The Bax subfamily of Bcl2-related proteins is essential for apoptotic signal transduction by c-Jun NH(2)-terminal kinase - PubMed (original) (raw)

Kui Lei et al. Mol Cell Biol. 2002 Jul.

Abstract

Targeted gene disruption studies have established that the c-Jun NH(2)-terminal kinase (JNK) signaling pathway is required for stress-induced release of mitochondrial cytochrome c and apoptosis. Here we demonstrate that activated JNK is sufficient to induce rapid cytochrome c release and apoptosis. However, activated JNK fails to cause death in cells deficient of members of the Bax subfamily of proapoptotic Bcl2-related proteins. Furthermore, exposure to stress fails to activate Bax, cause cytochrome c release, and induce death in JNK-deficient cells. These data demonstrate that proapoptotic members of the Bax protein subfamily are essential for JNK-dependent apoptosis.

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Figures

FIG. 1.

FIG. 1.

Characterization of MKK7-JNK fusion proteins. (A) Plasmid expression vectors were constructed to express fusion proteins with an NH2-terminal epitope tag (Flag). The structure of the fusion proteins is schematically illustrated. The MKK7-JNK fusion proteins contain residues 1 to 443 of MKK7β2 fused to JNK1α1, JNK2α2, and JNK3α2. The JNK1-MKK7 fusion protein contains residues 1 to 415 of JNK1α2 fused to MKK7β2. Point mutations were used to create kinase-negative MKK7 (Lys149 replaced with Ala) and phosphorylation-negative JNK (Thr180-Pro-Tyr182 replaced with Ala-Pro-Phe). (B) Measurement of JNK protein kinase activity. JNK1, JNK1 plus MKK7, and the MKK7-JNK fusion proteins were expressed in CHO cells and were detected by immunoblot (IB) analysis using an antibody to the Flag epitope tag (lower panel). Protein kinase assays were performed using an immune complex assay, with c-Jun and [γ-32P]ATP as the substrates. The reaction products were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The c-Jun protein was detected by Coomassie staining (middle panel) and phosphorylated c-Jun was detected by autoradiography (upper panel). The relative kinase activity was quantitated by phosphorimager analysis. The numbers on the left correspond to the migration of molecular mass standards (in kilodaltons). (C) The activation state of JNK was examined by immunoblot analysis using an antibody to phospho-JNK. The expression of JNK1, MKK7, and the MKK7-JNK fusion proteins in CHO cell lysates was examined by immunoblot analysis using an antibody to the Flag epitope tag (lower panel) and to phospho-JNK (upper panel). (D) Comparison of the wild-type MKK7-JNK1 fusion protein with the catalytically inactive mutant proteins MKK7(K>A)-JNK1 and MKK7-JNK1(APF). Upper panel, the phosphorylation of c-Jun was examined in an in vitro Flag-immune complex protein kinase assay. c-Jun was detected by staining with Coomassie blue. Phosphorylated c-Jun was detected by autoradiography. Lower panel, the activation state of JNK was examined by immunoblot analysis by probing with antibodies to the epitope tag (Flag) and phospho-JNK.

FIG. 2.

FIG. 2.

The MKK7-JNK fusion proteins increase AP-1 transcription activity. (A, C) AP-1 transcription activity was examined in a transfection assay using a reporter plasmid containing four AP-1 binding sites. CHO cells were cotransfected with the reporter plasmid p4XTRE-luciferase, a β-galactosidase expression vector, and the indicated plasmid expression vectors. The normalized luciferase activity detected in the cell lysates is presented. The data shown are the means ± SD for three independent experiments. (B, D) ATF2 transcription activity was monitored in transfection assays using a GAL4-ATF2 fusion protein. CHO cells were cotransfected with Gal4-ATF2(1-109), reporter plasmid pG5E1bLuc, a β-galactosidase expression vector, and the indicated plasmids. The normalized luciferase activity detected in the cell lysates is presented. The data shown are the means ± SD for three independent experiments. (E) Immunoblot analysis of transfected CHO cells. Triplicate samples from one experiment (panel C) were probed with antibody to JNK1/2 (lower panel) and phospho-JNK (upper panel).

