Rapid turnover of mcl-1 couples translation to cell survival and apoptosis

Abstract

Inhibition of translation plays a role in apoptosis induced by a variety of stimuli, but the mechanism by which it promotes apoptosis has not been established. We have investigated the hypothesis that selective degradation of anti-apoptotic regulatory protein(s) is responsible for apoptosis resulting from translation inhibition. Induction of apoptosis by cycloheximide was detected within 2-4 h and blocked by proteasome inhibitors, indicating that degradation of short-lived protein(s) was required. Caspase inhibition and overexpression of Bcl-x(L) blocked cycloheximide-induced apoptosis. In addition, cycloheximide induced rapid activation of Bak and Bax, which required proteasome activity. Mcl-1 was degraded by the proteasome with a half-life of approximately 30 min following inhibition of protein synthesis, preceding Bak/Bax activation and the onset of apoptosis. Overexpression of Mcl-1 blocked apoptosis induced by cycloheximide, whereas RNA interference knockdown of Mcl-1 induced apoptosis. Knockdown of Bim and Bak, downstream targets of Mcl-1, inhibited cycloheximide-induced apoptosis, as did knockdown of Bax. Apoptosis resulting from inhibition of translation thus involves the rapid degradation of Mcl-1, leading to activation of Bim, Bak, and Bax. Because of its rapid turnover, Mcl-1 may serve as a convergence point for signals that affect global translation, coupling translation to cell survival and the apoptotic machinery.

Fig. 1

Inhibition of the proteasome blocks apoptosis induced by cycloheximide. A, Cultures of Rat-1, PC12, U937, or T98G cells were left untreated or treated with 10 μM MG132 for 30 min prior to treatment with 10 μg/ml cycloheximide (CHX). Cytosolic nucleic acids were isolated after the indicated time of cycloheximide treatment and DNA fragmentation assessed by gel electrophoresis. B, U937 cells were harvested at the indicated times after cycloheximide treatment, stained with propidium iodide, and analyzed for DNA content by flow cytometry. The percentage of cells with sub-G₁ DNA content is indicated in each panel. C, The percentage of apoptotic cells from panel B is plotted as a function of time of cycloheximide treatment. D, U937 cells were treated with 10 μM MG132, 30 μM MG115, or 30 μM PSI for 30 min prior to treatment with cycloheximide. DNA fragmentation was assayed after 6 h of cycloheximide treatment.

Fig. 2

Apoptosis induced by translation inhibition is dependent on caspase activity and blocked by Bcl-x_L overexpression. A, U937 cells were left untreated or treated with 10μM zVAD-fmk for 30 min prior to treatment with 10 μg/ml cycloheximide for 6 h. Cells were harvested for immunoblot analysis to detect PARP. B, Cells were treated with zVAD-fmk (Rat-1 and T98G cells, 100 μM; U937 cells, 10 μM) or left untreated for 30 min prior to treatment with 10 μg/ml cycloheximide for 4 h. Cytosolic nucleic acids were isolated and DNA fragmentation assessed by gel electrophoresis. C, HeLa cells were transfected with 1μg pCMV-DsRed expression construct and 1 μg pcDNA3 empty vector or pSG5-Bcl-x_L expression contruct. 48 h after transfection, cells were treated with 10 μg/ml cycloheximide for 18 h, trypsinized, and apoptosis was assessed by TUNEL analysis. Transfected cells were identified by flow cytometry and data are presented as the percentage of transfected cells that were TUNEL-positive. The data represent average values of two independent cultures ± SD.

Fig 3

Translation inhibition induces Bak and Bax activation. A, U937 cells were left untreated or treated with 10 μg/ml cycloheximide for 8 h. Mitochondria were isolated and cross-linking reactions performed using the cross-linker BMH, followed by SDS-PAGE and immunoblot analysis to visualize oligomeric Bak and Bax. B, Duplicate cultures of U937 cells were left untreated or pretreated with 10 μM MG132 for 30 min prior to treatment with 10 μg/ml cycloheximide for 6 h. Cells were analyzed as described in A.

