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. 2013 Mar 15;288(11):7606-7617.
doi: 10.1074/jbc.M112.424655. Epub 2013 Jan 22.

p53 and translation attenuation regulate distinct cell cycle checkpoints during endoplasmic reticulum (ER) stress

Sally E Thomas  1 , Elke Malzer  2 , Adriana Ordóñez  1 , Lucy E Dalton  1 , Emily F A van T Wout  1 , Elizabeth Liniker  1 , Damian C Crowther  3 , David A Lomas  1 , Stefan J Marciniak  4
Affiliations

p53 and translation attenuation regulate distinct cell cycle checkpoints during endoplasmic reticulum (ER) stress

Sally E Thomas et al. J Biol Chem. .

Abstract

Cell cycle checkpoints ensure that proliferation occurs only under permissive conditions, but their role in linking nutrient availability to cell division is incompletely understood. Protein folding within the endoplasmic reticulum (ER) is exquisitely sensitive to energy supply and amino acid sources because deficiencies impair luminal protein folding and consequently trigger ER stress signaling. Following ER stress, many cell types arrest within the G(1) phase, although recent studies have identified a novel ER stress G(2) checkpoint. Here, we report that ER stress affects cell cycle progression via two classes of signal: an early inhibition of protein synthesis leading to G(2) delay involving CHK1 and a later induction of G(1) arrest associated both with the induction of p53 target genes and loss of cyclin D(1). We show that substitution of p53/47 for p53 impairs the ER stress G(1) checkpoint, attenuates the recovery of protein translation, and impairs induction of NOXA, a mediator of cell death. We propose that cell cycle regulation in response to ER stress comprises redundant pathways invoked sequentially first to impair G(2) progression prior to ultimate G(1) arrest.

