在程序性细胞死亡过程中，CHK1的切割受到半胱天冬酶和非半胱天冬酶家族蛋白酶的复杂调控。

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

作者信息

Okita Naoyuki, Yoshimura Miyuki, Watanabe Kazuhito, Minato Shota, Kudo Yuki, Higami Yoshikazu, Tanuma Sei-ichi

机构信息

Department of Molecular Pathology and Metabolic Disease, Tokyo University of Science, Chiba, Japan.

出版信息

Biochim Biophys Acta. 2013 Jan;1830(1):2204-13. doi: 10.1016/j.bbagen.2012.10.009. Epub 2012 Oct 16.

DOI:10.1016/j.bbagen.2012.10.009

PMID:23085068

Abstract

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 (296)SNLD(299) and (348)TCPD(351), 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 (320)EPRT(323) 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 (320)EPRT(323). Additionally, the cleavage catalyzed by the (320)EPRT(323) 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 "(320)EPRT(323) protease(s)." Furthermore, (320)EPRT(323) 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.

摘要

背景

CHK1是一种调节细胞周期检查点的重要效应激酶。此前，我们发现CHK1在DNA损伤诱导的程序性细胞死亡（PCD）过程中以半胱天冬酶（CASP）依赖的方式被切割，并研究了其生理作用。

方法与结果

在本研究中，我们调查了CHK1在PCD中的行为。首先，我们发现CHK1在PCD中有三个切割位点，所有切割均被泛CASP抑制剂或丝氨酸蛋白酶抑制剂的联合处理所抑制。我们还表明，CHK1被识别（296）SNLD（299）和（348）TCPD（351）的CASP3和/或CASP7切割，并且这种切割导致CHK1激酶活性增强。此外，通过定点诱变对切割位点进行表征以及使用缺失突变体进行分析的结果，我们确定（320）EPRT（323）为另一个切割识别序列。考虑到被CASP切割的共有序列，CHK1可能被识别（320）EPRT（323）的非CASP家族蛋白酶切割。此外，当通过顺铂处理诱导同步进入G1期的U2OS细胞发生PCD时，由（320）EPRT（323）蛋白酶催化的切割显著且特异性增加。