Oops, you're using an old version of your browser so some of the features on this page may not be displaying properly.

MINIMAL Requirements: Google Chrome 24+Mozilla Firefox 20+Internet Explorer 11Opera 15–18Apple Safari 7SeaMonkey 2.15-2.23

Poster Display session 1

4793 - Bub3 is phosphorylated by the Ataxia-Telangiectasia Mutated Kinase in mitosis and required for activation of the mitotic spindle checkpoint in Breast Cancer

Date

28 Sep 2019

Session

Poster Display session 1

Presenters

Mingming Xiao

Citation

Annals of Oncology (2019) 30 (suppl_5): v1-v24. 10.1093/annonc/mdz238

Authors

M. Xiao, B. Xu

Author affiliations

  • Key Laboratory Of Breast Cancer Prevent And Therapy, Tianjin Medical University Cancer Institute & Hospital, 300060 - Tianjin/CN
More

Resources

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.

This site uses cookies. Some of these cookies are essential, while others help us improve your experience by providing insights into how the site is being used.

For more detailed information on the cookies we use, please check our Privacy Policy.

Customise settings