Oxidative stress promotes SIRT1 recruitment to the GADD34/PP1α complex to activate its deacetylase function

Abstract

Phosphorylation of the eukaryotic translation initiation factor, eIF2α, by stress-activated protein kinases and dephosphorylation by the growth arrest and DNA damage-inducible protein (GADD34)-containing phosphatase is a central node in the integrated stress response. Mass spectrometry demonstrated GADD34 acetylation at multiple lysines. Substituting K³¹⁵ and K³²² with alanines or glutamines did not impair GADD34's ability to recruit protein phosphatase 1α (PP1α) or eIF2α, suggesting that GADD34 acetylation did not modulate eIF2α phosphatase activity. Arsenite (Ars)-induced oxidative stress increased cellular GADD34 levels and enhanced Sirtuin 1 (SIRT1) recruitment to assemble a cytoplasmic complex containing GADD34, PP1α, eIF2α and SIRT1. Induction of GADD34 in WT MEFs paralleled the dephosphorylation of eIF2α (phosphoserine-51) and SIRT1 (phosphoserine-47). By comparison, eIF2α and SIRT1 were persistently phosphorylated in Ars-treated GADD34-/- MEFs. Expressing WT GADD34, but not a mutant unable to bind PP1α in GADD34-/- MEFs restored both eIF2α and SIRT1 dephosphorylation. SIRT1 dephosphorylation increased its deacetylase activity, measured in vitro and in cells. Loss of function of GADD34 or SIRT1 enhanced cellular p-eIF2α levels and attenuated cell death following Ars exposure. These results highlighted a novel role for the GADD34/PP1α complex in coordinating the dephosphorylation and reactivation of eIF2α and SIRT1 to determine cell fate following oxidative stress.

Figure 1

GADD34 is acetylated. (a) Schematic of the GADD34 protein with acetylated lysines (K). The N-terminal ER localization sequence is denoted by yellow box. The central PEST repeats are represented as green boxes and the C-terminal blue box represents the PP1-binding domain. (b) Mass spectrometry of FLAG-GADD34 (WT) or FLAG-GADD34 (KARA) immunoprecipitates (IP) from cells treated with or without 5 mM nicotinamide for 6 h identified acetylated peptides with sequences as shown. (c) HEK293 cells expressing FLAG-GADD34 (WT) and mutants, FLAG-K312A/K322A and FLAG-K315Q/K322Q, were subjected to immunoprecipitation using anti-FLAG antibody. Immunoprecipitates (IP) and whole cell lysates (WCL) were analyzed for endogenous eIF2α, PP1α and FLAG-GADD34 by immunoblotting. Molecular weight markers (kDa) are shown. (d) HEK293 cells expressing the FLAG epitope and increasing amounts of FLAG-GADD34 WT or FLAG-K315A/K322A and FLAG-K315Q/K322Q mutation were analyzed for eIF2α dephosphorylation. Cells were immunoblotting for eIF2α, p-eIF2α, FLAG-GADD34 and tubulin. (e) Quantitation of p-eIF2α/eIF2α ratio in cells expressing increasing amounts of the WT FLAG-GADD34, FLAG-K315A/K322A and FLAG-K315Q/K322Q (expressed in arbituary units, A.U.) from 4 independent experiments each consisting of 12 data points

