Chk1-Mad2 interaction

Abstract

Chk1 is implicated in several checkpoints of the cell cycle acting as a key player in the signal transduction pathway activated in response to DNA damage and crucial for the maintenance of genomic stability. Chk1 also plays a role in the mitotic spindle checkpoint, which ensures the fidelity of mitotic segregation during mitosis, preventing chromosomal instability and aneuploidy. Mad2 is one of the main mitotic checkpoint components and also exerts a role in the cellular response to DNA damage. To investigate a possible crosslink existing between Chk1 and Mad2, we studied Mad2 protein levels after Chk1 inhibition either by specific siRNAs or by a specific and selective Chk1 inhibitor (PF-00477736), and we found that after Chk1 inhibition, Mad2 protein levels decrease only in tumor cells sensitive to Chk1 depletion. We then mapped six Chk1’s phosphorylatable sites on Mad2 protein, and found that Chk1 is able to phosphorylate Mad2 in vitro on more than one site, while it is incapable of phoshorylating the Mad2 form mutated on all six phosphorylatable sites. Moreover our studies demonstrate that Chk1 co-localizes and physically associates with Mad2 in cells both under unstressed conditions and after DNA damage, thus providing new and interesting evidence on Chk1 and Mad2 crosstalk in the DNA damage checkpoint and in the mitotic spindle checkpoint.

Keywords: :

• Chk1
• Mad2
• DNA damage checkpoint
• mitotic spindle checkpoint

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The generous contribution of the Italian Association for Cancer Research (AIRC), Milan, Italy, is gratefully acknowledged. We thank J.D. Baggott that kindly edited the paper. Thanks to Alessandra Basana, an undergraduate student that helped with interest and passion while she was learning how to work in a laboratory. A special thanks to Faina Vikhanskaya, who has always pushed me up in performing one more additional recombinant protein extraction and kinase assay after every bad result. She will always be a great example of scientist to me.

Supplemental Materials

Supplemental materials may be found here: 
www.landesbioscience.com/journals/cc/article/24090