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Figures

FIGURE 1.
FIGURE 1.
Translation attenuation activates CHK1. A, HCT116 cells were treated for 1 h with thapsigargin (500 nm), cycloheximide (50 μg/ml), anisomycin (5 μg/ml), puromycin (75 μg/ml), or emetine (100 μg/ml). Postnuclear lysates were separated by 10% SDS-PAGE, transferred to nitrocellulose, and immunoblotted for P317-CHK1, total CHK1, p53, cyclin D1, and actin. B, HCT116, HeLa, COS-7, and 293T cells were treated with cycloheximide (50 μg/ml) for 1 h and analyzed by immunoblot for total P317-CHK1, total CHK1, and eIF2α. C, HCT116 cells were treated with cycloheximide (50 μg/ml), puromycin (75 μg/ml), anisomycin (5 μg/ml), or emetine (100 μg/ml) for 1 h or irradiated with 30 or 120 J/m2 UV light. Total cell lysates were analyzed by immunoblot for total P317-CHK1, P345-CHK1, total CHK1, P68-CHK2, and total CHK2. D, HCT116 cells were irradiated with UV light at 0, 8, 10, 12, or 15 J/m2 or incubated in the presence or absence of 500 nm thapsigargin (Tg) for 1 h. Total cell lysates were analyzed by immunoblot for P317-CHK1, total CHK1, P139-H2AX (γH2AX), and H2AX. E, graphical representation of data from D showing P317-CHK1 band intensity corrected for total CHK1 band intensity and represented as percent maximum signal. F, graphical representation data from D showing γH2AX band intensity corrected for total H2AX. Data are mean ± S.E. (error bars). n = 3; *, p < 0.05; ns, not significant.
FIGURE 2.
FIGURE 2.
ATR and ATM are not required for CHK1 activation during translation attenuation. A, representative light micrographs of eyes from GMR-GAL4>dPERK Drosophila or GMR-GAL4>dPERK animals expressing UAS-shRNAi against grp/CHK1, CHK2, dATR, and dATM (upper row) or w1118 strain control expressing GMR-GAL4 driver alone or GMR-GAL4 with each of the specified shRNAi (lower row). All flies were raised at 18 °C. B, ATR+/+ and ATR−/− lymphoblasts were treated for 1 h with cycloheximide (50 μg/ml), and whole cell lysates were separated by 10% SDS-PAGE, transferred to nitrocellulose, and immunoblotted for ATR, P317-CHK1, total CHK1, and eFI2α. A graphical summary of P317-CHK1 from three independent repeats normalized to untreated for each genotype is shown. C, ATM+/+ and ATM−/− fibroblasts were treated for 1 h with cycloheximide (50 μg/ml), and postnuclear lysates were separated by 10% SDS-PAGE, transferred to nitrocellulose, and immunoblotted for ATM, P317-CHK1, total CHK1, and eFI2α. A graphical summary of P317-CHK1 from three independent repeats normalized to untreated for each genotype is shown. Data in B and C are mean ± S.E. (error bars).
FIGURE 3.
FIGURE 3.
G2 delay during endoplasmic reticulum stress is enhanced in p53 mutant cells. A, representative FACS histograms of HCT116 cells of the indicated genotypes expressing either scrambled or CHK1 shRNAi and treated with thapsigargin (500 nm) for 0, 16, or 24 h; permeabilized; and then stained with propidium iodide. B, cell lysates were prepared from HCT116 cells of the indicated genotypes expressing either scrambled or CHK1 shRNAi. These were separated by 10% SDS-PAGE, transferred to nitrocellulose, and immunoblotted for total CHK1. eFI2α served as a loading control. C, graphs of G2/G1 normalized to untreated cells from A. Data are mean ± S.E. (error bars). n = 5; *, p < 0.05. D, graphs of sub-G1 cells from A. Data are mean ± S.E. (error bars). n = 5; *, p < 0.05. E, experiments were repeated identically, but cells were stained with propidium iodide without prior fixation to identify late cell death. Data are mean ± S.E. (error bars). n = 5; *, p < 0.05. F, representative FACS histograms of p53 wild type HCT116 cells expressing either scrambled or CHK1 shRNAi and treated with thapsigargin (500 nm) for 0, 16, or 24 h; permeabilized; and then stained with propidium iodide. G, graphs of G2/G1 normalized to untreated cells from F. Data are mean ± S.E. (error bars). n = 3; *, p < 0.05.
FIGURE 4.
FIGURE 4.
p53 genotype affects CHOP and GADD34 protein accumulation. A, schematic representation of domain structure of p53 and p53/47 isoforms. TA, transactivation domain; P, polyproline-rich domain (PXXP); DBD, DNA binding domain; NLS, nuclear localization sequence; OD, oligomerization domain; Epitope 1 within TA1, p53 Ab6 DO1 antibody; Epitope 2 within TA2, p53 pAb1801 antibody. B, postnuclear lysates from HCT116 cells of the indicated genotypes were analyzed by immunoblot using a polyclonal antibody against full-length p53 (polyclonal rabbit; catalog number 9282, Cell Signaling Technology), Epitope 1 (p53 Ab6 DO1), and Epitope 2 (p53 pAb1801). C, postnuclear lysates from HCT116 cells of the indicated genotypes that had been treated with thapsigargin (Tg) at 500 nm for the indicated times were analyzed by immunoblot for total p53, CHOP, GADD34, p21, cyclin D1, NOXA (note that the upper band is a nonspecific signal marked by an asterisk, and the specific band marked by an arrowhead), Mcl-1, PUMA, BAX, and eIF2α. D and E, postnuclear supernatant (cytosol) and high salt nuclear protein extract (nuclear) from HCT116 cells of the indicated genotypes treated with 400 nm thapsigargin (Tg) or 2.5 μg/ml tunicamycin (Tm) were analyzed by immunoblot; cytosol was blotted for p53, BiP, and eIF2α; and nuclear proteins were blotted for p53 and CHOP.
FIGURE 5.
FIGURE 5.
CHOP and GADD34 are not direct targets of p53. A, HCT116 cells of the specified genotypes were treated with thapsigargin (500 nm) for 4 h, and mRNA was extracted, reverse transcribed using oligo(dT) primers, analyzed by quantitative PCR, normalized to actin mRNA, and expressed as -fold change relative to HCT116 p53+/+ untreated cells. Data are mean -fold change ±S.E. (error bars). n = 3 independent experiments performed in triplicate. B, experiment performed as in A but with tunicamycin (2.5 μg/ml) in place of thapsigargin.
FIGURE 6.
FIGURE 6.
Recovery of translation is reduced in p53 mutant cells. A, HCT116 cells of the specified genotypes were treated with thapsigargin (Tg) (500 nm) for the indicated times and labeled with 35S-labeled methionine and cysteine for 15 min, and then equal quantities of postnuclear lysate were separated by 10% SDS-PAGE, fixed, stained, dried, and exposed to a phosphorimaging screen. B, pooled data from three independent repeats of A expressed as percent maximum signal. Data are mean ± S.E. (error bars). n = 3. C, HCT116 cells of the specified genotypes were co-transfected with p5×ATF6-Luc encoding firefly luciferase under the control of a UPR response element and with pRL-TK Renilla luciferase as a normalization control. Data presented as firefly luciferase activity normalized to Renilla luciferase are mean ± S.E. (error bars). n = 3 independent repetitions. D, XBP1s mRNA level normalized to the mRNA levels of actin and RPL13A mRNA (normalized expression). HCT116 cells of the specified genotypes were treated with thapsigargin (500 nm) for the indicated times. mRNA levels were determined by qPCR. Data are mean ± S.E. (error bars). n = 3 independent repetitions; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (analysis of variance with Bonferroni post hoc test).
FIGURE 7.
FIGURE 7.
GADD34 stability is affected by p53 genotype. A, HCT116 cells of the specified genotypes were treated with or without thapsigargin (Tg) (500 nm) for 24 h and/or with lactacystin (5 μm). Cell lysates were analyzed by 10% SDS-PAGE, transferred to nitrocellulose, and immunoblotted for GADD34 and eIF2α. B, graphical representation of A. Immunoblots were analyzed using ImageJ software. Data presented as percent GADD34 band intensity relative to eIF2α loading control are mean ± S.E. (error bars). n = 3 independent repeats; *, p < 0.05; **, p < 0.01.
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