Intrinsic capacities of molecular sensors of the unfolded protein response to sense alternate forms of endoplasmic reticulum stress

Abstract

The unfolded protein response (UPR) regulates the protein-folding capacity of the endoplasmic reticulum (ER) according to cellular demand. In mammalian cells, three ER transmembrane components, IRE1, PERK, and ATF6, initiate distinct UPR signaling branches. We show that these UPR components display distinct sensitivities toward different forms of ER stress. ER stress induced by ER Ca2+ release in particular revealed fundamental differences in the properties of UPR signaling branches. Compared with the rapid response of both IRE1 and PERK to ER stress induced by thapsigargin, an ER Ca2+ ATPase inhibitor, the response of ATF6 was markedly delayed. These studies are the first side-by-side comparisons of UPR signaling branch activation and reveal intrinsic features of UPR stress sensor activation in response to alternate forms of ER stress. As such, they provide initial groundwork toward understanding how ER stress sensors can confer different responses and how optimal UPR responses are achieved in physiological settings.

Figure 1.

The ATF6 branch of the UPR is most responsive to the accumulation of unfolded proteins disrupted by disulfide bond formation in the ER. (A) ATF6 processing during the UPR induction. ATF6 (p90ATF6) is proteolytically cleaved during UPR induction. The transcriptional transactivation domain (p50ATF6) is released in the cytoplasm and subsequently migrates into the nucleus. (B) Conversion of p90ATF6 to p50ATF6 upon DTT treatment of CHO cells. Total cell lysate prepared from CHO cells pretreated with proteasome inhibitor was incubated with (lane 2; 2 mM for 1 h) or without DTT (lane 1) and analyzed by immunoblotting using anti-ATF6 antiserum raised against the N-terminal portion of ATF6 (Imgenex) and β-actin as a control. (C) Immunoblots of ATF6 from lysates of CHO cells treated with 2 mM DTT, 200 nM thapsigargin (Tg), and 10 μg/ml tunicamycin (Tm) for the indicated amount of time. At each time point, treated cells were collected and divided into two samples (for protein and RNA analysis) before quick freezing in liquid nitrogen. β-Actin immunoblotting of the same lysate, from each time course was carried out by reprobing blots with anti-β-actin antibody. (D) Quantitation of p90ATF6 cleavage over the time courses shown in C. p90ATF6 cleavage was quantitated with a Typhoon 9400 phosphorimager (Amersham Biosciences) and normalized with levels of β-actin from corresponding treatments. Percent cleaved ATF6 was calculated by subtracting the p90ATF6 at each time point from the level of p90ATF6 at time zero. The graph shown represents three independent time-course experiments carried out with each drug. Differences in the initial phases of the responses were shown with the close-up of the first hour of treatment; DTT (black solid line), Tg (black dashed line), and Tm (gray solid line). Untreated is represented as a solid black line with open circles.

Figure 2.

Rate of p90 ATF6 disappearance during the UPR is similar with or without proteasome inhibitor, MG132. (A) Immunoblots of ATF6 from HeLa cell lysates treated with 2 mM DTT, or 200 nM thapsigargin (Tg) in the presence of proteasome inhibitor, MG132, (lanes 1–4) or without MG132 (lane 5–8). β-Actin immunoblotting of the same lysates was used to normalize p90ATF6 quantitation. (B) Quantitation of p90ATF6 shown in A. Levels of p90ATF6 were quantitated using a Typhoon phosphorimager and normalized with levels of β-actin. Percent cleaved ATF6 was calculated as described in Figure 1 and were shown during DTT treatment with (●) or without (○) MG132 or during Tg treatment with (■) or without (□) MG132.

Figure 3.

