Cross-talk between sirtuin and mammalian target of rapamycin complex 1 (mTORC1) signaling in the regulation of S6 kinase 1 (S6K1) phosphorylation

Abstract

p70 ribosomal S6 kinase (S6K1), a major substrate of the mammalian target of rapamycin (mTOR) kinase, regulates diverse cellular processes including protein synthesis, cell growth, and survival. Although it is well known that the activity of S6K1 is tightly coupled to its phosphorylation status, the regulation of S6K1 activity by other post-translational modifications such as acetylation has not been well understood. Here we show that the acetylation of the C-terminal region (CTR) of S6K1 blocks mTORC1-dependent Thr-389 phosphorylation, an essential phosphorylation site for S6K1 activity. The acetylation of the CTR of S6K1 is inhibited by the class III histone deacetylases, SIRT1 and SIRT2. An S6K1 mutant lacking acetylation sites in its CTR shows enhanced Thr-389 phosphorylation and kinase activity, whereas the acetylation-mimetic S6K1 mutant exhibits decreased Thr-389 phosphorylation and kinase activity. Interestingly, relative to the acetylation-mimetic S6K1 mutant, the acetylation-defective mutant displays higher affinity toward Raptor, an essential scaffolding component of mTORC1 that recruits mTORC1 substrates. These observations indicate that sirtuin-mediated regulation of S6K1 acetylation is an additional important regulatory modification that impinges on the mechanisms underlying mTORC1-dependent S6K1 activation.

Keywords: Acetylation; Phosphorylation; Rapamycin; S6 Kinase; Sirt1; Sirtuins; mTOR; mTOR Complex (mTORC).

FIGURE 1.

Nicotinamide specifically inhibits S6K1 phosphorylation. COS-7 cells were treated with 100 nm Torin 1, 5 mm NAM, 5 nm TSA, or NT for the indicated times. Total cell lysates were prepared and assessed by immunoblotting (WB) with the indicated antibodies. A, NAM, TSA, and NT induce cellular protein acetylation. Samples treated for 3 h from C–E were analyzed by immunoblotting with anti-acetyl-lysine and actin antibodies. B, Torin 1, a specific mTOR kinase inhibitor, suppresses both Thr-389 phosphorylation of S6K1 (pT389-S6K1) and Thr-37/46 and Ser-65 phosphorylation of 4EBP1 (pT37/46–4EBP1 and pS65–4EBP1) in a similar dose-dependent manner. C, NAM specifically attenuates S6K phosphorylation. pS235/236-S6, Ser-235/236 phosphorylation of S6. D, inhibition of Class I/II HDACs by TSA does not affect S6K1 phosphorylation. E, inhibitory effect of nicotinamide on S6K1 phosphorylation is augmented by TSA co-treatment.

FIGURE 2.

Inhibition of class III HDACs, the sirtuins, by nicotinamide induces acetylation of S6K1. A, S6K1 but not S6K2 acetylation (Ac-S6K) is induced by NT treatment. COS-7 cells were transfected with HA-S6K1 or HA-S6K2 plasmid. 48 h after transfection, the cells were treated with NT for 3 h. HA-S6Ks were IPed, and levels of acetylation were analyzed by immunoblotting with anti-acetyl-lysine antibody. B and C, NAM, but not TSA, induces S6K1 acetylation. HA-S6K1 was expressed in COS-7 (B) or HEK293T cells (C). Cells were treated with NAM, TSA, or NT for 3 h. Levels of S6K1 acetylation were monitored as in A. D and E, acetylation of endogenous S6K1 was enhanced by NT treatment. HEK293T (D) or COS-7 cells (E) were treated with or without NT for 3 h. Endogenous S6K1 was IPed, and levels of S6K1 acetylation were monitored as in A. Abs, antibodies; ctr IgG, control IgG.

FIGURE 3.

