Inhibition of protein degradation induces apoptosis through a microtubule-associated protein 1 light chain 3-mediated activation of caspase-8 at intracellular membranes - PubMed (original) (raw)

Inhibition of protein degradation induces apoptosis through a microtubule-associated protein 1 light chain 3-mediated activation of caspase-8 at intracellular membranes

Ji-An Pan et al. Mol Cell Biol. 2011 Aug.

Abstract

The accumulation of damaged or misfolded proteins, if unresolved, can lead to a detrimental consequence within cells termed proteotoxicity. Since cancerous cells often display elevated protein synthesis and by-product disposal, inhibition of the protein degradation pathways is an emerging approach for cancer therapy. However, the molecular mechanism underlying proteotoxicity remains largely unclear. We show here that inhibition of proteasomal degradation results in an increased oligomerization and activation of caspase-8 on the cytosolic side of intracellular membranes. This enhanced caspase-8 oligomerization and activation are promoted through its interaction with the ubiquitin-binding protein SQSTM1/p62 and the microtubule-associated protein light chain 3 (LC3), which are enriched at intracellular membranes in response to proteotoxic stress. Silencing LC3 by shRNA, or the LC3 mutants defective in membrane localization or p62 interaction fail to induce caspase-8 activation and apoptosis. Our results unveiled a previously unknown mechanism through which disruption of protein homeostasis induces caspase-8 oligomerization, activation, and apoptosis.

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Figures

Fig. 1.

MG132 induces caspase-8-dependent apoptosis in human breast cancer cells. Human breast cancer cells—MDA-MB-231, MDA-MB-468, and MCF-7—were treated with MG132 (0.5 μM). At the indicated time points, cells were collected. (A) Cell death was measured by PI exclusion. The averages of four independent assays ± the SEM are shown. (B) Cell lysates were examined by immunoblotting with the indicated antibodies. (C) The activities of caspase-8 were assayed by using the Caspase-Glo 8 luminogenic assay kit. The fold increase of caspase-8 activity relative to that of the respective untreated cells is shown. The average of four independent assays ± the SEM is shown. (D to F) Cells were infected with lentiviral nontargeting control (shNTC) and caspase-8 (shC8) shRNAs. Cells with caspase-8 knockdown were treated with MG132 (0.5 μM). (D) Knockdown of the caspases was verified by immunoblotting. (E) Caspase-8 activity was measured using the Caspase-Glo 8 luminogenic assay kit. The fold increase in the caspase-8 activity relative to that of the respective untreated cells is shown. The averages of four independent assays ± the SEM are shown. **, P < 0.01. (F) Cell death was measured by PI exclusion. The averages of four independent assays ± the SEM are shown. *, P < 0.05.

Fig. 2.

MG132 induces caspase-dependent apoptosis in cells deficient in mitochondrial apoptosis. _bax_−/− _bak_−/− BMK cells were treated with indicated concentration of MG132. (A) At the indicated time points cells were collected and cell death measured by PI exclusion. The averages of three independent assays ± the SEM are shown. (B) Cells untreated or treated with MG132 for 24 h were stained with Hoechst 33342 and observed with a fluorescence microscope. Representative images are shown. Note the nuclear condensation and fragmentation in MG132-treated cells. (C) Cells untreated or treated with MG132 for 24 h were lysed and probed for indicated proteins by immunoblotting. (D) The pan-caspase inhibitor z-VAD was added to cell culture. Note that z-VAD protected cells from MG132 treatment. The averages of three independent assays ± the SEM are shown. (E) Upon treatment of MG132 at indicated time points, caspase-8 activity was assayed using the Caspase-Glo 8 luminogenic assay kit. The averages of three independent assays plus the SEM are shown.

Fig. 3.

MG132 induces caspase-8 oligomerization and aggregation. (A) Schematic representation of the full-length and truncated caspase-8 BiFC constructs. (B) _bax_−/− _bak_−/− BMK cells were transiently transfected with plasmids encoding C8 (C360S)-VN and C8 (C360S)-VC for 18 h, followed by treatment with MG132 for an additional 18 h. Representative fluorescence images were taken. (C) Cells untransfected, or transfected with the Venus pairs of C8 (C360S) were left untreated or were treated with MG132 for 18 h. The percentages of Venus green cells were analyzed by flow cytometry. The number of Venus-positive cells in MG132-treated samples was normalized against that of the untreated samples. The average of four experiments plus the SEM are shown. (D) Lysates from untreated or MG132-treated _bax_−/− _bak_−/− BMK cells were resolved by size exclusion chromatography on a Superdex 200 column. Eluate was collected in 0.5-ml fractions, concentrated by acetone precipitation, and probed for indicated proteins. The molecular masses of the protein standards used to indicate the approximate sizes of the fractions are indicated in kilodaltons.

