TTK/hMps1 participates in the regulation of DNA damage checkpoint response by phosphorylating CHK2 on threonine 68

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• CHK2 (hCds1) is a key kinase in the DNA damage response, regulating cell cycle checkpoints by phosphorylating targets like Cdc25 and p53.
• Understanding the CHK2 signaling pathway is essential for comprehending cellular responses to DNA damage.

Purpose of the Study:

• To identify novel proteins interacting with CHK2 using a yeast two-hybrid screen.
• To elucidate the role of TTK/hMps1 in the CHK2 signaling pathway and its involvement in DNA damage-induced cell cycle arrest.

Main Methods:

• Yeast two-hybrid screening to identify CHK2-interacting proteins.
• In vitro kinase assays to assess TTK/hMps1 phosphorylation of CHK2.
• Expression of a kinase-dead TTK mutant (TTK(D647A)) to study its effects on cell cycle arrest.
• Small interfering RNA (siRNA) to ablate TTK expression and analyze downstream effects.

Main Results:

• TTK/hMps1 was identified as a novel binding partner of CHK2.
• TTK/hMps1 directly phosphorylates CHK2 at Thr-68 in vitro.
• Expression of a dominant-negative TTK mutant impaired G(2)/M arrest induced by ionizing radiation and UV light.
• TTK ablation via siRNA reduced CHK2 Thr-68 phosphorylation and impaired DNA damage-induced growth arrest.
• Downstream events of CHK2, including cyclin B1 accumulation and Cdc2 Tyr-15 phosphorylation, were affected by TTK manipulation.

Conclusions:

• TTK/hMps1 acts upstream of CHK2 in the DNA damage response pathway.
• TTK/hMps1 plays a critical role in mediating DNA damage-induced cell cycle arrest.
• These findings suggest functional cross-talk between the spindle assembly checkpoint and the DNA damage checkpoint pathways.