CrmA/SPI-2 Inhibition of an Endogenous ICE-related Protease Responsible for Lamin A Cleavage and Apoptotic Nuclear Fragmentation

doi:10.1074/jbc.271.51.32487

CrmA/SPI-2 Inhibition of an Endogenous ICE-related Protease Responsible for Lamin A Cleavage and Apoptotic Nuclear Fragmentation*

From the ^‡ Institute of Cell and Molecular Biology, University of Edinburgh, Michael Swann Building, The King's Buildings, Mayfield Road, Edinburgh EH9 3JR, Scotland, United Kingdom,
^§ the Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, Florida 32610-0266, and the
^∥ Poly(ADP-Ribose) Metabolism Group, the Department of Molecular Endocrinology, Centre Hospitalier de l'Université Laval Research Center and Laval University, Sainte-Foy, Quebec, G1V 4G2, Canada

** To whom correspondence should be addressed. Tel.: 44-131-650-7101; Fax: 44-131-650-7100; E-mail: bill.earnshaw{at}ed.ac.uk

Abstract

CrmA, a poxvirus gene product with a serpin-like structure, blocks a variety of apoptotic death events in cultured cells. Based on the ability of CrmA to inhibit the interleukin-1β converting enzyme in vitro, it has been speculated that interleukin-1β converting enzyme-related proteases (caspases) essential for apoptosis are the cellular targets of CrmA. Here we found that rabbitpox virus CrmA/SPI-2 inhibits the cleavage of lamin A mediated by a caspase in our cell-free system of apoptosis. In the presence of CrmA/SPI-2, nuclear apoptosis in vitro was blocked at an intermediate stage after collapse of the chromatin against the nuclear periphery and before nuclear shrinkage and disintegration into apoptotic body-like fragments. Using N-(acetyltyrosinylvalinyl-N^ϵ-biotinyllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy] methyl ketone, which derivatizes the active forms of caspases, we could show that one of five caspases active in the extracts is inhibited both by CrmA/SPI-2 and by a peptide spanning the lamin A apoptotic cleavage site. These results reveal that CrmA/SPI-2 can inhibit a caspase responsible both for lamin A cleavage and for the nuclear disintegration characteristic of apoptosis.

Footnotes

↵^¶ Supported by a “Programa Gulbenkian de Doutoramento em Biologia e Medicina” studentship.
↵* This work was supported by a grant from the Wellcome Trust (to W. C. E.). Other support included National Institutes of Health Grants CA69008 (to W. C. E.) and AI 15722 (to R. W. M.), as well as grants from the Canadian Medical Research Council and the National Cancer Institute of Canada (to G. G. P.). 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.
↵¹ The abbreviations used are:

ICE

interleukin-1β converting enzyme

Me₂SO

dimethyl sulfoxide

MDB

mitotic dilution buffer

PARP

poly(ADP-ribose) polymerase

RPV

rabbitpox virus

YV(bio)KD-aomk

N-(acetyltyrosinylvalinyl-N^e-biotinyllysyl) aspartic acid [(2,6-dimethylbenzoyl)oxy] methyl ketone

VV

vaccinia virus

TLCK

Tos-LysCH₂Cl)1-chloro-3-tosylamido-7-amino-2-heptanone.
↵² R. W. Moyer, unpublished observations.
- Received May 20, 1996.
- Revision received September 26, 1996.