SIRT1 associates with eIF2-alpha and regulates the cellular stress response

Abstract

SIRT1 is a NAD+ dependent protein deacetylase known to increase longevity in model organisms. SIRT1 regulates cellular response to oxidative and/or genotoxic stress by regulating proteins such as p53 and FOXO. The eukaryotic initiation factor-2, eIF2, plays a critical role in the integrated stress response pathway. Under cellular stress, phosphorylation of the alpha subunit of eIF2 is essential for immediate shut-off of translation and activation of stress response genes. Here we demonstrate that SIRT1 interacts with eIF2α. Loss of SIRT1 results in increased phosphorylation of eIF2α. However, the downstream stress induced signaling pathway is compromised in SIRT1-deficient cells, indicated by delayed expression of the downstream target genes CHOP and GADD34 and a slower post-stress translation recovery. Finally, SIRT1 co-immunoprecipitates with mediators of eIF2α dephosphorylation, GADD34 and CreP, suggesting a role for SIRT1 in the negative feedback regulation of eIF2α phosphorylation.

Figure 1. (A) and (B) Co-immunoprecipitation of endogenous SIRT1-eIF2α: Whole cell extracts from HeLa cells were immunoprecipitated for eIF2α or SIRT1 using respective antibodies.

Samples were then probed for SIRT1 and eIF2α respectively, by Western blotting. (C) and (D) Co-immunoprecipitation of epitope tagged SIRT1-eIF2α: HeLa cells were transfected with flag-tagged SIRT1 and myc-tagged eIF2α. 24 hr post transfection, whole cell extracts were immunoprecipitated for SIRT1 or eIF2α using flag or myc antibodies respectively, followed by Western blot analysis using anti-myc and anti-flag antibodies respectively. IgG antibody was used as a negative control for immunoprecipitation and 25–50μg of whole cell protein lysate was used as input. The data presented is a representative of multiple independent experiments.

Figure 2. (A) eIF2α phosphorylation in SIRT1 deficient HeLa cells in response to stress: SIRT1 depleted and matched-control HeLa cells were treated as indicated for 1 hr.

The whole cell lysates were analyzed by Western blotting using phosphorylated and total protein antibodies for eIF2α. Tubulin was used as a loading control. –leu: leucine stravation, -glc: glucose starvation, -serum: serum starvation, +TM: 2μg/ml tunicamycin, H₂O₂ : 500 nM hydrogen peroxide, +: Negative control RNAi HeLa cells, -: SIRT1 RNAi HeLa cells. (B) and (C) Kinetics of eIF2α phosphorylation in SIRT1 depleted HeLa cells (B) and SIRT1-/- MEFs (C): SIRT1-deficient and matched control cells were subjected to ER stress (thapsigargin) or leucine starvation for indicated time periods. The bar graphs represent the relative band intensities (mean ± SD from two independent experiments). Combined two-tailed p value across all group sets was calculated using student's t-test D. Basal eIF2α phosphorylation level in WT, SIRT1 null, and stably SIRT1 over-expressing null MEFs. Whole cell lysates were analyzed by Western blot analysis using phospho- or total protein antibody for eIF2α. +: Negative control RNAi HeLa cells or SIRT1+/+ MEFs, -: SIRT1 RNAi HeLa cells or SIRT1-/- MEFs.

Figure 3. CHOP expression in SIRT1 deficient cells in response to ER stress (A) and amino acid (B) starvation: SIRT1 knock-out and WT MEFs were treated with thapsigargin (ER stress) or leucine starvation for indicated time period.

Whole cell lysates were analyzed by western blot analysis. The bar graph represents the relative band intensities (mean ± SD from two independent experiments). Combined two-tailed p value across all group sets was calculated using student's t-test. (C) mRNA expression of CHOP gene in SIRT1-deficient MEFs: WT and SIRT1 null MEFs treated with TG (2 uM) for specified time-period and analyzed by qPCR. Data represent expression levels relative to untreated WT MEFs (assigned an arbitrary value of 1).

Figure 4. (A) Gene expression of GADD34 in SIRT1-deficient MEFs.: WT and SIRT1 null MEFs treated with or without TG (2 uM) for 1 h, and cells were analyzed after specified time-period by qPCR.

Data represent expression levels relative to untreated WT MEFs (assigned an arbitrary value of 1). The two-tailed p value for specified groups were calculated using student's t-test. (B) Post-stress translation recovery in SIRT1 deficient cells in response to ER stress: Wild-type and SIRT1 null mouse embryonic fibroblasts were treated with 2 μM thapsigargin for 1 hr. Cells were then pulsed with 200 μCi/ml ³⁵S labeled methionine for 30 min at each time point and new protein synthesis was assessed by incorporation of ³⁵S in cellular protein. Actin was used as the loading control. +: SIRT1+/+ MEFs, -: SIRT1-/- MEFs. (C) The graph represents the relative band intensities of the blot in ‘B' calculated using Image J software after normalization with the loading control actin. The combined two-tailed p value across all group sets was calculated using student's t-test.

Figure 5. SIRT1-eIF2α association under stress conditions: (A) and (B) HeLa cells were transfected with flag-tagged SIRT1 and myc-tagged eIF2α expression plasmids.

24 h post-transfection, cells were either treated with vehicle alone or –leucine media or tunicamycin (2 μg/ml) for 1 hr. Whole cell lysates were used for immunoprecipitation of eIF2α or SIRT1 using anti-myc-tag or anti-flag-tag antibody respectively and Western blotted with flag for SIRT1 or eIF2α antibody. IgG antibody was used as negative control of immunoprecipitation and 25 μg whole cell lysate was used as input. The blots were also probed for the immunoprecipitated proteins. (C) Co-immunoprecipitation of SIRT1 with the phosphorylation mutant and mimetic eIF2α. (D) Co-immunoprecipitation of the catalytic-mutant-SIRT1 with eIF2α: HeLa cells were transfected with the indicated expression plasmids. 24 h post-transfection whole cell lysates were extracted and used for immunoprecipitation of eIF2α using anti-myc-tag antibody and Western blotted with flag antibody for SIRT1. IgG antibody was used as negative control of immunoprecipitation and 25 μg whole cell lysate was used as input. The data presented is a representative of multiple independent experiments.

Figure 6. SIRT1 interacts with the proteins that mediate eIF2α dephosphorylation: 293T cells were transfected either with flag-tagged GADD34 and myc-tagged SIRT1, or with flag-tagged CReP and myc-tagged SIRT1 expression plasmids.

48 h post-transfection whole cell lysates were used for immunoprecipitation of (A) GADD34 or CReP using anti-flag-tag antibody or (B) SIRT1 using anti-SIRT1 antibody and Western blotted with SIRT1 or flag respectively. IgG antibody was used as negative control of immunoprecipitation and 10–30 μg whole cell lysate was used as input. Lanes 1& 3: cells transfected with flag-CReP and myc-SIRT1, Lanes 7&9: with flag-GADD34 and myc-SIRT1. The data presented is a representative of two independent experiments.