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. 2021 Jul 1;297(2):100929. doi: 10.1016/j.jbc.2021.100929

Figure 1.

Figure 1

LKB1-dependent phosphorylation is required for resveratrol-induced Sirt1 activation.A, phosphorylation of Sirt1 in cells treated with resveratrol. HEK293T cells were infected with lentivirus-based particles expressing FLAG-Sirt1 for 12 h, and 48 h later cells were treated with 25 μM resveratrol for 6 h. Cells were collected at indicated time points and FLAG-Sirt1 proteins were immunoprecipitated by anti-FLAG and immunoblotted with anti-phospho-serine/threonine. Immunoprecipitated FLAG-Sirt1 proteins treated with lambda protein phosphatase at 30 °C for 30 min were negative controls. For C2C12 myoblasts, cells were infected with virus particles for 12 h. After infection, cells were continued to culture with fresh medium for 48 h and then were grown in DMEM with 2% horse serum for 4 days to generate C2C12 myotubes. The next steps were same as the mentioned process for HEK293T cells. Representative of three independent experiments. B, in vitro Sirt1 deacetylase activity assay. HEK293T cells were infected with lentivirus-based particles expressing FLAG-Sirt1 for 12 h, and 48 h later cells were treated with 25 μM resveratrol for 6 h. FLAG-Sirt1 proteins were immunoprecipitated by anti-FLAG and eluted by 3×FLAG peptide. Then 100 ng eluted FLAG-Sirt1 was incubated with 1 mM NAD+ and 2 μg GST-tagged K382ac p53 peptide from E. coli at 37 °C for 30 min in 40 μl Sirt1 assay buffer. The acetylation level of K382 site was analyzed by using anti-acetyl-p53 K382 antibody. The precipitated FLAG-Sirt1 pretreated with lambda protein phosphatase and then subjected to in vitro deacetylation assay was the negative control. Representative of three independent experiments. p53 K382 is the deacetylation site of Sirt1 and ack382-p53 is the marker of Sirt1 activity (6, 86). C, phosphorylation of Sirt1 in gene-depleted HEK293T cells treated with resveratrol. HEK293T cells were infected with lentivirus-based particles expressing shRNA control, CAMKKβ shRNA, AMPK shRNA, DYRK1A shRNA, DYRK3 shRNA, JNKs (JNK1 and JNK2) shRNAs, CK2α shRNA, or LKB1 shRNA for 12 h, and 48 h later cells were treated with 25 μM resveratrol for 6 h. Sirt1 proteins were immunoprecipitated by anti-Sirt1 antibody and immunoblotted with anti-phospho-serine/threonine. Representative of three independent experiments. D, phosphorylation of Sirt1 in resveratrol-treated LKB1-depleted HEK293T cells expressing FLAG-tagged WT LKB1. HEK293T cells were infected with lentivirus-based particles expressing shRNA control, AMPK shRNA, or LKB1 shRNA for 12 h, and 36 h later LKB1-depleted cells were infected with virus particles expressing FLAG-LKB1 for 12 h. After 36 h, cells were treated with 25 μM resveratrol for 6 h. Sirt1 proteins were immunoprecipitated by anti-Sirt1 antibody and immunoblotted with anti-phospho-serine/threonine. Representative of three independent experiments. E, the deacetylase activity of Sirt1 in resveratrol-treated LKB1-depleted cells. HEK293T cells were infected with lentivirus-based particles expressing shRNA control, AMPK shRNA, or LKB1 shRNA for 12 h. After 48 h, cells were pretreated with 1 μM doxorubicin for 1 h to increase in vivo K382 acetylation of p53 (deacetylation site of Sirt1) and then were treated with 25 μM resveratrol for 6 h. The whole-cell lysate (WCL) was immunoblotted with anti-acetyl-p53 K382. For C2C12 myoblasts, cells were first were infected with virus particles for 12 h. After infection, cells were continued to culture in fresh DMEM for 48 h and then were grown in DMEM with 2% horse serum for 4 days to generate myotubes. The next steps are same as the mentioned process for HEK293T cells. Representative of three independent experiments. (44). The p53 K382 is the deacetylation site of Sirt1 and ack382-p53 is the marker of Sirt1 activity (6, 86). F, the deacetylase activity of Sirt1 in resveratrol-treated LKB1-depleted HEK293T cells expressing WT LKB1 or kinase-dead mutant. HEK293T cells were infected with lentivirus-based particles expressing shRNA control or LKB1 shRNA for 12 h, and 36 h later LKB1-depleted cells were infected with virus particles expressing FLAG-LKB1 (WT or the KD mutant) for 12 h. After 36 h, cells were treated with 25 μM resveratrol for 6 h. WCL were immunoblotted with anti-acetyl-p53 K382. Endo LKB1, endogenous LKB1; KD, kinase dead; LKB1, Lys78Met; NAD, nicotinamide adenine dinucleotide, Sirt1 cofactor; pT172-AMPK, marker of LKB1 activity; RSV, resveratrol; (44).