FIG. 3.

FIG. 3.

Activated JNK causes cell death. (A) CHO cells were cotransfected with a GFP expression vector together with expression vectors for the MKK7-JNK1 fusion protein (wild type and phosphorylation defective) or an active fragment of the BH3-only protein Bid (tBid). The cells were examined at 40 h posttransfection by fluorescence microscopy. The data presented are representative of three independent experiments. (B) CHO cells were transfected with empty vector or with the indicated expression plasmids. The cells were harvested after 40 h and stained with crystal violet. The data shown are the means ± SD for three independent experiments. (C, D) CHO cells were cotransfected with the indicated expression vectors and a luciferase expression vector (pCDNA3-Luc) to monitor cell viability. The cells were harvested at 40 h posttransfection and luciferase activity was measured. The data shown are the means ± SD for three independent experiments.

FIG. 4.

FIG. 4.

JNK-stimulated apoptosis is inhibited by Akt. (A) CHO cells were transfected with vector control, Akt, or activated Akt∗ (myristoylated Akt) together with the indicated plasmid expression vectors. Decreased cell survival caused by activated JNK (MKK7-JNK) was prevented by activated Akt. (B) The cell lysates were examined by immunoblot analysis using antibodies to Akt and phospho-Ser473-Akt.

FIG. 5.

FIG. 5.

Activated JNK causes decreased ERK activity. (A) CHO cells were transfected with an empty expression vector (control) or with an expression vector for activated MEK1 (HA-ΔN3-MEK1 [S218E, S222D]) together with the indicated expression vectors. The cells were harvested at 40 h posttransfection. Endogenous ERK1 and ERK2 were examined by immunoblot analysis using an antibody to ERK and phospho-ERK. The expression of transfected MEK1 was examined by immunoblot analysis using an antibody to the HA epitope tag. (B) Activated JNK does not cause decreased p38 MAP kinase activity. CHO cells were transfected with an empty expression vector (control) or with an expression vector for activated MKK6 (Flag-MKK6 [S207E, T211E]) together with the indicated expression vectors. The cells were harvested at 40 h posttransfection. Endogenous p38 MAP kinase was examined by immunoblot analysis using an antibody to p38 and phospho-p38.

FIG. 6.

FIG. 6.

Activated JNK causes apoptosis. (A) Cell lysates prepared at 40 h posttransfection were examined by immunoblot analysis using antibodies to the Flag epitope tag (lower panel), activated caspase-3 (middle panel), and caspase-3 (upper panel). Equal amounts of total cell lysate (50 μg) were examined in each lane. The effect of exposure of the cells to UV radiation (80 J/m2) 12 h prior to harvesting was examined. The numbers on the left correspond to the migration of molecular mass standards (in kilodaltons). (B) CHO cells were transfected with empty vector (control) or the indicated expression vectors. Nucleosomal DNA fragmentation was examined at 40 h posttransfection. The data presented are the means ± SD of the results obtained for three independent experiments. (C) Activated JNK causes cytochrome c release from the mitochondria. CHO cells were cotransfected with GFP and the indicated expression vectors. The cells were fixed after 24 h, processed for immunofluorescence analysis, and stained with DAPI. GFP (green), DNA (blue), and cytochrome c (red) are illustrated. The cells were incubated with 100 μM zVAD-fmk to inhibit apoptosis. Transfected cells (green) show punctate mitochondrial cytochrome c (red) in experiments using the kinase-negative fusion protein MKK7-JNK1(APF). Expression of tBid or MKK7-JNK1 caused the release of cytochrome c from the mitochondria and the redistribution of cytochrome c throughout the cytoplasm and nucleus.

FIG. 7.

FIG. 7.

