Extracellular signal-regulated kinase (ERK) 1/2 signaling is involved in tumor cell survival through the regulation of Bcl-2 family members. To explore this further and to demonstrate the central role of the mitochondria in the ERK1/2 pathway we used the HeLa cellular model where apoptosis was induced by tumor necrosis factor (TNF) and cycloheximide (CHX). We show that HeLa cells overexpressing ERK-1 displayed resistance to TNF and CHX. HeLa cells overexpressing a kinase-deficient form of ERK-1 (K71R) were more sensitive to TNF and CHX. In the ERK-1 cells, Bad was phosphorylated during TNF + CHX treatment. In the HeLa wt cells and in the K71R clones TNF and CHX decreased Bad phosphorylation. ERK-1 cells treated with TNF and CHX did not release cytochrome c from the mitochondria. By contrast, HeLa wt and K71R clones released cytochrome c. Bax did not translocate to the mitochondria in ERK-1 cells treated with TNF + CHX. Conversely, HeLa wt and K71R clones accumulated Bax in the mitochondria. In the HeLa wt cells and in both ERK-1 transfectants Bid was cleaved and accumulated in the mitochondria. The caspase-8 inhibitor IETD-FMK and the mitochondrial membrane permeabilization inhibitor bongkrekic acid (BK), partially prevented cell death by TNF + CHX. Anisomycin, a c-Jun N-terminal kinases activator, increased TNF-killing. The ERK-1 cells were resistant to TNF and anisomycin, whereas K71R clones resulted more sensitive. Our study demonstrates that in HeLa cells the ERK-1 kinase prevents TNF + CHX apoptosis by regulating the intrinsic mitochondrial pathway through different mechanisms. Inhibition of the intrinsic pathway is sufficient to almost completely prevent cell death. © 2009 Wiley-Liss, Inc.

ERK-1 MAP kinase prevents TNF-induced apoptosis through bad phosphorylation and inhibition of bax translocation in HeLa cells

Russo, Matteo A.;
2009-01-01

Abstract

Extracellular signal-regulated kinase (ERK) 1/2 signaling is involved in tumor cell survival through the regulation of Bcl-2 family members. To explore this further and to demonstrate the central role of the mitochondria in the ERK1/2 pathway we used the HeLa cellular model where apoptosis was induced by tumor necrosis factor (TNF) and cycloheximide (CHX). We show that HeLa cells overexpressing ERK-1 displayed resistance to TNF and CHX. HeLa cells overexpressing a kinase-deficient form of ERK-1 (K71R) were more sensitive to TNF and CHX. In the ERK-1 cells, Bad was phosphorylated during TNF + CHX treatment. In the HeLa wt cells and in the K71R clones TNF and CHX decreased Bad phosphorylation. ERK-1 cells treated with TNF and CHX did not release cytochrome c from the mitochondria. By contrast, HeLa wt and K71R clones released cytochrome c. Bax did not translocate to the mitochondria in ERK-1 cells treated with TNF + CHX. Conversely, HeLa wt and K71R clones accumulated Bax in the mitochondria. In the HeLa wt cells and in both ERK-1 transfectants Bid was cleaved and accumulated in the mitochondria. The caspase-8 inhibitor IETD-FMK and the mitochondrial membrane permeabilization inhibitor bongkrekic acid (BK), partially prevented cell death by TNF + CHX. Anisomycin, a c-Jun N-terminal kinases activator, increased TNF-killing. The ERK-1 cells were resistant to TNF and anisomycin, whereas K71R clones resulted more sensitive. Our study demonstrates that in HeLa cells the ERK-1 kinase prevents TNF + CHX apoptosis by regulating the intrinsic mitochondrial pathway through different mechanisms. Inhibition of the intrinsic pathway is sufficient to almost completely prevent cell death. © 2009 Wiley-Liss, Inc.
2009
Anisomycin; Bad phosphorylation; Bax; Erk-1; TNF; Amino Acid Substitution; Apoptosis; Caspase 8; Cycloheximide; Gene Targeting; HeLa Cells; Humans; JNK Mitogen-Activated Protein Kinases; Mitochondria; Mitogen-Activated Protein Kinase 3; Phosphorylation; Protein Transport; Signal Transduction; Tumor Necrosis Factors; bcl-2-Associated X Protein; bcl-Associated Death Protein; Biochemistry; Cell Biology; Molecular Biology
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.12078/2990
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