Figure 3. SIRT2 binds to, deacetylates and activates GSK3β.

(A) Western blot analysis of acetylated GSK3β in heart samples of 9 months old WT and SIRT2-KO littermates. GSK3β was immunoprecipitated from heart tissue lysates of WT and SIRT2-KO mice using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnolgy), and the affinity resin immobilized with protein A/G. Western blotting was performed to detect GSK3β acetylation by anti-Ac-Lysine antibody. IgG was used as a negative control. Whole cell lysates (WCL) were probed for the SIRT2 and GAPDH by western blotting. n = 4 mice per group. (B) Histogram showing relative acetylated GSK3β in 9 months old WT and SIRT2-KO mice heart tissues, as measured from Figure 3A. Signal intensities of acetylated GSK3β and GSK3β were measured by densitometry analysis (ImageJ software). n = 4 mice per group. Data is presented as mean ± s.d, *p<0.05. Student’s t test was used to calculate the p values. (C) GSK3β was immunoprecipitated from heart tissue lysates of 8 weeks old 129/Sv mice using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnolgy ), and the affinity resin immobilized with protein A/G. GSK3β interaction with SIRT2 was tested by western blotting using anti-SIRT2 antibody. IgG was used as negative control. Heart lysates was probed for indicated proteins by western blotting. (D) In vitro binding assay to test the interaction between GSK3β and SIRT2. Flag-SIRT2 was overexpressed in 293 cells by a plasmid encoding human Flag-SIRT2. Recombinant His or His-GSK3β was purified from E. coli BL21 (DE3) by Ni-NTA affinity chromatography and were incubated with 293 T cell lysates overexpressing human Flag-SIRT2. Interaction between GSK3β and SIRT2 was tested by western blotting. # marked western images denotes SIRT2 antibody used in this assay detects single band. (E) In vitro deacetylation assay showing SIRT2 as GSK3β deacetylase. Human HA-GSK3β was overexpressed in HeLa cells by transfection of the plasmid pcDNA3-HA-GSK3β. HA-GSK3β was immunoprecipitated using HA-coupled agarose beads (Sigma-Aldrich) and the HA-GSK3β was acetylated by recombinant p300 (Millipore), in the presence or absence of Acetyl-CoA (Ac-CoA) in HAT buffer. The acetylated HA-GSK3β was further incubated with either Flag-tagged SIRT2 or SIRT2-H187Y, which were immunoprecipitated from HEK 293 cell lysates overexpressing respective plasmids encoding Flag-tagged WT or SIRT2-H187Y using agarose beads conjugated to anti-Flag antibody (Sigma A2220). The deacetylation reaction was carried out in the presence or absence of NAD⁺ in a HDAC buffer. GSK3β acetylation was analyzed by western blotting using anti-Ac-Lysine antibody. # marked western images denotes SIRT2 antibody used in this assay detects single band. (F) In vitro kinase assay depicting the activity of acetylated and deacetylated GSK3β. Human HA-GSK3β was overexpressed in HeLa cells by transfection of the plasmid pcDNA3-HA-GSK3β. Recombinant HA-GSK3β was immunoprecipitated using HA-coupled beads and was acetylated by recombinant p300 in the presence or absence of Acetyl-CoA (Ac-CoA) in HAT buffer. Acetylated GSK3β was further deacetylated by either Flag-tagged WT or SIRT2-H187Y (SIRT2-HY), a catalytic inactive mutant of SIRT2, which was immunoprecipitated from HEK 293 cells, overexpressed with plasmid encoding Flag-tagged WT or SIRT2-H187Y using agarose beads conjugated to anti-Flag antibody (Sigma A2220). The deacetylation reaction was carried out in the presence or absence of NAD⁺ in a HDAC buffer and further enzymatic activity of GSK3β was measured against glycogen synthase (GS)-peptide, as described in the Materials and methods section. n = 5. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values. (G) Western blot analysis of acetylated GSK3β from control or SIRT2-depleted (SIRT2-KD) cardiomyocytes. Neonatal rat cardiomyocytes were transfected with either non-targeting (control) or siRNA targeting SIRT2 using Lipofectamine RNAiMAX reagent for 72 hr. SIRT2 depletion was confirmed by Western blotting. Total cellular acetylation was probed by anti-Ac-Lysine antibody to test the effect of SIRT2 depletion in cardiomyocytes. GSK3β was immunoprecipitated from these cell lysates using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechnolgy), and the affinity resin immobilized with protein A/G. Western blotting was performed to detect acetylation of GSK3β by anti-Ac-Lysine antibody. Cell lysates (WCL) from control and SIRT2-KD cardiomyocytes were probed for indicated proteins by western blotting. (H) Western blotting analysis of hearts lysates from 9 months old WT and SIRT2-KO mice littermates for indicated proteins. n = 4 mice per group. (I) Histogram showing activity of GSK3β in WT and SIRT2-KO mice hearts at 9 months of age. GSK3β was immunoprecipitated from the heart lysates of WT and SIRT2-KO mice using anti-GSK3β antibody, clone GSK-4B (Sigma). The immunoprecipitated GSK3β was incubated with the peptide substrate in the presence of γ−³²P-ATP. The incorporation of ³²P into the GSK3β Peptide Substrate, which contains specific phosphorylation residue of GSK3β was measured. n = 6 mice per group. Data is presented as mean ± s.d. *p<0.05. Student’s t test was used to calculate the p values. (J) In vitro deacetylation assay to test whether SIRT2 deacetylates K183 residue of GSK3β. HA-tagged GSK3β or GSK3β-K183R was overexpressed in HeLa cells and was immunoprecipitated using HA-coupled beads. HA-tagged WT-GSK3β or GSK3β-K183R were incubated with Flag-SIRT2 immunoprecipitated from HEK 293 T cells using agarose beads conjugated to Anti-Flag antibody (Sigma A2220). The deacetylation reaction was carried out in the presence or absence of NAD⁺ in a deacetylation buffer. Acetylation status of GSK3β was analyzed by western blotting. # marked western images denotes SIRT2 antibody used in this assay detects single band. (K) Histogram showing relative acetylation of HA-tagged GSK3β or GSK3β-K183R, which was incubated with Flag-SIRT2. The data is generated from Figure 3J. Signal intensities of acetylated-GSK3β and GSK3β were measured by densitometry analysis (ImageJ software). n = 4 independent experiments. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values. (L) Histogram showing binding of γ−³²P-ATP to acetylated and deacetylated His-GSK3β. Recombinant His-GSK3β was purified from E. coli BL 21 (DE3) by Ni-NTA affinity chromatography. Purified His-GSK3β was acetylated by recombinant p300 in the presence of Ac-CoA in HAT buffer. Acetylated His-GSK3β was further deacetylated by Flag-SIRT2 immunoprecipitated from HEK 293 T cells. The binding of γ−³²P-ATP to acetylated and deacetylated His-GSK3β was assessed by the protocol described in Materials and methods section. n = 4. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values. (M) Histogram showing activity of WT or mutants of GSK3β. HA-tagged WT or mutants of GSK3β was immunoprecipitated from HeLa cells transfected with respective plasmids using HA-coupled agarose beads. The enzymatic activity of GSK3β was measured against glycogen synthase (GS)-peptide, as described in the Materials and methods section. n = 4. Data is presented as mean ± s.d. *p<0.05. One-way ANOVA was used to calculate the p values.

