its kinase activity can stabilize the correct kinetochore–microtubule attachments. Genomic stability is a hallmarks of breast cancer, and the level of Bub3 in breast cancer are higher than that in normal tussue. Understanding how the ATM-Bub3 signal contributes to kinetochore-based signaling may help us to recognize the occurrence of breast cancer.
Methods
Stable Isotope Labeling with Amino Acid in Cell Culture (SILAC) to identify ATM interation protein in breast cancer. Bioinformatics analyze the phosphorylation site of Bub3. Biacore assay and in vitro kinase assay to validate the phosphorylation site. Flow cytometry assay to check the mitotic index. Mutation of the phosphorylation site of bub3 and Co-IP were proformed to identify the interation of Bub3 and Bub1 or ZNF207, and western blot to be used to test the activity of Bub1.
Results
Through Stable Isotope Labeling with Amino Acid in Cell Culture (SILAC), we identified additional mitotic proteins that interact and can be phosphorylated by mitotically activated ATM kinase. We provide both in vitro and in vivo evidence that ATM phosphorylates Bub3 on Serine 135. This phosphorylation event promotes Bub1 activity in the mitotic spindle checkpoint. Further, we find that phosphorylation of Bub3 on Ser135 is required for formation of the Bub3-Bub1 complex, which is essential for Bub1 Ser314 phosphorylation. Mutation of Ser135 to alanine of Bub3 resulted in an enhanced interaction with BuGZ/ZNF207 and reduced interaction with Bub1, which lead to a spindle checkpoint defect.
Conclusions
Our findings highlight the functional significance of ATM-mediated kinetochore protein phosphorylation and elucidate a detailed regulatory mechanism of the SAC in breast cancer.
Clinical trial identification
Editorial acknowledgement
Legal entity responsible for the study
The authors.
Funding
Has not received any funding.
Disclosure
All authors have declared no conflicts of interest.
Resources from the same session
785 - JAK-STAT inhibitor overcomes interferon γ-reduced, NK cell-mediated cytotoxicity in non-small-cell lung cancer cells
Presenter: Riki Okita
Session: Poster Display session 1
Resources:
Abstract
2326 - Low LATS2 expression is associated with poor prognosis in non small cell lung cancer
Presenter: Si-hyong Jang
Session: Poster Display session 1
Resources:
Abstract
5960 - Application of ESCAT and OncoKB scales in Liquid biopsy (LB) in Advanced NSCLC patients (pts): Is it feasible and reliable?
Presenter: Michael McCusker
Session: Poster Display session 1
Resources:
Abstract
4855 - IDH1R132H mutation induces a less aggressive phenotype of glioma cells and affects the radiosensitivity by interacting with Wnt/β-catenin signaling
Presenter: Xuetao Han
Session: Poster Display session 1
Resources:
Abstract
2641 - Impact of Angiopoietin-2 on glioblastoma response to combined chemo-radiotherapy
Presenter: Charly Helaine
Session: Poster Display session 1
Resources:
Abstract
5743 - The Discovery of RNA-aptamers That Selectively Bind and Inhibit Glioblastoma Stem Cells by targeting EphA2
Presenter: Alessandra Affinito
Session: Poster Display session 1
Resources:
Abstract
4160 - Impact of tumor reoxygenation by nanoparticles on Tumor Associated Macrophages (TAMs)
Presenter: Aurélie Ferré
Session: Poster Display session 1
Resources:
Abstract
2474 - Prognostic significance of c-Rel/p50 heterodimer in the tumor microenvironment of uveal melanoma
Presenter: Seema Kashyap
Session: Poster Display session 1
Resources:
Abstract
1769 - Synergistic role of BAP1 and DNA damage response pathway in uveal melanoma and its prognostic significance.
Presenter: JAYANTI JHA
Session: Poster Display session 1
Resources:
Abstract
5037 - CXCR4, CCR2 and CCR5 expression in subsets of tumor cells with stem and/or EMT features
Presenter: Olga Savelieva
Session: Poster Display session 1
Resources:
Abstract