Both SIRT1 and SIRT2 are responsible for deacetylation of S6K1. A, overexpressed S6K1 interacts with exogenous SIRT1 and SIRT2. COS-7 cells were transfected with either HA-SIRT1 or HA-SIRT2 along with FLAG-S6K1. After 48 h, cells were treated with NT for 3 h, and FLAG-S6K1 was IPed. Immunoprecipitates were analyzed by immunoblotting using FLAG and HA antibodies. B, endogenous S6K1 interacts with endogenous SIRT1 and SIRT2. COS-7 cells were treated with NT for 3 h, and endogenous S6K1 was IPed. Immunoprecipitates were analyzed by immunoblotting using the indicated antibodies (Abs). Arrows indicate SIRT1 or SIRT2. C, S6K1 acetylation (Ac-S6K1) induced by NT treatment was suppressed by overexpression of SIRT1 or SIRT2. COS-7 cells were transfected with Myc-S6K1 together with HA-SIRT1 or HA-SIRT2 as indicated. SIRT1 HY and SIRT2 HY denote the deacetylase-inactive mutants of SIRT1 and SIRT2, respectively. Cells were treated with NT for 3 h and Myc-S6K1 was IPed. Levels of acetylation of Myc-S6K1 were determined. D, both endogenous SIRT1 and endogenous SIRT2 are responsible for S6K1 deacetylation. COS-7 cells were transfected with HA-S6K1 together with the indicated siRNAs against SIRT1, SIRT2, or GFP (Control). TSA treatment was for 3 h, as indicated. E, the effect of cytoplasmic HDAC6 knockdown on S6K1 acetylation. The indicated siRNAs were co-transfected with HA-S6K1. NT treatment (3 h) was as indicated. F, knockdown efficiency of the indicated siRNAs in COS-7 cells was examined by quantitative RT-PCR and immunoblotting. Error bars indicate mean ± S.D.

FIGURE 4.

Inhibition of sirtuins induces acetylation of lysines 484, 485, and 493 of S6K1. A, the CTR of S6K1 possesses NT-sensitive acetylation sites. COS-7 cells were transfected with full-length wild type (FL 1–502 amino acids) and two C-terminal truncation dC-S6K1 mutants (1–399 amino acids and 1–417 amino acids), and treated with NT for 3 h. Levels of acetylation (Ac) in the indicated S6K1s were determined. B and C, mutations of lysine 484 and 485 to arginine (DKR) largely inhibit NT-induced S6K1 acetylation. COS-7 cells were transfected with the indicated S6K1 KR mutants, and levels of acetylation induced by NT were monitored. 427R, 484R, 485R, 484/485R, and 493R denote KR mutation of the indicated residues. D, mutations of lysine 484, 485, and 493 to arginine (TKR) abolished NT-induced S6K1 acetylation. COS-7 cells were transfected with DKR or TKR and treated with NT for 3 h. An anti-acetyl lysine 493 antibody was used to determine the level of S6K1 acetylation.

FIGURE 5.

SIRT1 and SIRT2 enhance S6K1 phosphorylation and its kinase activity. A, COS-7 cells were transfected with the siRNAs against SIRT1 and SIRT2 or GFP (Control). 72 h after transfection, cells were starved with growth factors and nutrients with Hanks' balanced salt solution for 1 h and stimulated with the culture medium (10% FBS in DMEM) for the indicated times. Total cell lysates were prepared, and acetylated S6K1 was IPed by Ac-Lys-493-S6K1 antibody (Ac-K493-S6K1). Immunoprecipitates and lysates were analyzed by immunoblotting with the indicated antibodies. Levels of Thr-389 phosphorylation (pT389, left panel) were quantified with ImageJ (right panel). *, p < 0.05, versus 90 min control; mean ± S.D., n = 3. A. U., arbitrary units. B, transient overexpression of SIRT1 or SIRT2 increases S6K1 phosphorylation. COS-7 cells were co-transfected with HA-S6K1 and HA-SIRT1 or HA-SIRT2. After 48 h, total cell lysates were prepared, and levels of S6K1 phosphorylation were monitored by immunoblotting with phospho-specific Thr-389 (pT389), Thr-229 (pT229), and Thr-421/Ser-424 (pT421/S424) S6K1 antibodies. C, SIRT1 and SIRT2 overexpression enhances S6K1 kinase activity. COS-7 cells were co-transfected with Myc-S6K1 and HA-SIRT1 or HA-SIRT2. After 48 h, Myc-S6K1 was IPed and subjected to an in vitro kinase assay. GST-S6 was prepared from bacteria and used for the in vitro kinase assay as a substrate. The incorporated radioactivity into GST-S6 was quantified. Data were expressed as mean ± S.D., *, p < 0.05 versus control (without sirtuins) (n = 3). D and E, the effect of SIRT1 or SIRT2 on AKT or AMP-activated protein kinase (AMPK) phosphorylation. COS-7 cells were transfected with HA-S6K1 and GST-AKT or Myc-S6K1 and HA-AMP-activated protein kinase together with either HA-SIRT1 or HA-SIRT2. Experiments were performed as in panel A. Levels of AKT and AMP-activated protein kinase phosphorylation were monitored by immunoblotting with the indicated phospho-specific antibodies. pS473, Ser-473 phosphorylation; pT172, Thr-172 phosphorylation. F and G, activation of the NAD biosynthetic pathway stimulates S6K1 phosphorylation. Myc-nicotinamide phosphoribosyltransferase (Myc-NAMPT) (F) or Myc-nicotinamide mononucleotide adenylyltransferase (Myc-NMNAT) (G) was co-expressed with HA-S6K1 (upper panels) or GST-AKT (lower panels) in COS-7 cells. Levels of S6K1 or AKT phosphorylation were monitored by immunoblotting with the indicated phospho-specific antibodies.

