S6K1 regulates GSK3 under conditions of mTOR-dependent feedback inhibition of Akt - PubMed (original) (raw)

S6K1 regulates GSK3 under conditions of mTOR-dependent feedback inhibition of Akt

Hui H Zhang et al. Mol Cell. 2006.

Erratum in

Abstract

Feedback inhibition of the PI3K-Akt pathway by the mammalian target of rapamycin complex 1 (mTORC1) has emerged as an important signaling event in tumor syndromes, cancer, and insulin resistance. Cells lacking the tuberous sclerosis complex (TSC) gene products are a model for this feedback regulation. We find that, despite Akt attenuation, the Akt substrate GSK3 is constitutively phosphorylated in cells and tumors lacking TSC1 or TSC2. In these settings, GSK3 phosphorylation is sensitive to mTORC1 inhibition by rapamycin or amino acid withdrawal, and GSK3 becomes a direct target of S6K1. This aberrant phosphorylation leads to decreased GSK3 activity and phosphorylation of downstream substrates and contributes to the growth-factor-independent proliferation of TSC-deficient cells. We find that GSK3 can also be regulated downstream of mTORC1 in a HepG2 model of cellular insulin resistance. Therefore, we define conditions in which S6K1, rather than Akt, is the predominant GSK3 regulatory kinase.

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Figures

Figure 1

Figure 1. GSK3 phosphorylation is constitutive in Tsc2 null cells and is elevated in TSC tumors

(A) Differential regulation of the Akt substrates FOXO3a and GSK3 in Tsc2−/− MEFs. Littermate-derived Tsc2+/+ (+) and Tsc2−/− (−) MEFs were cultured in the absence (−ser) or presence (+ser) of serum for 16 h, or serum starved then stimulated for 15 min with 50 ng/ml IGF1 or 5 ng/ml. (B) GSK3 phosphorylation is elevated in liver hemangiomas from Tsc2+/− mice. A representative example from five tumors obtained from five different Tsc2+/− mice aged 12 months is shown at 200X magnification; inset with adjacent normal tissue is shown at 1000X. Phospho-GSK3 is depicted in brown; nuclei are counterstained in blue. (C) GSK3 phosphorylation is elevated in cortical tuber giant cells from a TSC patient. A representative giant cell from a cortical tuber resection is shown stained with vimentin (green) and phospho-GSK3 (red). Bar: 20 μM.

Figure 2

Figure 2. Loss of the TSC1-2 complex leads to wortmannin-resistant rapamycin-sensitive phosphorylation of GSK3

(A) Littermate-derived Tsc1+/+ and Tsc1−/− MEFs were serum starved for 16 h and then pretreated for 15 min with 100 nM wortmannin (w) or 20 nM rapamycin (r) then stimulated for 15 min with 25 ng/ml IGF1, where indicated. Lysates were then immunoblotted with the indicated phospho-specific antibodies or antibodies to total GSK3 and eIF4E as loading controls. (B) Cells were treated as described in A, but using littermate-derived Tsc2+/+ and Tsc2−/− MEFs. (C) Rescue of the alternative regulation of GSK3 in Tsc2−/− MEFs with wild-type human TSC2. Retrovirus-infected pools of Tsc2−/− MEFs stably expressing empty vector, human TSC2, or a patient-derived mutant of TSC2 (P419S) were treated as described in A. (D) TSC2 knockdown in HeLa cells leads to constitutive rapamycin-sensitive GSK3 phosphorylation. 48 hours post-transfection with control or TSC2 siRNAs, HeLa cells were serum starved for 16 h and treated for 15 min with 20 nM rapamycin, where indicated.