Figure 3—figure supplement 1. Western blotting analysis showing acetylation and phosphorylation of GSK3β and its downstream target GS in HeLa cells overexpressing the Sirtuin isoforms, SIRT1-SIRT7.

Cells were transiently overexpressed with either pcDNA-Flag or pcDNA-Flag-SIRT1-7 plasmid for 48 hr, and western blotting was performed to detect the indicated proteins. HeLa cell lysates (WCL) were probed with Flag-antibody for detecting the expression of Sirtuins. GSK3β was immunoprecipitated from these overexpressed lysates using anti-GSK3β antibody (sc-9166, Santa Cruz Biotechology) and tested for its acetylation by western blotting using anti-Ac-Lys antibody.

Figure 3—figure supplement 2. Western blotting analysis of vehicle or SIRT2 inhibitor, AGK2-(10 µM, 12 hr) treated rat neonatal cardiomyocytes for indicated proteins.

Acetylation of tubulin at K40, which is a specific deacetylation target of SIRT2 was probed to test the efficacy of SIRT2 inhibition. GSK3β was immunoprecipitated from these cell lysates using anti-GSK3β antibody, and the affinity resin immobilized with protein A/G. GSK3β acetylation was detected by Western blotting.

Figure 3—figure supplement 3. Representative confocal images of vehicle or AGK2 (10 µM, 12 hr) treated HeLa cells stained with GSK3β (Green).

MnSOD was used to localize the mitochondria (Red). DAPI was used to stain the nucleus (Blue).

Figure 3—figure supplement 4. Representative confocal images of HA-tagged WT or mutants of GSK3β transiently overexpressed in GSK3β-deficient mouse embryonic fibroblasts.

Immunostaining was performed using HA antibody to localize WT and mutants of GSK3β (Red).

Figure 3—figure supplement 5. Western blot analysis of 9 months old WT and SIRT2-KO mice heart samples for indicated proteins.

n = 4 mice per group.