FIGURE 6.

S6K1 acetylation attenuates mTORC1-dependent S6K1 phosphorylation. A, acetylation-defective mutant (TKR) showed higher levels of S6K1 phosphorylation. Wild type or TKR S6K1 mutant was expressed in COS-7 cells. After 48 h, cells were treated with either NAM or NT for 3 h before harvest. Levels of Thr-389 phosphorylation (pT389) were monitored by immunoblotting (left panel) and quantified by ImageJ (right panel). (*, p < 0.05, **, p < 0.01 versus control (Ctr), N.S. indicates not significant; mean ± S.D., n = 3). A. U., absorbance units. B, characterization of the acetylation-defective mutant (TKR) and acetylation-mimetic mutants (TKA, TKQ, and TKT) of S6K1. COS-7 cells were transfected with wild type, TKR, TKA, TKQ, or TKT S6K1. Levels of Thr-389 phosphorylation of S6K1 were monitored (left panel) and quantified (right panel) (*, p < 0.05 versus WT; mean ± S.D., n = 3). C, acetylation-defective mutant (TKR) shows higher kinase activity as compared with that of acetylation-mimetic mutant (TKA) in vitro. COS-7 cells were transfected with either HA-S6K1 TKR or TKA mutant. After 48 h, the HA-S6K1 mutants were IPed and subjected to an in vitro kinase assay. GST-S6 was used as a substrate. The incorporated radioactivity into GST-S6 was quantified. Data were expressed as mean ± S.D., *, p < 0.05 versus TKR (n = 3). D, proteasome inhibition induces acetylation of S6K1 concomitantly decreasing S6K1 phosphorylation. COS-7 cells were transfected with either wild type HA-S6K1 or acetylation-defective mutant (TKR). Cells were treated with 10 μm MG132 for the indicated times, and total cell lysates were analyzed by immunoblotting using the indicated antibodies. Ac-K493-S6K1, Ac-Lys-493-S6K1 antibody; pT421/S424, phosphorylation of Thr-421 and Ser-424. E, Thr-389 phosphorylation status does not affect S6K1 acetylation. Wild type, T389E (phosphorylation-mimetic mutant), or T389A S6K1 (phosphorylation-defective mutant) was expressed in COS-7 cells. 48 h after transfection, cells were treated with NT for 3 h. HA-S6K1s were IPed with HA antibody, and immunoprecipitates were analyzed by immunoblotting with the indicated antibodies (top panel). Levels of S6K1 acetylation were quantified (right panel). N.S. indicates not significant versus WT treated with NT; mean ± S.D., n = 3. F, TKR S6K1 shows higher affinity for mTORC1 as compared with TKA S6K1. COS-7 cells were co-transfected with Myc-Raptor together with either wild type HA-S6K1, HA-TKR, or HA-TKA S6K1 mutant. Levels of co-IPed HA-S6K1 were determined. G, schematic model of the effect of sirtuins on S6K1 phosphorylation by mTORC1. Deacetylation of S6K1 by sirtuins enhances mTORC1-dependent S6K1 phosphorylation (Thr-389). HATs indicate histone acetyltransferases.