JNK causes rapid release of cytochrome c and apoptosis. (A) Activated JNK causes cytochrome c release from the mitochondria. CHO cells were microinjected with the MKK7-JNK1 expression plasmid together with dog IgG. The injected cells were visualized by staining with fluorescein-conjugated anti-dog IgG (green, indicated by arrowheads) and cytochrome c was stained with mouse anti-cytochrome c and rhodamine-conjugated goat anti-mouse antibodies. Cells releasing cytochrome c showed diffuse cytoplasmic and nuclear staining (red). Punctate mitochondrial staining was observed in the noninjected cells. (B) Time course of apoptosis induced by microinjection of MKK7-JNK1. CHO cells were microinjected with the indicated plasmids and apoptosis was evaluated by membrane blebbing and cell detachment. The data presented represent the means ± SD of data obtained for four independent experiments.

FIG. 8.

FIG. 8.

Regulation of Bcl2 by activated JNK. (A) Activated JNK causes Bcl2 phosphorylation in vivo. CHO cells were transfected without and with a plasmid expression vector for human Bcl2 (20 ng). The cells were cotransfected with the empty expression vector or expression vectors for JNK1 and MKK7-JNK1 fusion proteins. The cells were harvested at 40 h posttransfection and the expression of Bcl2 was examined by immunoblot analysis. (B) Bcl2 phosphorylation is suppressed by increased expression of Bcl2. CHO cells were cotransfected with an expression vector for the Flag-MKK7-JNK1 fusion protein together with increasing amounts of an expression vector for human Bcl2 (0, 10, 20, 40, 60, and 80 ng). The cells were harvested at 40 h posttransfection and the expression of Bcl2 and Flag-MKK7-JNK1 was examined by immunoblot analysis. (C) CHO cells were transfected with an empty vector (control) or an expression vector for the MKK7-JNK1 fusion protein. The effect of expression of Bcl2 was examined (20 ng of plasmid). Cell viability at 40 h posttransfection was examined using a luciferase reporter plasmid. The data presented represent the means ± SD of data obtained for three independent experiments.

FIG. 9.

FIG. 9.

The proapoptotic Bcl2 family protein Bid is not required for JNK-dependent apoptosis. (A) Bid is not required for JNK-dependent apoptosis. Wild-type and _Bid_−/− mouse fibroblasts were transfected with empty vector (control) or expression vectors for MKK7-JNK fusion proteins. Cell viability at 40 h posttransfection was examined using a luciferase reporter plasmid. The data presented represent the means ± SD of data obtained for three independent experiments. (B) Endogenous JNK is not required for apoptosis caused by ectopic expression of activated JNK. Wild-type and _Jnk1_−/− _Jnk2_−/− mouse fibroblasts were transfected with empty vector (control) or expression vectors for MKK7-JNK fusion proteins. Cell viability at 40 h posttransfection was examined using a luciferase reporter plasmid. The data presented represent the means ± SD of data obtained for three independent experiments.

FIG. 10.

FIG. 10.

The proapoptotic Bcl2 family proteins Bax and Bak are required for JNK-dependent apoptosis. (A) Bax and Bak are required for JNK-dependent apoptosis. Wild-type and _Bax_−/− _Bak_−/− mouse fibroblasts were transfected with empty vector (control) or expression vectors for JNK1 or MKK7-JNK fusion proteins. Cell viability at 40 h posttransfection was examined using a luciferase reporter plasmid. The data presented represent the means ± SD of data obtained for three independent experiments. (B) Endogenous JNK is not required for apoptosis caused by Bax and Bak. Wild-type and _Jnk1_−/− _Jnk2_−/− mouse fibroblasts were transfected with empty vector (control) or expression vectors for Bax, Bak, or tBid. Cell viability at 40 h posttransfection was examined using a luciferase reporter plasmid. The data presented represent the means ± SD of data obtained for three independent experiments. (C) Bax is not activated by UV radiation in JNK-deficient murine fibroblasts. Wild-type and _Jnk1_−/− _Jnk2_−/− fibroblasts were exposed to UV radiation (80 J/m2) and incubated (24 h). The cells were fixed and stained with a conformation-specific antibody to Bax and Texas Red-conjugated goat anti-mouse antibody. The Bax antibody stains activated and membrane-targeted Bax but not cytoplasmic Bax. DNA was stained with DAPI (blue).

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