The 24-kD apoptotic protease (AP24) is a serine protease that is activated during apoptosis and has the capacity to activate internucleosomal DNA fragmentation in isolated nuclei. This study examined the following: ( a ) the functional relationship between AP24 and the CPP32-like proteases of the caspase family; and ( b ) whether activation of CPP32-like proteases is sufficient to commit irreversibly a cell to apoptotic death. In three different leukemia cell lines, we showed that agents that directly (carbobenzoxy-Ala-Ala-borophe (DK120) or indirectly inhibit activation of AP24 (protein kinase inhibitors, basic fibroblast growth factor, tosylphenylalaninechloromethylketone, and caspase inhibitors) protected cells from apoptosis induced by TNF or UV light. Only the caspase inhibitors, however, prevented activation of CPP32-like activity as revealed by cleavage of the synthetic substrate, DEVD-pNa, by cell cytosols, and also by in vivo cleavage of poly (ADP-ribosyl) polymerase, a known substrate of CPP32. Activation of DEVD-pNa cleaving activity without apoptosis was also demonstrated in two variants derived from the U937 monocytic leukemia in the absence of exogenous inhibitors. Cell-permeable peptide inhibitors selective for CPP32-like proteases suppressed AP24 activation and apoptotic death. These findings indicate that CPP32-like activity is one of several upstream signals required for AP24 activation. Furthermore, activation of CPP32-like proteases alone is not sufficient to commit irreversibly a cell to apoptotic death under conditions where activation of AP24 is inhibited. Footnotes 1 Abbreviations used in this paper: ADPRT, ADP ribosyl transferase; CaM-KII, calcium/calmodulin-dependent protein kinase II; cpDEVD-CHO, cell-permeable Asp-Glu-Val-Asp-aldehyde; DEVD(meth)-cmk, side chain fully methylated carbobenzoxy-Asp-Glu-Val-Asp-chloromethylketone; ICE, interleukin-1β converting enzyme; PARP, poly (ADP-ribosyl) polymerase; SMase, sphingomyelinase; TPCK, tosylphenylalaninechloromethylketone; VAD-cmk, acetyl-Val-Ala-Asp-chloromethylketone. Submitted: 3 June 1997 Revision received 5 August 1997
The Journal of Experimental Medicine – Rockefeller University Press
Published: Oct 6, 1997
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