Figure 2

Ars enhances SIRT1 association with GADD34. (a) HEK293 cells expressing FLAG-GADD34 were subjected to the following stresses for 1 h (hr): TG, TN, Etop, H₂O₂, Ars and MG132 before immunoprecipitations (IP) using anti-FLAG antibody. IP and WCL were immunoblotted for GFP-SIRT1, FLAG-GADD34, eIF2α and PP1α. Molecular weight markers (kDa) are shown. (b) HEK293 cells expressing the FLAG peptide or FLAG-GADD34 were subjected to increasing Ars concentrations for 1 h before IP using anti-FLAG. IP and WCL were immunoblotted for SIRT1, eIF2α, PP1α and FLAG-GADD34. (c) Anti-GADD34 immunoprecipitates from WT MEFs treated with Ars (50 μM, 5 h) or UT were immunoblotted for SIRT1 and GADD34. Control IgG antibody was included for control IP. Tubulin was used as loading control. (d) HEK293 cells, exposed to antioxidant, NAC (20 mM, 2 h), or Ars (0.5 mM, 1 h) or NAC (20 mM, 1 h) before Ars (0.5 mM, 1 h), were stained using mitochondrial ROS-detecting dye, MitoSOX. MitoSOX-stained cells were quantified by FACS and represented as fold change compared with UT HEK293 (mean±S.E.M., n=3). Sidak’s multiple comparisons test was used after two-way ANOVA to generate P-values (**P<0.01). (e) Anti-FLAG immunoprecipitations from HEK293 cells co-expressing FLAG-GADD34 and GFP-SIRT1 WT either UT (−) or following treatment with NAC, Ars or NAC and Ars as described in d were analyzed. IP and WCL were immunoblotted for GFP-SIRT1 and FLAG-GADD34. Tubulin was used as a loading control. Molecular weight markers (kDa) are shown

Figure 3

Cytosolic SIRT1 Binds GADD34. (a) Subcelullar distribution of GFP-SIRT1 wild-type (WT) in HeLa cells is shown under UT conditions and after Ars (0.5 mM, 1 h) treatment. LMB (10 nM, 3 h), or LMB (10 nM, 2 h) pretreatment before Ars (0.5 mM, 1 h) exposure are also shown. GFP-SIRT1 is shown in green with nuclear staining by Hoescht 33342 in blue. Scale bar, 30 μm. (b) Anti-FLAG immunoprecipitations from HEK293 cells co-expressing FLAG-GADD34 and GFP-SIRT1, either UT (−) or following treatment with LMB, Ars or both LMB and Ars, as described in a, were analyzed for GFP-SIRT1, FLAG-GADD34, eIF2α and PP1α by immunoblotting IPs and WCL with appropriate antibodies. (c) Subcellular distribution of GFP-SIRT1 wild-type (WT) and mutant GFP-SIRT1 lacking the NLS (mt NLS) are shown in HeLa cells either UT or after Ars (0.5 mM, 1 h) treatment. GFP-SIRT1 is shown in green and nuclear staining by Hoescht 33342 in blue. Scale bar, 30 μm. (d) HEK293 cells coexpressing FLAG-GADD34 and either GFP-SIRT1 WT or GFP-SIRT1 mt NLS UT or following Ars treatment (0.5 mM, 1 h) were subjected immunoprecipitations using anti-FLAG. The GFP-SIRT1 proteins and FLAG-GADD34 in IP and WCL were analyzed by immunoblotting as described in methods. (e) Anti-GADD34 immunoprecipitates from WT MEFs either UT (−) or following treatment with Ars (50 μM, 5 h) or LMB (10 nM, 1 h) pretreatment before exposure to Ars (50 μM, 5 h) were immunoblotted with anti-SIRT1 and anti-GADD34 antibodies. Control IgG was included as control. Tubulin was used a loading control

Figure 4

GADD34 contributes to SIRT1 localization in cytoplasm. (a) Immunohistochemistry assessed the subcellular distribution of SIRT1 (green) in WT and GADD34−/− MEFs either UT or following treatment with 50 μM Ars for 4 h. The nuclei were stained with Hoescht 33342 (blue) and cytoplasm outlined using Rhodamine-Phalloidin to stain filamentous actin (red). Scale bar, 30 μm. (b) The intensity of SIRT1 staining in nucleus and cytoplasm of WT and GADD34−/− MEFs before and after Ars treatment was quantified. (Mean±S.E.M., n≥15 cells). Tukey multiple comparison test was used after two-way ANOVA to generate P-values (****P<0.0001). (c) Immunohistochemistry assessed the subcellular distribution of SIRT1 (green) in WT MEFs either UT or NAC (20 mM, 5 h) or Ars (50 μM, 4 h), or pretreatment of NAC (20 mM, 1 h) before exposure to 50 μM Ars for 4 h. The nuclei were stained with Hoescht 33342 (blue) and cytoplasm outlined using Rhodamine-Phalloidin (red). Scale bar, 30 μm. (d) The intensity of SIRT1 staining in the nucleus and cytoplasm of WT MEFs subjected to various treatments as shown in c was quantified (mean±S.E.M., n≥70 cells). Tukey multiple comparison test was used after two-way ANOVA to generate P-values (****P<0.0001)