Fig. 4.

Proteasome inhibition promotes apoptosis by enhancing the interaction of caspase-8 and SQSTM1/p62. (A) _bax_−/− _bak_−/− BMK cells were transiently transfected with plasmid encoding His-tagged ubiquitin (His-Ub). At 24 h posttransfection, cells were treated with MG132 for the indicated periods of time and subjected to His pulldown. The pulldown samples were probed with an anti-His and an anti-caspase-8 antibody. The asterisk indicates a nonspecific band. The samples subjected to His pulldown were probed with an anti-caspase-8 antibody. (B) _bax_−/− _bak_−/− BMK cells were left untreated or were treated with MG132. Cell lysates were subjected to IP with rat IgG or an anti-caspase-8 antibody. IP samples and cell lysates were probed for caspase-8 or p62. (C) _bax_−/− _bak_−/− BMK cells stably expressing Flag-p62 were left untreated or were treated with MG132 for 18 h. Immunofluorescence analysis was performed using anti-Flag (green) and anti-caspase-8 (red) antibodies. DAPI was used to visualize the nucleus. (D) _bax_−/− _bak_−/− BMK cells were left untreated or were treated with MG132 for 18 h. Immunofluorescence was performed using anti-p62 (green) and anti-caspase-8 (red) antibodies. DAPI was used to visualize the nucleus. (E) _bax_−/− _bak_−/− BMK cells were stably infected with lentivirus encoding nontarget control shRNA (shNTC), caspase-8 shRNA (shC8), or p62 shRNA (shp62). The expression of p62 or caspase-8 was examined by immunoblotting. (F and G) Cells with different shRNAs were treated with MG132 (0.5 μM). Cell death was determined by PI exclusion (G) or immunoblotting (G). Note that the lower amount of cleaved PARP and C3 in shNTC at 48 h is due to excessive cell death. The averages of four independent assays plus the SEM is shown for the cell death curve. (H to J) Characterization of the p62 dependence of MG132-induced apoptosis. (H) Wild-type p62 and mutants deficient in dimerization (D69A), ubiquitin binding (I431A), and LC3-interaction (Δ321-342) were expressed in _bax_−/− _bak_−/− BMK cells stably expressing shp62. (I) Cells were treated with MG132 (0.5 μM) for the indicated periods of time, and cell death was determined by PI exclusion. The averages of four experiments ± the SEM are shown. (J) Cell lysates were probed for cleaved caspase-3 (C-C3) and tubulin.

Fig. 4.

Fig. 5.

LC3 forms a complex with p62 and caspase-8, which is enhanced by proteasome inhibition. (A) Caspase-8, p62, and LC3 form a complex, which is enhanced by proteasome inhibitor treatment. HEK293 cells were transfected with plasmids encoding Flag-caspase-8, together with GFP-LC3 or a GFP control (GFP-tPLD). At 24 h posttransfection, cells with GFP-LC3 were treated with MG132 for the indicated periods of time. Cell lysates were subjected to IP with GFP or Flag antibodies and probed for the indicated proteins. (B) LC3 colocalizes with caspase-8. _bax_−/− _bak_−/− BMK cells stably expressing Flag-p62 and GFP-LC3, untreated or treated with MG132 for 18 h, were stained with an anti-caspase-8 antibody. DAPI was used to visualize the nucleus. (C) LC3 colocalizes with p62. Flag-p62 was transfected into _bax_−/− _bak_−/− BMK cells expressing GFP-LC3. Cells were left untreated or were treated with MG132 (0.5 μM) for 18 h. Immunofluorescence analysis was performed using an anti-Flag antibody. DAPI was used to visualize the nucleus. (D) p62 knockdown abolishes caspase-8 and LC3 interaction. Lentivirus encoding control shRNA (shNTC) or p62 shRNA (shp62) were used to infect _bax_−/− _bak_−/− BMK cells stably expressing HA-caspase-8 (C360S) and GFP-LC3. Cells were treated with MG132 for indicated periods of time. Cell lysates were subjected to IP with GFP and probed for indicated proteins.

