We demonstrate that members of the erk-encoded family of mitogen-activated protein (MAP) kinases (pp44/421w/*) and members of the rsk-encoded protein kinases (RSKs or pp9Ok) are present in the cytoplasm and nucleus of HeLa cells. Addition of growth factors to serum-deprived cells results in increased tyrosine and threonine phosphorylation and in the activation of cytosolic and nuclear MAP phosphorylation, suggesting that protein-SerlThr kinases exist upstream in the signaling pathway that regulate these enzymes and that may in turn be regulated by proteintyrosine (Tyr) kinases or protein kinase C (13, 14, 52). At present, no evidence exists for the putative pp70S6K-protein kinase. However, recent studies have identified mitogenregulated, pp90Ysk-activating protein kinases (16,51).These studies showed that a 42-kDa insulin-stimulated, microtubule-associated protein 2 (MAP2) kinase could partially reactivate protein phosphatase-inactivated S6 protein kinase II (51), a Xenopus homolog of the pp9orsk family (3,32

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We have identified and characterized a novel cysteine protease named CMH-1 that is a new member of the interleukin 1␤ converting enzyme (ICE) family of proteases with substrate specificity for Asp-X. CMH-1 has the highest similarity to CPP32 (52% amino acid identity) and MCH2 (31% identical). CMH-1 shares conserved amino acid residues that form the core structure of ICE as well as those residues involved in catalysis and in the P1 aspartate binding. Overexpression of CMH-1 in COS cells resulted in the processing of CMH-1 and the induction of apoptosis of transfected cells. Coexpression of CMH-1 with poly(ADP-ribose) polymerase (PARP) also resulted in a specific cleavage of PARP. Purified recombinant CMH-1 cleaved PARP but not interleukin 1␤ precursor in vitro.

The proteolytic cleavage of poly(ADP-ribose) polymerase (PARP) is an early biochemical event, which occurs during apoptosis. A recent study suggested that PARP cleavage can be mediated by a novel cytosolic protease (prICE) that resembles interleukin-1 beta converting enzyme (ICE), but cannot be mediated by ICE itself (Lazebnik, Y.A., Kaufmann, S.H., Desnoyers, S., Poirier, G.G., and Earnshaw, W.C. (1994) Nature 371, 346-347). We have used a COS cell co-transfection assay to investigate if ICE or any known ICE-like protease is active in PARP cleavage within the cell. Here we report that co-expression of human PARP with human ICE, or the ICE homologs TX and Nedd-2, resulted in a cleavage of PARP identical to that observed in apoptotic cells. Experiments with purified recombinant human ICE indicated that PARP polypeptide can be specifically cleaved in vitro by ICE in a time- and enzyme concentration-dependent manner. PARP cleavage, however, requires a 50-100-fold higher ICE concentration than does processing of the interleukin-1 beta precursor at an equivalent substrate concentration. The abilities of ICE, TX, and Nedd-2, when expressed at high intracellular concentrations, to cleave PARP are consistent with their induction of apoptosis in transfected cells.

Granzyme B plays an essential role in cytotoxic T lymphocyte (CTL)-mediated cell killing. Recent studies suggest that granzyme B may exert its effect by cleaving and activating CPP32, a member of the interleukin-1␤-converting enzyme/Ced-3 family of cysteine proteases. We have examined the processing and activation of CMH-1, a close homologue of CPP32, by granzyme B in vitro. We have found that granzyme B specifically cleaves CMH-1 at Asp -Ser199 between the p20 and p12 and activates the cysteine protease. Cleavage between p20 and the prosequence of CMH-1 at Asp 23 -Ala 24 is autocatalytic and is not required for CMH-1 activity in vitro. The cleavage and activation of CMH-1 by granzyme B in vitro suggest that, in addition to CPP32, CMH-1 may also play a role in CTL-mediated cell killing.