Figure 5

Domains of GADD34 and SIRT1 that mediate their association. (a) Schematic of SIRT1 and its deletion mutants used to map the GADD34-binding site. HA-GADD34 was coexpressed with either empty FLAG vector, full-length FLAG-SIRT1 (1-737) and selected SIRT1 fragments in HEK293 cells, which were treated with Ars (0.5 mM, 1 h). Following immunoprecipitations using anti-FLAG, IP and WCL were immunoblotted for HA-GADD34, FLAG-SIRT1, eIF2α and PP1α. Molecular weight markers (kDa) are shown. (b) Schematic of GADD34 and its deletion mutants used to map the SIRT1-binding site. FLAG-GADD34 polypeptides were immunoprecipitated from HEK293 cells treated with 0.5 mM Ars for 1 h using anti-FLAG. IP and WCL were immunoblotted for SIRT1, eIF2α, PP1α and FLAG-GADD34. (c) Schematic summarizing the binding sites on GADD34 for SIRT1, eIF2α and PP1α is shown. The N-terminal ER localization sequence in GADD34 is shown as yellow box, the central PEST repeats as green boxes and the C-terminal PP1-binding domain as a blue box

Figure 6

GADD34 promotes dephosphorylation of phosphoserine-47 on SIRT1. (a) WT or GADD34−/− MEFs expressing GFP-SIRT1 were treated with 50 μM Ars for 0, 2 and 4 h before immunoblotting for phosphoserine-47 SIRT1 (pSer47-SIRT1), GFP-SIRT1, phosphoserine-51 eIF2α (p-eIF2α), eIF2α, GADD34 and tubulin. (b) Ratio of pSer47-SIRT1/GFP-SIRT1 level in WT or GADD34−/− MEFs treated with Ars for indicated times is shown as a bar graph (mean±S.E.M., n=3 independent experiments). Tuke’s multiple comparison test was used after two-way ANOVA to generate P-values (*P<0.05, NS denotes not significant). (c) Fold change of p-eIF2α/eIF2α in WT or GADD34−/− MEFs treated with Ars for indicated times is shown as a bar graph (mean±S.E.M., n=3 independent experiments). Dunnett’s multiple comparisons test was used after two-way ANOVA to generate P-values (*P<0.05, NS denotes not significant). (d) HEK293 cells expressing FLAG empty vector, FLAG-GADD34 or FLAG KARA were either UT (−) or Ars-treated (+)before immunoprecipitations using anti-FLAG conjugated beads. IP and WCLs were immunoblotted for SIRT1, eIF2α, PP1α and the FLAG-GADD34. (e) GFP-SIRT1 was coexpressed with either empty FLAG vector, FLAG-GADD34 or FLAG-KARA in GADD34−/− MEFs. As control, GFP-SIRT1 was co-expressed with empty FLAG vector in WT MEFs. Transfected MEFs were UT (−) or treated with 50 μM Ars for 4 h and immunoblotted as described in a. (f) Ratio of pSer47-SIRT1/GFP-SIRT1 in WT or GADD34−/− MEFs expressing various FLAG plasmids with or without Ars treatment (mean±S.E.M., n=3). Sidak multiple comparison test was used after two-way ANOVA to generate P-values (*P<0.05)

Figure 7

GADD34 enhances SIRT1’s deacetylase activity. (a) HEK293 cells expressing FLAG-GADD34 were exposed to increasing concentrations of Ars (1 h) and immunoblotted for acetylated p53 (ac-p53), p53, SIRT1, eIF2α, p-eIF2α, FLAG-GADD34 and Tubulin. (b) Quantification of the fold-change in ac-p53/p53 in HEK293 cells expressing FLAG or FLAG-GADD34 in the presence of increasing Ars concentrations (mean±S.E.M., n=3). Sidak’s multiple comparison test was used after two-way ANOVA to generate P-values (*P<0.05). (c) HEK293 cells expressing either FLAG or increasing amounts of the FLAG-GADD34 plasmid DNA were treated either without or with Ars (0.5 mM, 1 h) before immunoblotting for ac-p53, p53, SIRT1, eIF2α, p-eIF2α and FLAG-GADD34. (d) Ratio of ac-p53/p53 level in HEK293 cells expressing FLAG or increasing amounts of FLAG-GADD34 either UT (Ars−) or Ars-treated (+). (Mean±S.E.M., n=3). Tukey’s multiple comparison test was used after two-way ANOVA to generate P-values (*P<0.05 and ***P<0.001)

