Activation of stress-activated protein kinase/c-Jun NH2-terminal kinase and p38 kinase in calphostin C-induced apoptosis requires caspase-3-like proteases but is dispensable for cell death

Apoptosis was induced in human glioma cell lines by exposure to 100 nM calp hostin C, a specific inhibitor of protein kinase C, Calphostin C-induced ap optosis was associated with synchronous down-regulation of Bcl-2 and Bcl-x( L), as well as activation of caspase-3 but not caspase-1, The exposure to c alphostin C led to activation of stress-activated protein kinase/c-Jun NH2- terminal kinase (SAPK/JNK) and p38 kinase and concurrent inhibition of extr acellular signal-regulated kinase (ERK). Upstream of ERK, Shc was shown to be activated, but its downstream Raf1 and ERR were inhibited. The pretreatm ent with acetyl-Tyr-Val-Ala-Asp-aldehyde, a relatively selective inhibitor of caspase-3, or benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fm k), a broad spectrum caspase inhibitor, similarly inhibited calphostin C-in duced activation of SAPK/JNK and p38 kinase as well as apoptotic nuclear da mages (chromatin condensation and DNA fragmentation) and cell shrinkage, su ggesting that caspase-3 functions upstream of SAPK/JNR and p38 kinase, but did not block calphostin C-induced surface blebbing and cell death. On the other hand, the inhibition of SAPK/JNK by transfection of dominant negative SAPK/JNK: and that of p38 kinase by SB203580 induced similar effects on th e calphostin C-induced apoptotic phenotypes and cell death as did z-VAD.fmk and acetyl-Tyr-Val-Ala-Asp-aldehyde, but the calphostin C-induced PARP cle avage was not changed, suggesting that SAPK/JNK and p38 kinase are involved in the DNA fragmentation pathway downstream of caspase-3. The present find ings suggest, therefore, that the activation of SAPK/JNK and p38 kinase is dispensable for calphostin C-mediated and z-VAD.fmk-resistant cell death.