Obesity-Linked Phosphorylation of SIRT1 by Casein Kinase 2 Inhibits Its Nuclear Localization and Promotes Fatty Liver

S164-SIRT1 phosphorylation inhibits its nuclear localization. (A) Illustration of the locations of S172 (mS164), E230 (mE222), and NLS2 in human SIRT1. The structure of human SIRT1 from residues 174 to 658 (Protein Data Bank entry 5BTR ) is shown. For illustration purposes, Ser-Ser has been added at the N terminus to represent S172 and S173. The image on the right has been rotated 90° relative to that on the left. (B) Levels of endogenous SIRT1 and p-S164–SIRT1 in cytoplasmic (C) and nuclear (N) fractions were detected in duplicate by IB from mice fed an ND or HFD. Relative (Rel.) levels for each sample are shown below the blot, and the averages are plotted at the right. (C) Endogenous SIRT1 and p-S164–SIRT1 in liver sections of mice fed an ND or HFD were detected by IF. (D and E) SIRT1-WT, S164A-SIRT1, or S164D-SIRT1 was adenovirally expressed in hepatocytes from SIRT1-LKO mice, and the subcellular localization of SIRT1 was examined by biochemical fractionation (D) and IF (E) studies.

S164-SIRT1 phosphorylation inhibits its deacetylase activity. (A) Flag-SIRT1-WT, S164A-SIRT1, and S164D-SIRT1 were expressed in Cos-1 cells. Fluorescently labeled Ac-p53 peptide was incubated with Flag-SIRT1 proteins that were immunoprecipitated from whole-cell extracts, and SIRT1 deacetylase activity was measured as described in Materials and Methods. Results are shown as standard errors of the means (SEM) (n = 3); *, P value of <0.05 for Ad-SIRT(S164A) versus Ad-SIRT1 (164D). (B) Flag-SIRT1 proteins bound to M2 agarose were incubated with acetylated histone H3 in vitro, in the presence of NAD⁺ and an inhibitor of SIRT1, nicotinamide (NAM), as indicated, and levels of acetylated histone H3 and input proteins were detected by IB (SEM [n = 3]; *, P value of <0.05 for Ad-SIRT1 [164A] versus Ad-SIRT1 [164D]). Statistical significance was determined by one-way ANOVA with Tukey's test. (C and D) In-cell deacetylation assays. Cos-1 cells were transfected with plasmids as indicated, and acetylated levels of PGC-1α (C) and SREBP-1 (D) were measured by IP/IB. Relative amounts are indicated below the blot. Consistent results were observed from two independent deacetylation assays.

Obesity-induced CK2 mediates phosphorylation of SIRT1 at S164. (A) Primary mouse hepatocytes (PMH) were infected with Ad-Flag-SIRT1-WT and treated with cytokines as indicated for 1 h, and p-S164–SIRT1 and SIRT1 levels were measured by IB. Veh, vehicle; TNF-α, tumor necrosis factor alpha; IFN-γ, gamma interferon; LPS, lipopolysaccharide. (B) PMH were infected with Ad-Flag-SIRT1-WT; treated with TBB for CK2, SB203580 for mitogen-activated protein kinase, SP600125 for JNK, and PD98059 for MEK and then treated with IL-1β (10 ng/ml) for 1 h; and p-S164–SIRT1 levels were measured. DMSO, dimethyl sulfoxide. (C) PMH were transfected with two different siRNAs for CK2 or with control siRNAs for GFP. Twenty-four hours later, the cells were infected with Ad-Flag-SIRT1-WT or S164A, and 36 h later the cells were treated with IL-1β for 1 h and p-S164–SIRT1 levels were measured. (D) Immunoprecipitated Flag-SIRT1-WT or Flag-S164A-SIRT1, expressed in Cos-1 cells, was incubated with CK2, and p-S164–SIRT1 levels were detected. (E) CK2 interaction with GST fusion proteins containing different domains of SIRT1. Positions of S164 and the acidic cluster are shown. Bound CK2 was detected by IB. (F) Mice were fed an ND or HFD for 16 weeks, and the interaction of SIRT1 with CK2 was determined by CoIP using whole-cell liver extracts. (G) PMH were infected with adenovirus as indicated and treated with IL-1β, and interaction between SIRT1 and CK2 using whole-cell extracts was measured by CoIP. (H) Cellular localization of CK2 and p-S164–SIRT1 in liver sections from mice fed an ND or HFD diet for 16 weeks was examined by IF. (I and J) Hepatic protein (left) and mRNA (right) levels of CK2 in mice fed an ND or HFD for 16 weeks (I) or PMH treated with IL-1β or Fsk (10 μM) for 3 h (J) were determined. Below the CK2 protein blots, the CK2 levels relative to actin, with the first lane set to 1, are indicated. For the protein, 3 independent determinations are shown, and for mRNA, means ± SEM (n = 3) are shown. (K) Flag-SIRT1 was expressed in Cos-1 cells. (Left) Flag-SIRT1 immunoprecipitated from nuclear extracts by M2 agarose was incubated with CK2 in vitro, and SIRT1 and p-S164–SIRT1 levels were detected by IB. (Right) M2-agarose-bound Flag-SIRT1 then was washed with deacetylation buffer, and deacetylase activity was determined using fluorescently labeled Ac-p53 peptide (n = 6; results are means ± SEM; *, P < 0.05) as described in Materials and Methods. Statistical significance was determined by the Student t test. (L) Mice (n = 5/group) were fed an HFD for 16 weeks and then were treated with vehicle (Veh) or a CK2 inhibitor, CX-4945 (50 mg/kg of body weight), over a period of 40 days as previously described (28). Protein levels of endogenous SIRT1 and p-S164–SIRT1 in liver extracts were determined by IB. Normal levels of SIRT1 in livers from ND mice are shown for comparison.