Figure 3

Figure 3. mTORC1-dependent phosphorylation of GSK3 in Tsc2 null cells

(A) S6K1 and GSK3 phosphorylation are more resistant to wortmannin in Tsc2−/− cells. Tsc2+/+ and Tsc2−/− MEFs were serum starved for 16 h and then pretreated for 15 min with 20 nM rapamycin or increasing doses of wortmannin (50 nM up to 5 μM) then stimulated for 15 min with 50 ng/ml IGF1. (B) Amino acid-dependent phosphorylation of GSK3 in Tsc2−/− cells. Tsc2+/+ and _Tsc2−/−_MEFs were serum starved for 16 h and then put in fresh media with or without amino acids for an additional 2 hours prior to 15 min stimulation with 50 ng/ml IGF1, where indicated. Immunoblots for phospho and total Akt and GSK3 were performed together. (C) Growth factor-responsive phosphorylation of Akt and GSK3 is restored to _Tsc2−/−_cells after prolonged rapamycin treatment. Tsc2−/− MEFs were serum starved for 24 h in the presence of 20 nM rapamycin for the specified duration prior to 15 min stimulation with 25 ng/ml IGF1, where indicated. (D) Prolonged rapamycin reduces basal GSK3 phosphorylation in Tsc2−/− cells. _Tsc2−/−_MEFs were serum starved for 48 h in the presence of 20 nM rapamycin for the specified duration. (E) Prolonged rapamycin treatment restores wortmannin-sensitive GSK3 phosphorylation to Tsc2−/− cells. Tsc2−/− MEFs were serum starved for 24 h in the presence or absence of 20 nM rapamycin, were pretreated for 15 min with 20 nM rapamycin or 100 nM wortmannin, where indicated, and then stimulated for 15 min with 25 ng/ml IGF1.

Figure 4

Figure 4. S6K1 phosphorylates GSK3 in TSC-deficient cells

(A) The corresponding phosphorylation sites on GSK3 and the S6K1 substrates eIF4B and S6 are similar. The residues flanking GSK3α-S21, GSK3β-S9, eIF4B-S422, and ribosomal S6-S236 are aligned. (B) S6K1 from serum-starved Tsc2−/− MEFs, but not wild-type MEFs, can phosphorylate GSK3β-S9 in vitro. Tsc2+/+ and Tsc2−/− MEFs were serum starved for 16 h and then treated for 15 min with 20 nM rapamycin, where indicated. Immunoprecipitated S6K1 was used in an in vitro kinase assay with bacterially produced GSK3β as the substrate. GSK3 phosphorylation on S9 was detected using a phospho-specifc antibody. (C) S6K1 from serum-starved HeLa cells treated with TSC2 siRNAs, but not control siRNAs, can phosphorylate GSK3β-S9 in vitro. 24 h post-transfection with control or _TSC2_-targetting siRNAs, HeLa cells were treated as described in B. (D) S6K1 knockdown in Tsc1−/− cells blocks the constitutive phosphorylation of GSK3. 24 hours post-transfection with the indicated doses of control or S6K1-targetting siRNAs, Tsc1−/− MEFs were serum starved for 16 hours. (E) Tsc2−/− MEFs were treated as described in D.

Figure 5

Figure 5. GSK3 activity is decreased in Tsc2 null cells and reactivated by rapamycin

(A) GSK3 kinase activity is lower in serum-starved Tsc2−/− cells and is activated by rapamycin treatment. Tsc2+/+ and Tsc2−/− MEFs were serum starved for 16 h and then treated for 30 min with 20 nM rapamycin (Rap), where indicated. The activity of immunoprecipitated GSK3 was assayed using a primed phospho-peptide derived from glycogen synthase. Data are presented as mean ± SEM activity relative to untreated wild-type cells. *1 versus 3, P<0.01; **3 versus 4, P<0.001. (B) Phosphorylation of the GSK3 sites on glycogen synthase and c-Myc are decreased in Tsc2−/− cells and restored by rapamycin. Tsc2+/+ and Tsc2−/− MEFs were serum starved for 6 h and then treated for 30 min with Rap, where indicated. Total levels of eIF4E are provided as a loading control. (C) Rescue of GSK3-dependent glycogen synthase phosphorylation in Tsc2−/− MEFs by human TSC2. Retrovirus-infected pools of Tsc2−/− MEFs were serum starved for 4 h and treated with 10 μM SB216763 and/or Rap, where indicated. Total levels of actin are provided as a loading control. (D) Rescue of c-Myc phosphorylation and degradation in Tsc2−/− MEFs by human TSC2. Two independent samples each of retrovirus-infected pools of Tsc2−/− MEFs stably expressing empty vector, human TSC2, or a patient-derived mutant of TSC2 (P419S) were serum starved for 4 h prior to lysis. *Indicates cross-reacting band in both B and D.

