Chaperone-mediated autophagy prevents apoptosis by degrading BBC3/PUMA

Abstract

Autophagy is a potentially inimical pathway and together with apoptosis, may be activated by similar stress stimuli that can lead to cell death. The molecular cues that dictate the cell fate choice between autophagy and apoptosis remain largely unknown. Here we report that the proapoptotic protein BBC3/PUMA (BCL2 binding component 3) is a bona fide substrate of chaperone-mediated autophagy (CMA). BBC3 associates with HSPA8/HSC70 (heat shock 70kDa protein 8), leading to its lysosome translocation and uptake. Inhibition of CMA results in stabilization of BBC3, which in turn sensitizes tumor cells to undergo apoptosis. We further demonstrate that upon TNF (tumor necrosis factor) treatment, IKBKB/IKKβ (inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase β)-mediated BBC3 Ser10 phosphorylation is crucial for BBC3 stabilization via blocking its degradation by CMA. Mechanistically, Ser10 phosphorylation facilitates BBC3 translocation from the cytosol to mitochondria. BBC3 stabilization resulting from either Ser10 phosphorylation or CMA inhibition potentiates TNF-induced apoptotic cell death. Our findings thus reveal that the selective degradation of BBC3 underlies the prosurvival role of CMA and define a previously unappreciated proapoptotic role of IKBKB that acts through phosphorylation-mediated stabilization of BBC3, thereby promoting TNF-triggered apoptosis.

Keywords: BBC3; IKBKB; IKKβ; PUMA; TNF; apoptosis; chaperone-mediated autophagy.

Figure 1.

Inhibition of CMA leads to BBC3 induction. (A) Representative western blots and densitometric data (n = 3 or 4) showing that CMA ablation leads to BBC3 upregulation. The indicated tumor cells were infected with vectors containing control, LAMP2A, or HSPA8 shRNA and then lysed. (B) Representative immunoblots (of n ≥ 3) showing that loss of CMA stabilizes BBC3. H1299 cells expressing the indicated shRNAs were treated with 40 μg/ml CHX and then harvested at the indicated times. (C) Relative BBC3 protein level shown in (B) was quantified. (D) Representative Western blots (n = 3) showing that LAMP2A depletion further promotes BBC3 induction upon serum withdrawal. H1299 cells were infected with control or LAMP2A shRNA lentiviruses for 48 h followed by serum deprivation for 48 h. Cell lysates were harvested for immunoblotting analysis. (E) Western blotting analysis of BBC3, TP53, and CDKN1A levels in HCT116 cells. Cells first infected with vectors containing shCon or BBC3 shRNAs, then with shCon or LAMP2A shRNAs were treated with 0.5 μM DOX for 6 h and then harvested. (F) FACS analysis of cell death by PI staining in HCT116 cells stably expressing shRNA constructs as in (E) showing that CMA inhibition induces cell death in a BBC3-dependent manner. (G) Percentage of early apoptotic cells after 24 h of DOX exposure in cells stably expressing shRNA constructs as in (E) was determined by ANXA5 and PI staining, which shows CMA inhibition induces cell death upon DNA damage in a BBC3-dependent manner. Data were represented as mean ±SEM; P < 0.01** and 0.001***, n = 4, t test. Quantification of BBC3 protein levels was done relative to loading control.

Figure 2.

For figure legend, see page 1627.

Figure 3.

For figure legend, see page 1629.

Figure 4.

IKBKB-dependent phosphorylation of BBC3 at Ser10 enables maximum BBC3 induction upon TNF exposure. (A) Representative immunoblots of immunoprecipitation (of n = 3) using IgG or anti-BBC3 antibody showing that endogenous IKBKB associates with BBC3. (B) Representative Western blots (of n = 3) of BBC3 WT and BBC3^S10A HCT116 cells incubated with 10 ng/ml TNF for the indicated times showing that serine to alanine mutation results in compromised BBC3 accumulation upon TNF exposure. The ratio of BBC3 protein normalized to ACTB relative to control (marked as 1.0) is indicated below each lane. (C) Representative immunoblots with densitometric quantification following immunoprecipitation (of n = 4) using anti-BBC3 antibody in HCT116 cells treated with TNF  for 30 min following control or IKBKB shRNA lentivirus infection showing BBC3 Ser10 phosphorylation upon TNF treatment. Of note, BBC3 levels in the IP experiment do not change in this case in the TNF lane, owing to saturation of the anti-BBC3 antibody. The pSer10-BBC3 protein levels were quantified relative to BBC3 protein levels in the IP experiment. (D) Representative western blots of immunoprecipitation (of n = 3) in HCT116 cells showing that BBC3 Ser10 phosphorylation upon TNF treatment is IKBKB dependent. HCT116 cells were infected with vectors containing control or IKBKB shRNA first. At 24-h postinfection, cells were incubated with TNF for the indicated times and then harvested for IP experiment. Of note, BBC3 levels in the coIP experiment do not change in this case in the TNF and IKBKB shRNA lanes, owing to saturation of the anti-BBC3 antibody. (E) Representative Western blots of mitochondria fractionation (of n = 3) in HCT116 cells showing that depletion of IKBKB results in an increase of cytosolic BBC3 upon TNF treatment. HCT116 cells were coinfected with shIKBKB and shHSPA8 lentivirus first. At 48-h postinfection, cells were treated with TNF for 30 min and then harvested. (F) The ratio of BBC3 protein normalized to TUBB (for Cytosolic fraction) in (E) relative to control (red for shCon and green for shHSPA8) is indicated. Statistics are depicted as mean ±SEM; P < 0.05 *, and 0.01**, n = 4, t test.

Figure 5.

BBC3 stabilization resulting from Ser10 phosphorylation or CMA blockage is essential for TNF-triggered apoptosis. (A) Representative western blots (n=3) of BBC3 WT and BBC3^S10A HCT116 cells cotreated with 10 ng/ml TNF and 2.5 μg/ml CHX for the indicated time periods. BBC3 is indicated with an arrow. SE, short exposure; LE, long exposure. Quantification of BBC3 protein levels was done relative to TUBB. (B) FACS analysis data detecting early apoptotic cells showing that BBC3^S10A cells confer resistance to TNF-triggered apoptosis under TNF and CHX cotreatment for 7 h. (C) Western blots showing shRNA knockdown efficiency of BBC3 in BBC3 WT HCT116 cells. (D) FACS analysis of early apoptotic cells in (C) showing that BBC3 is required for TNF-induced cytotoxicity under TNF and CHX cotreatment. BBC3 WT cells were infected with vectors containing control or BBC3 shRNA first, and 48 h after infection, cells were cotreated with TNF and CHX for 7 h before being harvested for FACS analysis. (E) Representative immunoblots (n = 3) of BBC3 WT HCT116 cells cotreated with TNF and CHX following infection with vectors containing the indicated shRNAs. BBC3 WT HCT116 cells first infected with vectors containing shCon or BBC3 shRNAs, then with shCon, LAMP2A or HSPA8 shRNAs were cotreated with TNF and CHX for 5 h and then harvested. The BBC3 protein levels were quantified relative to ACTB. (F) FACS analysis of early apoptotic cells in response to the treatment shown in (E) showing that CMA blockage could sensitize TNF-induced apoptosis,which is protected by BBC3 depletion. Statistics are depicted as mean ± SEM; P < 0.001***, n = 4, t test.

Figure 6.

Proposed model of CMA-dependent degradation of BBC3 and phosphorylation-mediated stabilization of BBC3 in response to TNF stimulation. Schematic models under basal conditions (A) and upon TNF treatment (B) are shown.