Figure 8

Loss of function of GADD34 or SIRT1 attenuates apoptosis induced by Ars. (a) Top: WT and GADD34−/− MEFs were treated with 50 μM Ars for the indicated duration (hours) and immunoblotted for caspase 3, cleaved-caspase 3 and tubulin. Bottom: viability of WT and GADD34−/− MEFs was monitored over the time-course of the treatment with 50 μM Ars. (Mean±S.E.M., n=3). Sidak’s multiple comparison test was used after two-way ANOVA to generate P-values (*P<0.05, **P<0.01). Top: WT and SIRT1−/− MEFs were treated with 50 μM Ars for the indicated duration (hours) and cell lysates were immunoblotted for SIRT1, caspase 3, cleaved-caspase 3 and tubulin. (b) Bottom: viability of WT and SIRT1−/− MEFs was monitored over the time-course of the treatment with 50 μM Ars. (Mean±S.E.M., n=4 independent experiments). Sidak’s multiple comparison test was used after two-way ANOVA to generate P-values (*P<0.05). (c) WT and GADD34−/− MEFs were treated with 50 μM Ars for the indicated duration (hours) and immunoblotted for SIRT1, GADD34, p-eIF2α, eIF2α, ATF4, CHOP and tubulin. (d) WT and SIRT1−/− MEFs were treated with 50 μM Ars for the indicated duration (hours) and immunoblotted for SIRT1, GADD34, p-eIF2α, eIF2α, ATF4, CHOP and tubulin. (e) WT MEFs were treated with either 50 μM Ars for the indicated duration (hours) or pretreatment of Nicotinamide (Nico, 5 mM, overnight) following Ars exposure for the indicated duration (hours). Treated MEFs were immunoblotted with SIRT1, GADD34, p-eIF2α, eIF2α, ATF4, CHOP, caspase 3, cleaved-caspase 3 and tubulin. (f) Fold change of p-eIF2α/eIF2α in treated MEFs as described in e is shown as a bar graph (mean±S.E.M., n=3 independent experiments). Sidak’s multiple comparisons test was used after two-way ANOVA to generate P-values (*P<0.05). (g) Fold change of cleaved-caspase 3/caspase 3 in treated MEFs as described in e is shown as a bar graph (mean±S.E.M., n=3 independent experiments). Sidak’s multiple comparisons test was used after two-way ANOVA to generate P-values (**P<0.01)

Figure 9

Oxidative stress regulates eIF2α phosphorylation and dephosphorylation (The 5-STEP Model). Oxidative stress promotes eIF2α phosphorylation at serine-51, which is enhanced by the acetylation of lysines-141/143. In addition to increasing p-eIF2α, oxidative stress engages other as yet unknown mechanisms to elevate cellular GADD34 to much higher levels than seen with other stresses (STEP 1). Assembly of the GADD34/p-eIF2α/PP1α complex (STEP 2) enables eIF2α dephosphorylation and terminates ISR signaling. To ensure adequate downstream ISR signaling, we propose that acetylation of eIF2α at lysines 141/143 impedes the dephosphorylation by GADD34-bound PP1α. However, oxidative stress also recruits p-SIRT1, whose phosphorylation at serine-47 is enhanced by stress (STEP 3). Assembly of the new SIRT1/GADD34/eIF2α/PP1α complex enables SIRT1 dephosphorylation by the GADD34-bound PP1α and the resulting SIRT1 activation (STEP 4) catalyzes eIF2α deacetylation. This removes the brake on GADD34-bound PP1α and accelerates eIF2α dephosphorylation (STEP 5) to terminate ISR signaling. Thus, the coordination of eIF2α deacetylation with its dephosphorylation generates a delay that ensures the activation of the complex transcriptional and translational program known as ISR that is critical for cell survival