S164-SIRT1 phosphorylation promotes pathological symptoms of liver steatosis. (A) C57BL/6J male mice were fed an ND or HFD for 16 weeks and injected via the tail vein with adenoviruses as indicated, and 2 weeks later the mice were sacrificed for analyses. (B) Protein levels of SIRT1-WT or S164 mutants and GFP (an indicator of viral infection) and p-S164–SIRT1 in whole-cell liver extracts are shown. (C) SIRT1 in liver sections was detected by IF, and merged images of SIRT1 with nuclear 4′,6-diamidino-2-phenylindole (DAPI) staining are shown. The scale bars indicate 50 μm. (D) Liver/body weight ratios. (E) Liver sections stained with H&E and Oil Red O. (F) Liver TG levels. (G) Fasting plasma levels of glucose, insulin, IL-6, and MCP-1. (H) Plasma glucose levels at the indicated times after i.p. injection of glucose are shown. (I) Plasma glucose levels at the indicated times after i.p. injection of insulin were measured, and areas under the curve (AUC) are shown. (J) Hepatic mRNA levels of the indicated genes were measured by qRT-PCR. For panels D and F to J, data shown are means ± SEM (n = 5 mice/group); *, P < 0.05; **, P < 0.01; ns, statistically not significant. Statistical significance was determined by one-way ANOVA with Tukey's posttest.

S164-SIRT1 phosphorylation inhibits β-oxidation and promotes liver steatosis. SIRT1-LKO mice fed an HFD for 6 weeks were infected with Ad-GFP, Ad-Flag-SIRT1-WT, or Ad-Flag-S164D-SIRT1 for 1 week. (A) Hepatic SIRT1 protein levels and p-S164–SIRT1 levels in SIRT1-LKO mice expressing SIRT1-WT or S164D-SIRT1 and control WT mice fed an ND (bottom) were determined by IB. (B) Relative liver weight as a percentage of total body weight (n = 4 mice/group). (C and D) Liver TG levels and liver sections stained with H&E and Oil Red O. (E) Plasma glucose levels at the indicated times after i.p. injection of glucose. (F) Hepatic mRNA levels of the indicated genes measured by qRT-PCR. (G and H) Serum β-hydroxyl butyrate and liver acylcarnitine levels determined by metabolomic analysis. (I and J) SIRT1-WT or S164D-SIRT1 was adenovirally expressed in primary mouse hepatocytes for 48 h, and palmitate oxidation (I) and the oxygen consumption rate (OCR) (J) were measured as described in Materials and Methods (means ± SEM; n = 6). Statistical significance was determined by one-way ANOVA with Tukey's posttest (B, C, and E to I) and by the Student t test (J).