Inhibitory protein-protein interactions of the SIRT1 deacetylase are choreographed by post-translational modification

Abstract

Regulation of SIRT1 activity is vital to energy homeostasis and plays important roles in many diseases. We previously showed that insulin triggers the epigenetic regulator DBC1 to prime SIRT1 for repression by the multifunctional trafficking protein PACS-2. Here, we show that liver DBC1/PACS-2 regulates the diurnal inhibition of SIRT1, which is critically important for insulin-dependent switch in fuel metabolism from fat to glucose oxidation. We present the x-ray structure of the DBC1 S1-like domain that binds SIRT1 and an NMR characterization of how the SIRT1 N-terminal region engages DBC1. This interaction is inhibited by acetylation of K112 of DBC1 and stimulated by the insulin-dependent phosphorylation of human SIRT1 at S162 and S172, catalyzed sequentially by CK2 and GSK3, resulting in the PACS-2-dependent inhibition of nuclear SIRT1 enzymatic activity and translocation of the deacetylase in the cytoplasm. Finally, we discuss how defects in the DBC1/PACS-2-controlled SIRT1 inhibitory pathway are associated with disease, including obesity and non-alcoholic fatty liver disease.

Keywords: CK2; DBC1; GSK3; NAFLD; PACS‐2; SIRT1; acetylation; insulin signaling; liver metabolism; obesity; post‐translational modification; protein–protein interactions.

FIGURE 1

Domain structure of the players in the DBC1, PACS‐2, SIRT1 protein interaction hub and protein constructs used in the present study. (a) Amino acid numbering is for the human proteins and the position of the N‐ and C‐terminal amino acids in the subcomponents are shown by superscripts after the names. For SIRT1 truncations marked with *, the amino acids from K506‐N640 have been replaced with a GGGSGGGS linker as per Dai et al. (2015). The rounded rectangles represent domains or structured elements (gray = structural data verifying the proposed domain is not yet available: LZ = Leucine Zipper, EF = EF Hand, CC = coiled‐coil). Magenta circles represent relevant phosphorylation sites for this manuscript and yellow circles denote acetylation sites. Molecular masses of proteins are shown along the right. (b) Respiratory quotient (RQ = VCO₂/VO₂) expressed as mean during the light and dark cycles over a 24 h period. (c) RT‐qPCR of liver Cpt1α isolated from WT or Pacs2 ^LKO mice that were fed or fasted for 14 h and then re‐fed ad libitum for 4 h. Data are mean + SD. n = 4 mice per group. (d) Isolated WT and Pacs2 ^LKO primary hepatocytes were starved overnight and treated for 6 h with 10 μM WY‐14643 with or without 100 nM insulin. Cpt1α was measured by qRT‐PCR. Data are mean ± SD; n = 3. (e) Primary hepatocytes from WT and Pacs2 ^LKO mice were isolated and subjected to overnight starvation. They were then treated with the PPARα agonist WY‐14643 for 6 h followed by treatment with 10 μM Ex‐527 for 1 h. Fgf21 was measured by qRT‐PCR. Data are mean ± SD; n = 3.

FIGURE 2

Identification of the SIRT1 binding region on the DBC1 S1‐L domain. (a) Superposition of ¹H,¹⁵N HSQC spectra of the DBC1 S1‐L in the presence (●) or absence (●) of 300 μM SIRT1 NTR. (b) Combined ¹H,¹⁵N CSPs for DBC1 S1‐L in the presence of 500 μM SIRT1 NTR. The line denotes the average CSP + 1 standard deviation. (c) Individual binding isotherms for six DBC1 S1‐L resonances that experience CSPs in the presence of the SIRT1 NTR. Global fitting yields a K _d value of 240 ± 20 μM for the interaction. (d) Ribbon representation of the DBC1 S1‐L x‐ray structure, with amino acid positions shown in magenta whose resonances exhibit significant CSPs in the presence of the SIRT1 NTR. The position of K112 is marked by a black dot.

FIGURE 3

The effect of the S1‐L^K112Q acetylation mimetic on SIRT1 binding. (a) Superposition of the ¹H,¹⁵N HSQC spectra of S1‐L (●) and S1‐L^K112Q (●) and co‐immunoprecipitation of SIRT1‐flag with ha‐tagged DBC1 or DBC1^K112Q (inset). (b) Superposition of the ¹H,¹⁵N HSQC spectra of S1‐L^K112Q in the presence (●) or absence (●) of 500 μM SIRT1 NTR. (c) Superposition of the ¹H,¹⁵N HSQC spectra of S1‐L in the presence (●) or absence (●) of 150 μM SIRT1^1‐655*. (d) Superposition of the ¹H,¹⁵N HSQC spectra of DBC1 S1‐L^K112Q in the presence (●) or absence (●) of 150 μM SIRT1^1‐655*.

FIGURE 4

Mapping of DBC1 S1‐L binding on the SIRT1 NTR. (a) Superposition of the ¹H,¹⁵N HSQC spectra of the SIRT1 NTR in the presence (●) or absence (●) of 100 μM DBC1 S1‐L and individual binding isotherms for seven resonances. Global fitting yielded a K _d value of 290 ± 40 μM. (b) Combined ¹H,¹⁵N CSPs for the 3HB region of the SIRT1 NTR in the presence of 500 μM DBC1 S1‐L. (c) Amino acid sequence of the SIRT1 NTR with residues that exhibit amide resonance CSPs in the presence of S1‐L highlighted by magenta boxes. In (b) and (c), the individual helices of the 3HB are indicated by brown bars above the sequence. (d) Superposition of the ¹H,¹⁵N HSQC spectra of the SIRT1^1‐124 in the presence (●) or absence (●) of 500 μM DBC1 S1‐L. The enlarged region depicts resonances from A73‐R77. (e) Superposition of the ¹H,¹⁵N HSQC spectra of the SIRT1 3HB in the presence (●) or absence (●) of 500 μM DBC1 S1‐L. In the inset, individual binding isotherms for four resonances are shown. Global fitting yields a K _d value of 1000 ± 300 μM.

