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

Abstract

Apoptosis was induced in human glioma cell lines by exposure to 100 nM calphostin C, a specific inhibitor of protein kinase C. Calphostin C-induced apoptosis 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 calphostin C led to activation of stress-activated protein kinase/c-Jun NH 2 -terminal kinase (SAPK/JNK) and p38 kinase and concurrent inhibition of extracellular signal-regulated kinase (ERK). Upstream of ERK, Shc was shown to be activated, but its downstream Raf1 and ERK were inhibited. The pretreatment with acetyl-Tyr-Val-Ala-Asp-aldehyde, a relatively selective inhibitor of caspase-3, or benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk), a broad spectrum caspase inhibitor, similarly inhibited calphostin C-induced activation of SAPK/JNK and p38 kinase as well as apoptotic nuclear damages (chromatin condensation and DNA fragmentation) and cell shrinkage, suggesting that caspase-3 functions upstream of SAPK/JNK 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 the calphostin Cinduced apoptotic phenotypes and cell death as did z-VAD.fmk and acetyl-Tyr-Val-Ala-Asp-aldehyde, but the calphostin C-induced PARP cleavage was not changed, suggesting that SAPK/JNK and p38 kinase are involved in the DNA fragmentation pathway downstream of caspase-3. The present findings suggest, therefore, that the activation of SAPK/JNK and p38 kinase is dispensable for calphostin C-mediated and z-VAD.fmk-resistant cell death.It has been expected that signal transduction pathways involving specific protein kinases are involved in mediating apoptosis. Extracellular signal-regulated kinase (ERK) 1 is most strongly stimulated by activation of protein-tyrosine kinase receptors (1) and also activated by both Ras-dependent (2-4) and Ras-independent signalings (5) in response to activation of G protein-coupled receptors, and common intermediates in intracellular signaling cascades are involved in diverse cellular functions including growth and differentiation (6). Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate results in the activation of Raf1 (7) and ERK (8 -11) within minutes, suggesting the involvement of PKC in the signaling pathway leading to ERK activation. In addition, Ras functions as an essential transducer of various physiological signals leading to cell growth and proliferation in a PKC-dependent or PKC-independent manner (12), and activated Ras also renders cells susceptible to apoptosis after depression of PKC activity (13,14).SAPK/JNK and p38 kinase have been proposed to mediate apoptosis, but a number of reports have challenged the notion that the activation of SAPK/JNK and/or p38 kinase is sufficient to induce apoptosis (15-21), and the integration and balance of SAPK/JNK and p38 pathways probably contribute to commitment to a...

Calphostin C-mediated translocation and integration of Bax into mitochondria induces cytochrome c release before mitochondrial dysfunction

“…Bcl-2 and Bcl-x L were downregulated nearly synchronously as early as 1 h after treatment with 100 nM calphostin C in both U-87MG and T98G cells ( Figure 1) and were barely detected as early as 4 h in U-87MG cells and 8 h in T98G cells after calphostin C treatment. 11 In addition, 29 kDa Bcl-x L isoform was denser than 31 kDa one before calphostin C treatment whereas both Bcl-x L isoforms became similar in density with each other as early as 2 h after 100 nM calphostin C treatment, indicating a mobility shift of Bcl-x L .…”

“…11 Immunoreactive 32 kDa procaspase-3 was degraded into active 12 kDa caspase-3 fragment as early as 6 h in U-87MG cells and 4 h in T98G cells after treatment with 100 nM calphostin C, when cytochrome c was released maximally from mitochondria. The generation of a COOHterminal 85 kDa PARP apoptosis fragment was shown to be associated with the production of active 12 kDa caspase-3 fragment ( Figure 5).…”

“…10,11 Calphostin C is a microbial compound isolated from Cladosporium cladosporioides and a highly potent and specific inhibitor of protein kinase C, interacting not with the catalytic domain, but with the regulatory domain. 12 Bcl-2 family proteins can have profound effects on apoptosis and play a pivotal role in controlling life and death.…”

“…Proapoptotic Bax plays a crucial role in the apoptotic process via a number of different mechanism, 13 ± 16 and the genetic approach using knockout mice has recently indicated that Bax can clearly promote apoptosis in the absence of Bcl-2. 17 The pretreatment with a pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk), inhibited calphostin C-induced activation of caspase-3 as well as apoptotic nuclear damages (chromatin condensation and DNA fragmentation) and cell shrinkage but did not block calphostin C-induced surface blebbing and cell death, 11 suggesting the involvement of Bax in calphostin C-induced apoptosis as indicated by Xiang et al 18 Bax has been shown to have both cytosolic and membrane-associated location. Death signals such as IL-3 withdrawal, staurosporine, dexamethasone, and girradiation induce the translocation of Bax protein from cytosol to mitochondrial membrane 15,19,20 ± 22 and subsequent all mitochondrial hallmarks of apoptosis.…”

A PI3K Pathway Mediates Hair Cell Survival and Opposes Gentamicin Toxicity in Neonatal Rat Organ of Corti

“…For all inhibitors used, the concentration was based on the dosages that produce robust suppression of activity mediated by the factor in question in other cell types (Kobayashi et al 1989;Ozaki et al 1999;da Rocha et al 2002;Montcouquiol and Corwin 2001;Vlahos et al 1994;Brunn et al 1996;Jin et al 1994;Kozikowski et al 2003). Unfortunately, no specific inhibitor of PDK1 is commercially available.…”