Cleavage of actin by interleukin 1(3-converting

Three of the predominant features of apoptosis are internucleosomal DNA fragmentation, plasma membrane bleb formation, and retraction ofcell processes. We demonstrate that actin is a substrate for the proapoptotic cysteine protease interleukin 1.8-converting enzyme. Actin cleaved by interleukin lp-converting enzyme can neither inhibit DNase I nor polymerize to its filamentous form as effectively as intact actin. These findings suggest a mechanism for the coordination of the proteolytic, endonucleolytic, and morphogenetic aspects of apoptosis. Programmed cell death is recognized as an integral part of the biological repertoire of multicellular organisms where an intrinsic suicide mechanism can be activated either as a response to an invading pathogen or to specific signals generated during the normal course of the development of the animal. Apoptosis is a morphologically distinct form of this death process, hallmarks of which include plasma membrane bleb formation, retraction of cellular processes (e.g., neurite retraction during neuronal apoptosis), decrease in cellular volume, cellular rounding, nuclear fragmentation, and cellular budding to produce apoptotic bodies (1). The biochemical alterations underlying these morphological changes are currently unknown. The internucleosomal fragmentation ofDNA that occurs as part of apoptosis in most cells (2, 3) has been shown to be due to DNase I (4-8), although this finding has not been universally accepted (9, 10), and evidence for the involvement of other endonucleases in apoptosis has been presented (11, 12). Programmed cell death in the nematode Caenorhabditis elegans requires the expression of the ced-3 and ced-4 genes (13). The ced-3 gene product is similar (28% of residues identical) to the mammalian cysteine protease interleukin 1,3 (IL-1p3)-converting enzyme (ICE; refs. 14-16), which was originally identified as the cytoplasmic protease that converts pro-IL-113 into the mature hormone by cleaving it at two sites, both containing an Asp residue at the P1 position (17, 18). Although many proteins have accessible Asp residues on their surfaces, the only natural protein substrate for ICE discovered to date, other than pro-IL-113, has been the precursor form of ICE itself (14, 15). These findings suggest that ICE has an unusually high substrate specificity. Recent findings demonstrated that ICE is currently the only purified and biochemically characterized member of a larger family of cysteine proteases (i.e., ICE/ced-3 family) whose other members identified to date include nedd-2 (Ich-1) and CPP32 (19-21). CrmA, a cowpox virus cytokine response modifier that specifically inhibits the proteolytic activity of ICE in vitro (22) also inhibits apoptosis when expressed in primary neuronal cultures (23), Rat-1 fibroblasts (20), and mammary epithelial cells (24). Furthermore, overexpression ofICE has been shown to induce apoptosis in Rat-1 fibroblasts (25) and HeLa cells The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 2234 (20). These and similar recent reports (19-21) with other members of the ICE/ced-3 family indicate that the proteolytic activity of ICE or an ICE-like protease is required for apoptosis. In many tissues and cell types that can undergo apoptosis but do not express IL-1,B, ICE has been shown to be constitutively expressed, often simultaneously with other members of the ICE/ced-3 family (19-21). Therefore, the expression data alone do not allow a definite conclusion as to which family member(s) may actually be involved in apoptosis. At this stage, it is also conceivable that more than one family member may jointly contribute to the death process and that the extent and nature of a possible cooperation among the family members may be dependent on the cell type and/or the apoptotic pathway activated upstream from the ICE/ced-3 family. Consistent with this possibility, recent reports demonstrate that thymocytes from mice deficient in ICE are sensitive to apoptosis induced by dexamethasone or ionizing radiation but are resistant to apoptosis induced by Fas antibodies (26, 27). If ICE is indeed one of the family members that contributes to apoptosis in at least certain cases, as suggested by studies cited above (20, 23-25, 27), then its putative apoptotic substrates are still to be identified. In the absence of any further clues about the identity of such ICE substrates, in this study we undertook a general biochemical search, utilizing a highly purified enzymatically active form of this protease.t MATERIALS AND METHODS Materials. Human recombinant ICE (p20/plO) was a generous gift of M. Tocci and N. A. Thornberry (Merck); rabbit skeletal muscle a-actin was a generous gift of E. Reisler (University of California, Los Angeles). The anti-actin monoclonal antibody was from Boehringer Mannheim; the antiactin polyclonal antibody and all protease inhibitors were from Sigma. Cell Culture. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) with 10% (vol/vol) fetal bovine serum and 5% (vol/vol) horse serum (HS) on plastic dishes. At semiconfluency cells were washed with and switched to serumfree DMEM to induce apoptosis. Preparation and ICE Treatment of Cell Extracts. Cells were washed with ice-cold PBS twice and were extracted with 1% Triton X-100 in 10 mM Hepes (pH 7.5). Supernatants were treated with ICE as follows: 50 ,ug of cellular protein extracted from semiconfluent cells grown in serum-containing medium was incubated with 50 ng of highly purified human recombinant ICE at 37°C for 2 h in the presence of 10 mM dithiothreitol. Abbreviations: IL-1f3, interleukin 1(3; ICE, IL-11,-converting enzyme; G-actin, globular actin; F-actin, filamentous actin. *To whom reprint requests should be addressed. tThis work was cited by S. J. Martin and D. R. Green (44) as in press (1995). Publication was inadvertently delayed because the galley proof was lost in the mail. Proc. Natl. Acad. Sci. USA 93 (1996) 2235 Immunoblot Analysis. Immunoblot analysis of cell extracts and purified a-actin, after ICE treatment and SDS/PAGE, was achieved by using two actin-specific antibodies, (i) a monoclonal antibody that binds to actin residues 23-34 and (ii) a polyclonal antibody that recognizes the C-terminal 11 amino acids of actin. DNase I Assay. Intact actin or actin cleaved by ICE was incubated with DNase I [2 x 10-4 unit (Sigma)] in 20 mM Tris HCl, pH 7.5/1 mM CaCl2/1 mM MgCl2 for 5 min at 20°C. Plasmid DNA [pBluescript (Stratagene), 0.5 ,tg] was added and incubation was continued for 30 min at 37°C. DNase I activity was stopped by 10 mM EDTA and products were analyzed by electrophoresis on 1% agarose gels. Actin Polymerization Assay. Intact actin (control; 10 ,tM) and ICE-cleaved actin (10 ,tM) were polymerized by the addition of 2 mM MgCl2 and monitored by light scattering of the solution at 660 nm at 23°C essentially as described (28).