FIGURE 5

Intra‐molecular interactions in the unstructured region of the SIRT1 NTR mapped by NMR. (a) Superposition of the ¹H,¹⁵N HSQC spectra of the SIRT1 NTR (●) and that of SIRT1^1‐124 (●) (left) and SIRT1^109‐233 (●) (right). (b) Amino acid sequence of the SIRT1 NTR with residues whose amide resonance exhibit larger than average CSPs in the presence of SIRT1^1‐124 or SIRT1^109‐233 are highlighted by magenta boxes. Helices of the 3HB are indicated by brown bars above the sequence. (c) Combined amide CSPs of SIRT1^1‐54 in the presence of 500 μM SIRT1^109‐233. (d) Combined amide CSPs of SIRT1^1‐84 in the presence of 500 μM SIRT1^109‐233. (e) Combined amide CSPs of SIRT1^109‐233 in the presence of 500 μM SIRT1^1‐54.

FIGURE 6

Phosphorylation of SIRT1 on S162 and S172 impacts the 3HB. (a) Primary WT mouse hepatocytes expressing SIRT1‐FLAG were starved overnight and then treated with or without 100 nM insulin for 30 min. FLAG‐tagged SIRT1 was immunoprecipitated and pS¹⁶⁴‐SIRT1 was detected by western blot. (b) Immunoprecipitation of DBC1‐ha, co‐expressed with flag‐tagged SIRT1, SIRT1^S162D/S172D or SIRT1^S162A/S172A in HCT116 cells detected by western blotting (HA). Data are average ± SD, n = 3. (c) Superposition of ¹H,¹⁵N HSQC spectra of u¹⁵N labeled SIRT1 NTR prior to (●) and following incubation (●) with 100 nM CK2 (top), 500 nM GSK3β (middle), or both CK2 and GSK3β (bottom) for 6 h. (d) Quantification of resonance intensities for S162 and S172. (e) Isolated mouse primary hepatocytes co‐expressing SIRT1‐flag and PACS‐2‐ha were starved for 16 h and then treated with 10 μM TBB or 0.5 μM CHIR98014 for 1 h followed by 10 nM insulin treatment for 30 min. SIRT1‐flag was immunoprecipitated and bound PACS‐2‐ha was detected by western blot. Data are average ± SD, n = 3. (f) Amino acid sequence of the SIRT1 NTR with residues whose amide resonance experience larger than average CSPs upon S‐D substitution or CK2/GSK3β phosphorylation highlighted by magenta boxes. Helices of the 3HB are indicated by brown bars above the sequence.

FIGURE 7

Structure of the 3HB and the influence of alanine substitution on the DBC1 S1L binding. (a) Backbone ribbon representation of the 3HB in the x‐ray structure (PDB ID 4ZZH) (Dai et al., 2015). The side‐chains of L192 and M218 are shown in stick representation. (b) Superposition of the ¹H,¹⁵N HSQC spectra of the SIRT1 3HB, and (top) the M218A or (bottom) T219A variant in the presence (●) or absence (●) of 450 μM DBC1 S1‐L. Binding isotherms for individual resonances are shown in the insets. Global fitting yielded K _d values of 340 ± 30 μM and K _d = 320 ± 40 μM, respectively.

FIGURE 8

PACS‐2 facilitates SIRT1 cytoplasmic shuttling. (a) Primary WT and Pacs2 ^LKO hepatocytes were left untreated (fed) or serum‐starved overnight and then treated with or without 100 nM insulin for 30 min. Cells were fractionated into cytosolic and nuclear fractions and the nucleocytoplasmic distribution of SIRT1 was determined by western blotting. (b) Primary WT and Pacs2 ^LKO hepatocytes were transduced with adenoviruses expressing WT SIRT1, SIRT1^S164D or SIRT1^S164A. After 24 h, the cells were fractionated into cytosolic and nuclear fractions and the nucleocytoplasmic distribution of SIRT1 was determined by western blotting. Data are mean + SD. n = 3. (c) Active SIRT1 is primed for interaction with the DBC1 S1‐L by the sequential phosphorylation at S162 and S172 by CK2 and GSK3β, respectively. DBC1 binding alters the SIRT1 NTR, priming SIRT1 for interaction with PACS‐2 without disrupting the intramolecular communication network within the N‐terminus. PACS‐2 binding disrupts the 3HB and inactivates SIRT1. (d) Acetylation of K215 on DBC1 promotes SIRT1's nuclear retention (Hubbard et al., ; Zheng et al., 2013). Two phosphorylation events prime SIRT1 for interaction with DBC1. In response to metabolic cues, triggered by insulin, phosphorylation of hS162/mS154 by CK2 leads to phosphorylation on hS172/mS164 by GSK3β, enhancing the DBC1 S1‐L interaction with the SIRT1 NTR. PACS‐2, although predominantly cytoplasmic, shuttles between the cytoplasm and the nucleus (Atkins et al., 2014), requiring phosphorylation on S437 to interact with SIRT1 (Atkins et al., 2014). Following DBC1‐mediated exposure of the PACS‐2 bipartite binding site on SIRT1, PACS‐2^pS437 engages SIRT1 and disrupts the structure of the 3HB (Krzysiak et al., 2018) thereby lowering SIRT1 enzymatic activity. Subsequently, the complex containing PACS‐2/SIRT1 translocates from the nucleus to the cytoplasm.