PERK activation measured by autophosphorylation is responsive to ER stress caused by both ER calcium release and disruption of disulfide bonds. (A) PERK autophosphorylates during the UPR response. (B) Immunoblots, after immunoprecipitation of PERK from CHO cells treated with thapsigargin (Tg; 200 nM), tunicamycin (Tm; 10 μg/ml), and DTT (2 mM) for indicated amounts of time. Immunoprecipitated PERK using anti-PERK antibody from total cell lysate of time points from each treatment were Western blotted with anti-PERK antiserum. The slower mobility PERK formed during ER stress caused by DTT, Tg, and Tm treatment is indicated as p-PERK. (C) Quantitation of p-PERK appearance shown in B. DTT (black solid line), Tg (black dashed line), and Tm (gray solid line).

Figure 4.

Phosphorylation of eIF2α, a PERK kinase substrate, is most responsive to ER stress caused by ER calcium release. (A) PERK phosphorylates eIF2α during UPR induction. (B) Immunoblots of phosphorylated eIF2α (p-eIF2α) and β-actin from lysates of CHO cells treated with DTT, thapsigargin (Tg), and tunicamycin (Tm) for the indicated amounts of time. For direct comparisons with ATF6- and IRE1-signaling branch activation, the same cell lysates used for Figure 1 were examined for phosphorylation of eIF2α. (C) Quantitation of the increase in phosphorylated eIF2α (p-eIF2α) levels over the time course shown in B. The levels of p-eIF2α were quantitated with a Typhoon 9400 phosphorimager and normalized to levels of β-actin. Fold induction was calculated by taking the ratio between the levels of normalized p-eIF2α at time zero and each time point. A close-up of the first hour is shown for comparison in the initial phases of the responses. The graph represents three independent time course experiments carried out with DTT (black solid line), Tg (black dashed line), and Tm (gray solid line). Untreated is represented as a solid black line with open circles.

Figure 5.

PERK activation measured by dissociation of BiP correlates with PERK activation measured by PERK autophosphorylation. (A) Immunoprecipitation of BiP was specific to uninduced PERK. BiP was specifically immunoprecipitated with anti-PERK antibody from uninduced lysate (lane 2), but not with anti-myc antibody (lane 1). Furthermore, UPR induction upon Tg treatment diminished levels of BiP-associated PERK (lane 3). (B) BiP associated with PERK during treatment with DTT (lanes 1–4), Tg (lanes 5–8), and Tm (lanes 9–12) is shown. Levels of BiP associated with PERK in each immunoprecipitated fraction were detected by blotting the same membranes using anti-BiP antibody. (C) Quantitation of BiP associated with PERK. Levels of BiP associated with PERK were quantitated with a phosphorimager and normalized against immunoprecipitated PERK from CHO cells treated with DTT (black solid line), Tg (black dashed line), and Tm (gray solid line). Error bars and values of each time point were averaged over three independent experiments.

Figure 6.

eIF2α phosphorylation during UPR induction in NIH3T3 cells. (A) Immunoblots of phosphorylated eIF2α (p-eIF2α) and β-actin from lysates prepared from NIH3T3 cells treated with DTT, thapsigargin (Tg), or tunicamycin (Tm) for the indicated amounts of time. (B) Quantitation of the fold increase in p-eIF2α levels over the time course shown in A. The levels of p-eIF2α were quantitated as described in Figure 2 and fold induction was calculated by taking the ratio between the levels of normalized p-eIF2α at time zero and each time point. A close-up of the first hour is shown for comparisons in the initial phase of the responses. The graph represents three independent time course experiments carried out with DTT (black solid line), Tg (black dashed line), and Tm (gray solid line). Untreated is represented as a solid black line with open circles.

Figure 7.

