Direct Cleavage by the Calcium-activated Protease Calpain Can Lead to Inactivation of Caspases

Boon Tin Chua,Ke Guo,Peng Li

doi:10.1074/jbc.275.7.5131

Boon Tin Chua, Ke Guo + Show 1 more

Open Access

https://doi.org/10.1074/jbc.275.7.5131

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Abstract

Caspases, a unique family of cysteine proteases involved in cytokine activation and in the execution of apoptosis can be sub-grouped according to the length of their prodomain. Long prodomain caspases such as caspase-8 and caspase-9 are believed to act mainly as upstream caspases to cleave downstream short prodomain caspases such as caspases-3 and -7. We report here the identification of caspases as direct substrates of calcium-activated proteases, calpains. Calpains cleave caspase-7 at sites distinct from those of the upstream caspases, generating proteolytically inactive fragments. Caspase-8 and caspase-9 can also be directly cleaved by calpains. Two calpain cleavage sites in caspase-9 have been identified by N-terminal sequencing of the cleaved products. Cleavage of caspase-9 by calpain generates truncated caspase-9 that is unable to activate caspase-3 in cell lysates. Furthermore, direct cleavage of caspase-9 by calpain blocks dATP and cytochrome-c induced caspase-3 activation. Therefore our results suggest that calpains may act as negative regulators of caspase processing and apoptosis by effectively inactivating upstream caspases.

Highlights

Alterations in intracellular calcium (Ca2ϩ) homeostasis have been implicated both in promoting or inhibiting apoptosis in many cell types
Chelation of extracellular Ca2ϩ protects against apoptosis induced by glucocorticoids in thymocytes [2, 3] but leads to apoptosis in other cell types such as in cultured neuronal cells [4]
HeLa cell lysate was incubated with different amounts of recombinant calpain II at 30 °C for 20 min prior to the addition of dATP and cytochrome-c to activate caspase-3 as described by Li et al [10]

Summary

Introduction

Alterations in intracellular calcium (Ca2ϩ) homeostasis have been implicated both in promoting or inhibiting apoptosis in many cell types. HeLa cell lysate was incubated with different amounts of recombinant calpain II at 30 °C for 20 min prior to the addition of dATP and cytochrome-c to activate caspase-3 as described by Li et al [10]. To identify factors involved in Ca2ϩ-mediated apoptosis, an in vitro biochemical assay was established in which radiolabeled caspase-7 was added to MCF-7 cell lysates in the presence of Ca2ϩ (Fig. 1A).

Results

Conclusion