CHK1 cleavage in programmed cell death is intricately regulated by both caspase and non-caspase family proteases

Background

CHK1 is an important effector kinase that regulates the cell cycle checkpoint. Previously, we showed that CHK1 is cleaved in a caspase (CASP)-dependent manner during DNA damage-induced programmed cell death (PCD) and have examined its physiological roles.

Methods and results

In this study, we investigated the behavior of CHK1 in PCD. Firstly, we found that CHK1 is cleaved at three sites in PCD, and all cleavages were inhibited by the co-treatment of a pan-CASP inhibitor or serine protease inhibitors. We also showed that CHK1 is cleaved by CASP3 and/or CASP7 recognizing at ²⁹⁶SNLD²⁹⁹ and ³⁴⁸TCPD³⁵¹, and that the cleavage results in the enhancement of CHK1 kinase activity. Furthermore, as a result of the characterization of cleavage sites by site-directed mutagenesis and an analysis performed using deletion mutants, we identified ³²⁰EPRT³²³ as an additional cleavage recognition sequence. Considering the consensus sequence cleaved by CASP, it is likely that CHK1 is cleaved by non-CASP family protease(s) recognizing at ³²⁰EPRT³²³. Additionally, the cleavage catalyzed by the ³²⁰EPRT³²³ protease(s) markedly and specifically increased when U2OS cells synchronized into G1 phase were induced to PCD by cisplatin treatment.

Conclusion

CHK1 cleavage is directly and indirectly regulated by CASP and non-CASP family proteases including serine protease(s) and the “³²⁰EPRT³²³ protease(s).” Furthermore, ³²⁰EPRT³²³ cleavage of CHK1 occurs efficiently in PCD which is induced at the G1 phase by DNA damage.

General significance

CASP and non-CASP family proteases intricately regulate cleavage for up-regulation of CHK1 kinase activity during PCD.