Fig. 6.

LC3 promotes MG132-induced apoptosis. (A) GFP or GFP-LC3 was expressed in _bax_−/− _bak_−/− BMK cells. Cells were treated with indicated concentration of MG132, and cell death was determined by PI exclusion. The averages of four independent assays + the SEM are shown. (B) Cells expressing GFP or GFP-LC3 were treated with MG132 and probed for the indicated proteins. Note that the apoptotic cleavage of caspase-3 and PARP was more pronounced in GFP-LC3 cells. Note that the lower amount of cleaved PARP and caspase-3 at 36 h is due to excessive cell death. (C) _bax_−/− _bak_−/− BMK cells were infected with lentivirus encoding control shRNA (shNTC) or LC3 shRNA (shLC3). Two individual cell clones with LC3 knockdown are shown. (D and E) shNTC and the two shLC3 cell lines were treated with MG132. (D) Cell death was measured by PI exclusion. The averages of four independent assays + the SEM are shown. (E) Cell lysates from the shNTC and shLC3 #2 were probed for indicated proteins. Note the reduced apoptosis in shLC3 cells. (F) The parental, LC3-overexpressing, and shLC3 #2 _bax_−/− _bak_−/− BMK cells were treated with TNF-α plus cycloheximide for indicated periods of time. Cell death was determined by PI exclusion. The averages of four independent assays plus SEM are shown.

Fig. 7.

The proapoptotic activity of LC3 requires its p62 interaction and membrane localization. (A) _bax_−/− _bak_−/− BMK cells with stable LC3 knockdown were infected with retrovirus encoding GFP-LC3wt, GFP-LC3 F52A, or GFP-LC3 G120A. (B) Cells were left untreated or were treated with MG132. Cell lysates were subjected to IP with an anti-GFP antibody, and the IP samples were probed for GFP and p62. Note that while MG132 induced p62 interaction with LC3wt and LC3 G120A, the LC3 F52A mutant failed to interact with p62. (C) Cells were cultured in regular medium or serum-free medium for 18 h. Note the GFP-LC3 puncta formation in the LC3wt and the LC3 F52A mutant but not in the LC3 G120A mutant. (D and E) Cells were treated with MG132 for the indicated periods of time. Cell death was determined by PI exclusion. The averages of four independent assays and the SEM are shown (D) or immunoblotting for apoptotic cleavage of caspase-3 and PARP (E). (F and G) Chloroquine (CQ) enhances MG132-induced apoptosis. _bax_−/− _bak_−/− BMK cells were treated with the indicated conditions of MG132 and/or chloroquine. (F) Cell death was measured by PI exclusion. The averages of four independent assays plus the SEM are shown. (G) PARP, caspase-3, and caspase-8 cleavage was examined by immunoblotting.

Fig. 8.

Proteasome inhibition leads to caspase-8 aggregation and activation on the cytosolic side of intracellular membranes. (A) bax −/− bak −/− BMK cells stably expressing GFP-LC3 were left untreated (one well) or were treated with MG132 (two wells). At 16 h after treatment, one well of MG132-treated cells was treated with z-VAD for 2 h. All three wells were incubated with Red-IETD-FMK for 1 h. The cells were observed under a Zeiss inverted Axiovert 200M microscope. Note that MG132 induced caspase-8 activity that colocalized with GFP-LC3. (B) _bax_−/− _bak_−/− BMK cells were left untreated or were treated with MG132 (0.5 μM) for 18 h. Cells were homogenized and left untreated or were treated with trypsin (100 μg/ml) and fractionated. Heavy-membrane (HM) and light-membrane (LM) fractions were subjected to immunoblotting for the indicated proteins. (C) MG132-induced caspase-8 membrane recruitment is dependent on both p62 and LC3. _bax_−/− _bak_−/− BMK cells expressing control shRNA (shNTC), shLC3, or shp62 were left untreated or were treated with MG132 (0.5 μM) for 18 h. The cells were fractionated. The heavy-membrane (HM) and light-membrane (LM) fractions were probed with the indicated antibodies. Note that in the LC3 and p62 knockdown cells, MG132-induced increased membrane-localized caspase-8 was diminished.

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Inhibition of protein degradation induces apoptosis through a microtubule-associated protein 1 light chain 3-mediated activation of caspase-8 at intracellular membranes - PubMed (original) (raw)