The IRE1 signaling branch of the UPR can respond efficiently to all types of ER stress, but is most sensitive to the accumulation of unfolded proteins due to disrupted disulfide bonds in the ER. (A) IRE1 autophosphorylates upon UPR induction. On activation, IRE1 becomes oligomerized and is an active endoribonuclease in the splicing of XBP1 mRNA. Schematic representation of both the unspliced (U) and spliced (S) forms of XBP1 mRNA and the PCR primers used. (B) Immunoblots, after immunoprecipitation, of IRE1α from CHO cells treated with DTT (2 mM), thapsigargin (Tg; 200 nM), and tunicamycin (Tm; 10 μg/ml) for indicated amounts of time using anti-IRE1α antiserum raised against the C-terminal portion of IRE1α. The slower mobility IRE1α formed during ER stress caused by DTT, Tg, and Tm treatment is indicated as p-IRE1α. BiP associated with IRE1α was detected by blotting the same membrane using anti-BiP antibody. During the analyses, we consistently observed that the extent of IRE1α mobility shift was much more pronounced during DTT-induced ER stress than during other treatments. Currently, the molecular bases for these differences are not clear, although they may represent differential phosphorylation in response to the altered forms of ER stress. (C) Quantitation of immunoprecipitated BiP. Levels of BiP associated with IRE1α were quantitated with a phosphorimager and normalized against immunoprecipitated IRE1α (shown as a solid line). Error bars and values of each time point were averaged over three independent experiments. IRE1α RNase activity indicated as % spliced XBP1 is shown as dashed line. (see E for detail). (D) RT-PCR of RNA isolated from CHO cells treated with 2 mM DTT, 200 nM thapsigargin (Tg), and 10 μg/ml tunicamycin (Tm). Total RNA was isolated from samples collected for Figures 1 and 3. cDNA was prepared using oligodT primers. PCR with primers flanking the 26-nt UPR intron of hamster XBP1 RNA. PCR products were analyzed on 1.5% agarose gels and stained with ethidium bromide. DNA fragments derived from unspliced (U) and spliced (S) are indicated. Bands marked as (*) are nonspecific PCR fragments. (E) Quantitation of the spliced XBP1 shown in D. Both unspliced and spliced forms of XBP1 were quantitated with a phosphorimager. Percent spliced at each time point was calculated by S/(S + U) × 100. The graph represents three independent time-course experiments carried out with DTT (black solid line), thapsigargin (black dashed line), and tunicamycin (gray solid line). Untreated is represented as a solid black line with open circles. Quantitation of the spliced XBP1 for the entire 5-h time course was shown in C as dashed lines, whereas that for the first hour is shown in E.

Figure 8.

Preferential activation of UPR signaling branches by alternate types of ER stress. (A) ATF6 is activated quickly to disruption of disulfide bonds of ER proteins caused by DTT (black solid line), whereas both IRE1 and PERK are activated rapidly and extensively in response to ER stress caused by thapsigargin (black dashed line), an ER Ca²⁺ release agent. For all ER stress tested, IRE1 reacts relatively fast and efficiently, resulting in splicing of XBP1 mRNA. (B) Because all activation profiles for ATF6, PERK, and IRE1α described in Figures 1, 2, and 6 were performed with the same extract samples, the kinetic induction of the three UPR initiators was reanalyzed by the type of ER stress imposed. ER stress caused by DTT activated both IRE1α (blue) and ATF6 (green) rapidly and efficiently. UPR induced by thapsigargin was detected by both PERK (red) and IRE1 (blue), whereas all three UPR sensors respond to ER stress activated by tunicamycin with relatively similar kinetics.

Figure 9.

Differential activation of three UPR sensors is likely to reflect their intrinsic properties. (A) Activation kinetics of PERK, IRE1, and ATF6 signaling branches during DTT (2 mM) or tunicamycin (Tm; 10 μg/ml) treatment, or during treatment with both DTT (2 mM) and tunicamycin (Tm; 10 μg/ml) together for indicated amounts of time. Immunoblots, after immunoprecipitation, of PERK using anti-PERK antiserum, and of ATF6 using anti-ATF6 antibody and RT-PCR of XBP1 mRNA from total RNA isolated, are shown. (B) Quantitation of PERK autophosphorylation, ATF6 p90 fragment disappearance, and XBP1 mRNA splicing, shown in A during ER stress caused by DTT only (black solid line), both DTT and tunicamycin (black dashed line), and tunicamycin only (gray solid line).