Cleavage of PITSLRE Kinases by ICE/CASP-1 and CPP32/CASP-3 during Apoptosis Induced by Tumor Necrosis Factor

doi:10.1074/jbc.272.18.11694

Cleavage of PITSLRE Kinases by ICE/CASP-1 and CPP32/CASP-3 during Apoptosis Induced by Tumor Necrosis Factor*

From the Laboratory of Molecular Biology, Flanders Interuniversity Institute for Biotechnology and University of Ghent, B-9000 Ghent, Belgium and the ^§Department of Tumor Cell Biology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105

Abstract

Emerging evidence suggests that multiple aspartate-specific cysteine proteases (caspases (CASPs)) play a crucial role in programmed cell death. Many cellular proteins have been identified as their substrates and serve as markers to assay the activation of CASPs during the death process. However, no substrate has yet been unambiguously identified as an effector molecule in apoptosis. PITSLRE kinases are a superfamily of Cdc2-like kinases that have been implicated in apoptotic signaling and tumorigenesis. In this paper we report that tumor necrosis factor (TNF)-mediated apoptosis is associated with a CrmA- and Bcl-2-inhibitable cleavage of PITSLRE kinases, indicating a role for CASPs. Testing of seven murine CASPs for their ability to cleave p110 PITSLRE kinase α2-1 in vitrorevealed that only CASP-1 (ICE (interleukin-1β-converting enzyme)) and CASP-3 (CPP32) were able to produce the same 43-kDa cleavage product as observed in cells undergoing TNF-induced apoptosis. Mutational analysis revealed that cleavage of p110 PITSLRE kinase α2-1 occurred at Asp³⁹³ within the sequence YVPDS, which is similar to that involved in the CASP-1-mediated cleavage of prointerleukin-1β. TNF-induced proteolysis of PITSLRE kinases was still observed in fibroblasts from CASP-1^0/0 mice. These data implicate CASP-3 as a potentially important CASP family protease responsible for the cleavage of PITSLRE kinases during TNF-induced apoptosis.

Footnotes

↵* This work was supported by the Interuniversitaire Attractiepolen, the Fonds voor Geneeskundig Wetenschappelijk Onderzoek, the Vlaams Interuniversitair Instituut voor Biotechnologie, EC Biotech Program Grant BIO2-CT92-0316, EC Biomed Program Grant BMH4-CT96-0300, National Institutes of Health Grant GM44088, Cancer Center Core Grant CA21765, and by the American Lebanese Syrian Associated Charities (ALSAC).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ Postdoctoral Fellow with the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen.
↵¶ Research Assistant with the Fonds voor Wetenschappelijk Onderzoek-Vlaanderen.
↵‖ To whom correspondence should be addressed: Laboratory of Molecular Biology, K. L. Ledeganckstraat 35, B-9000 Ghent, Belgium. Tel.: 32-9-264-51-31; Fax: 32-9-264-53-48; E-mail:fiers{at}lmb1.rug.ac.be.
↵1 The abbreviations used are: ICE, interleukin-1β-converting enzyme; AMC, aminomethylcoumarin; CASP, caspase; IL-1β, interleukin-1β; TNF, tumor necrosis factor; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction.
↵2 R. Gururajan and V. J. Kidd, unpublished data.
- Received November 8, 1996.
- Revision received February 7, 1997.