¹Laboratory of Biomedical Research, Institute of Molecular and Cellular Biosciences, University of Tokyo, Tokyo 113, Japan

²Inflammation Research, Merck Research Laboratories, New Jersey 07065, USA

³Merck Frosst Centre for Therapeutic Research, Quebec, Canada H9R 4P8

⁴Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo 170, Japan

^aAuthor for correspondence

Interleukin-1-converting enzyme (ICE)/ced-3 family proteases play key roles in apoptosis. However, cellular substrates for ICE family proteases involved in apoptosis are not well understood. We previously showed that actin is cleaved in vitro by an ICE family protease, distinct from ICE itself, which is activated during VP-16-induced apoptosis. In this report, we demonstrate that the actin-cleaving ICE-family protease in the apoptotic cell extract is the activated CPP-32/apopain. CPP-32 effectively cleaves actin protein to 15 kDa and 31 kDa fragments. Studies with an antibody raised against Gly-Gln-Val-Ile-Thr peptide, the N-terminal sequence of the cleaved 15 kDa actin fragment, showed that actin is also cleaved in vivo during the development of apoptosis. Moreover, Benzyloxycarbonyl-Glu-Val-Asp-CH₂OC(O)-2,6,-dichlorobenzene (Z-EVD-CH₂-DCB), a selective inhibitor of CPP-32(-like) protease, efficiently inhibited the cleavage of actin and the apoptosis of VP-16-treated U937 cells. Our present results indicate that actin is the substrate of CPP-32/apopain(-like) protease both in vitro and in vivo and suggest the role of actin in the control of cell growth and apoptosis.

CPP-32/apopain; ICE; actin; apoptosis