Fig. 4

Mcl-1 is rapidly degraded following translation inhibition. Duplicate cultures of U937, T98G, and HeLa cells were harvested 0, 2, 4, 6, and 12 h after treatment with 10 μg/ml cycloheximide for immunoblot analysis of Mcl-1, Bcl-x_L, and Bcl-2.

Fig. 5

Mcl-1 degradation precedes several markers of apoptosis and is blocked by proteasome inhibition. A, U937 cells were either left untreated or pretreated with either 10 μM MG132 or 10 μM zVAD-fmk for 30 min prior to treatment with 10 μg/ml cycloheximide for 0 – 6 h. Each flask of cells was separated into two equal fractions, and processed to either assess DNA fragmentation by gel electrophoresis or Mcl-1 levels and PARP cleavage by SDS-PAGE and immunoblot analysis. B, The levels of Mcl-1 from three independent experiments with U937 cells were quantitated by densitometry and plotted (average ± SD) relative to untreated cells. C, U937 cells were left untreated or treated with 10 μg/ml cycloheximide for 1 – 6 h. Mitochondrial fractions were isolated and treated with cross-linker BMH followed by SDS-PAGE and immunoblot analysis to detect oligomeric Bak and Bax.

Fig. 6

Effect of Mcl-1, Bcl-x_L, and Bcl-2 overexpression on apoptosis induced by translation inhibition. A, HeLa cells were transfected with 1 μg pCMV-DsRed plus 1 μg pcDNA3 empty vector or pcDNA3-Mcl-1, -Bcl-x_L, or –Bcl-2 expression constructs. 24 or 48 h after transfection, cells were left untreated or treated with 10 μg/ml cycloheximide for 20 h and harvested for TUNEL analysis by flow cytometry. Data represent the percentage of transfected cells that were TUNEL-positive and are averages ± SD from four independent experiments. B, Immunoblot analysis of Mcl-1, Bcl-x_L and Bcl-2 expression in cells from one of the experiments shown in A.

Fig. 7

Mcl-1 knockdown induces apoptosis. A, HeLa and T98G cells were transfected with nonspecific (NS) siRNA or one of three siRNAs designed to knockdown Mcl-1, designated siRNAs 6314, 6126, and 42844. Cells were harvested 24 h after transfection and 20 μg protein subjected to SDS-PAGE and immunoblot analysis. B, T98G cells were left untransfected or transfected with nonspecific (NS) siRNA or Mcl-1 siRNA 6314. 48 h after transfection, cytosolic nucleic acids were isolated and subjected to gel electrophoresis to detect DNA fragmentation. C, T98G cells were transfected with NS siRNA or Mcl-1 siRNA 6314, 6126 or 42844. Cells were harvested 24 or 48 h after transfection for analysis of Mcl-1 protein levels by immunoblot and apoptosis by flow cytometry to quantitate the fraction of cells with sub-G₁ DNA content. Mcl-1 levels following transfection with Mcl-1 siRNAs relative to transfection with nonspecific siRNA for the same durations were determined by densitometry and normalized to β-actin and shown below the immunoblot as % Mcl-1 remaining. D, HeLa cells were transfected with nonspecific (NS) siRNA or Mcl-1 siRNA 6314. Cells were harvested 24 h after transfection and analyzed by flow cytometry to determine the fraction of cells with sub-G₁ DNA content.

Fig 8

Knockdown of Bak, Bax, or Bim partially blocks cycloheximide-induced apoptosis. A, HeLa cells were transfected for 72 h with nonspecific (NS), Bak, Bax, or Bim siRNA and harvested for immunoblot analysis. B and C, 72 h after transfection with the indicated siRNA, HeLa cells were either left untreated or treated with 10 μg/ml cycloheximide for an additional 24 h. Cells were stained with propidium iodide and the fraction of cells with sub-G₁ DNA content determined by flow cytometry. For Bak and Bax knockdowns (panel B), data are averages ± SD of ten independent cultures. Two different siRNAs (ID #'s 120199 and 120201) were used with comparable results. For Bim knockdowns (panel C), data are averages ± SD of seven independent cultures. *A Student's t-test comparing levels of apoptosis following cycloheximide treatment of cells transfected with nonspecific siRNA to cells transfected with Bak, Bax, or Bim siRNA all yielded a p-value < 0.001.