Figure 6

Figure 6. Aberrant regulation of GSK3 contributes to the serum-free proliferation property of TSC-deficient cells

(A) Proliferation of littermate-derived Tsc2+/+ (WT) and Tsc2−/− (KO) MEFs in full serum. Cells were cultured in 10% fetal bovine serum with fresh nutrients and serum provided daily in the presence of 0.1% DMSO (vehicle) or 20 nM rapamycin (Rap). Data are presented as mean ± SEM. (B) Cells were cultured as in A but under serum starvation conditions. (C) Pharmacological inhibition of GSK3 partially blocks the effects of rapamycin on the serum-free proliferation property of Tsc2−/− cells. Tsc2−/− MEFs were cultured for 48 h in serum free media in the presence of vehicle, Rap, or Rap plus either 10 mM LiCl or 10 μM SB216763. Data are presented as the mean ± SEM percentage of cells present relative to vehicle control, after subtracting the original cell number plated. *Rap versus Rap + LiCl or Rap + SB216763, P<0.05. (D) Cells were cultured as described in C but with BrdU added for the final 24 h. Data are presented as the mean ± SEM percentage of DAPI-stained nuclei (red) also positive for BrdU (yellow/green). *Rap versus Rap + LiCl, P<0.05. (E) SiRNA knockdown of GSK3α and β in Tsc2−/− MEFs. (F) SiRNA knockdown of GSK3α and β significantly blocks the effects of rapamycin on Tsc2−/− cells. 24 hours post-transfection with control or GSKα and β-targetting siRNAs, equal numbers of Tsc2−/− MEFs were reseeded and serum starved in the presence of vehicle or Rap for 24 h. Data are presented as in C. **control siRNAs + Rap versus GSK3α/β siRNAs + Rap, P<0.001. (G) GSK3β-S9A can suppress the growth factor-independent proliferation of Tsc2−/− cells. Retrovirus-infected pools of Tsc2−/− MEFs stably expressing empty vector (pBabe-puro) or pBabe-puro encoding GSK3β-S9A or TSC2 were plated in equal numbers and cultured in the absence of serum for 48 h.

Figure 7

Figure 7. mTORC1-dependent phosphorylation of GSK3 under conditions of cellular insulin resistance

(A) GSK3 phosphorylation is sensitive to rapamycin in insulin-resistant HepG2 cells. HepG2 cells were serum starved for 16 h in the absence or presence of 100 nM insulin and were treated for 15 min with 20 nM rapamycin (R) or 100 nM insulin (I), as indicated. (B) Rapamycin increases Akt phosphorylation but decreases GSK3 phosphorylation in insulin-resistant HepG2 cells. Cells were treated for 16 h with 100 nM insulin and then treated for 15 min with 20 nM rapamycin. Three independent lysates from each condition were then immunoblotted for phospho-Akt-S473, total Akt, phospho GSK3α/β-S21/S9, and total GSK3α/β, and the data were quantified using an infared imaging system. The data are expressed as the mean +/− SEM relative ratio of phospho-protein to total protein between untreated and rapamycin-treated samples. *p=0.0105; **p=0.0001; ***p=0.0439 (C) Differential regulation of GSK3 by the mitogen-stimulated PI3K-Akt pathway (green) and the nutrient-sensitive mTOR-S6K1 pathway (blue).

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