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The mTOR pathway controls cell proliferation by regulating the FoxO3a transcription factor via SGK1 kinase.

Mori S, Nada S, Kimura H, Tajima S, Takahashi Y, Kitamura A, Oneyama C, Okada M - PLoS ONE (2014)

Bottom Line: Although Akt was activated by mTORC1 inactivation, FoxO3a was upregulated via an epigenetic mechanism and hypophosphorylated at Ser314, which resulted in its nuclear accumulation.Consistently, mTORC1 inactivation induced downregulation of serum- and glucocorticoid-inducible kinase 1 (SGK1), the kinase responsible for Ser314 phosphorylation.SGK1 overexpression suppressed CDKI expression in p18-deficient cells, whereas SGK1 knockdown induced CDKI expression in wild-type cells, resulting in the suppression of cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, Japan.

ABSTRACT
The mechanistic target of rapamycin (mTOR) functions as a component of two large complexes, mTORC1 and mTORC2, which play crucial roles in regulating cell growth and homeostasis. However, the molecular mechanisms by which mTOR controls cell proliferation remain elusive. Here we show that the FoxO3a transcription factor is coordinately regulated by mTORC1 and mTORC2, and plays a crucial role in controlling cell proliferation. To dissect mTOR signaling, mTORC1 was specifically inactivated by depleting p18, an essential anchor of mTORC1 on lysosomes. mTORC1 inactivation caused a marked retardation of cell proliferation, which was associated with upregulation of cyclin-dependent kinase inhibitors (CDKIs). Although Akt was activated by mTORC1 inactivation, FoxO3a was upregulated via an epigenetic mechanism and hypophosphorylated at Ser314, which resulted in its nuclear accumulation. Consistently, mTORC1 inactivation induced downregulation of serum- and glucocorticoid-inducible kinase 1 (SGK1), the kinase responsible for Ser314 phosphorylation. Expression of FoxO3a mutated at Ser314 suppressed cell proliferation by inducing CDKI expression. SGK1 overexpression suppressed CDKI expression in p18-deficient cells, whereas SGK1 knockdown induced CDKI expression in wild-type cells, resulting in the suppression of cell proliferation. These results suggest that mTORC1, in coordination with mTORC2, controls cell proliferation by regulating FoxO3a gene expression and SGK1-mediated phosphorylation of FoxO3a at Ser314.

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Inactivation of mTORC1 upregulates mTORC2 and FoxO3a.(A) Western-blot analyses to detect the indicated signaling molecules, using total cell lysates from WT, p18Rev, p18NΔ5-CAAX, and p18KO cells. (B) Expression levels of mRNAs encoding Rictor, FoxO3a and FoxO1 in p18KO and WT cells were determined by quantitative real-time PCR. Means±SD were obtained from three independent assays. ***P<0.001 and **P<0.01 (Student's t-test).
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pone-0088891-g003: Inactivation of mTORC1 upregulates mTORC2 and FoxO3a.(A) Western-blot analyses to detect the indicated signaling molecules, using total cell lysates from WT, p18Rev, p18NΔ5-CAAX, and p18KO cells. (B) Expression levels of mRNAs encoding Rictor, FoxO3a and FoxO1 in p18KO and WT cells were determined by quantitative real-time PCR. Means±SD were obtained from three independent assays. ***P<0.001 and **P<0.01 (Student's t-test).

Mentions: To address the molecular mechanisms by which lysosomal mTORC1 regulates cell proliferation, we investigated changes in signaling molecules upstream and downstream of the mTOR pathway (Figure 3A). Western-blot analyses of total cell lysates revealed that expression levels of mTOR and Raptor were not significantly influenced by inactivation of mTORC1, whereas protein and mRNA levels of Rictor were significantly upregulated (Figures 3A and B). Consistent with this, phosphorylation of Akt at Ser473, a site specifically phosphorylated by mTORC2 [24], was elevated, indicating that mTORC2 is activated by mTORC1 inactivation. Activated S6K1 phosphorylates Grb10, which mediates negative-feedback regulation of growth-factor signaling [17]. Indeed, inactivation of S6K1 by mTORC1 inactivation induced a mobility shift of Grb10, indicating dephosphorylation of this protein. Consequently, phosphorylation of Akt at Thr308, a PDK1 phosphorylation site [32], was elevated, probably due to activation of PI3K. Although the reason remains unknown, the Grb10 expression level was slightly decreased in p18Rev cells compared with WT cells. However, the phosphorylation level of Akt Thr308 in these cells was almost comparable with that in WT cells, suggesting that perturbation of Grb10 expression had no significant effect on the Akt pathway. Activation of Akt was further confirmed by the elevated phosphorylation of an Akt substrate, TSC2. These results suggest that inactivation of mTORC1 strongly activated Akt via activation of mTORC2 and abrogation of the negative-feedback inhibition mediated by Grb10. Activated Akt phosphorylates FoxO transcription factors and suppresses their nuclear function [26]. In p18KO cells, the levels of FoxO3a protein and mRNA were significantly upregulated (Figures 3A and B). Although the level of FoxO1 protein, a close relative of FoxO3a, appeared unchanged (Figure 3A), the FoxO1 mRNA level was significantly upregulated (Figure 3B). Furthermore, both FoxO3a and FoxO1 proteins exhibited apparent gel-mobility shifts (Figure 3A), as discussed further below. These findings suggest that the expression and modification of FoxO transcription factors are regulated via the p18-mTORC1 pathway. Based on the observations that FoxO3a protein is more markedly upregulated in p18KO cells than FoxO1 and that FoxO3a is widely expressed in various tissues [33], we hereafter focused on the regulatory mechanism for FoxO3a function.


The mTOR pathway controls cell proliferation by regulating the FoxO3a transcription factor via SGK1 kinase.

Mori S, Nada S, Kimura H, Tajima S, Takahashi Y, Kitamura A, Oneyama C, Okada M - PLoS ONE (2014)

Inactivation of mTORC1 upregulates mTORC2 and FoxO3a.(A) Western-blot analyses to detect the indicated signaling molecules, using total cell lysates from WT, p18Rev, p18NΔ5-CAAX, and p18KO cells. (B) Expression levels of mRNAs encoding Rictor, FoxO3a and FoxO1 in p18KO and WT cells were determined by quantitative real-time PCR. Means±SD were obtained from three independent assays. ***P<0.001 and **P<0.01 (Student's t-test).
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3928304&req=5

pone-0088891-g003: Inactivation of mTORC1 upregulates mTORC2 and FoxO3a.(A) Western-blot analyses to detect the indicated signaling molecules, using total cell lysates from WT, p18Rev, p18NΔ5-CAAX, and p18KO cells. (B) Expression levels of mRNAs encoding Rictor, FoxO3a and FoxO1 in p18KO and WT cells were determined by quantitative real-time PCR. Means±SD were obtained from three independent assays. ***P<0.001 and **P<0.01 (Student's t-test).
Mentions: To address the molecular mechanisms by which lysosomal mTORC1 regulates cell proliferation, we investigated changes in signaling molecules upstream and downstream of the mTOR pathway (Figure 3A). Western-blot analyses of total cell lysates revealed that expression levels of mTOR and Raptor were not significantly influenced by inactivation of mTORC1, whereas protein and mRNA levels of Rictor were significantly upregulated (Figures 3A and B). Consistent with this, phosphorylation of Akt at Ser473, a site specifically phosphorylated by mTORC2 [24], was elevated, indicating that mTORC2 is activated by mTORC1 inactivation. Activated S6K1 phosphorylates Grb10, which mediates negative-feedback regulation of growth-factor signaling [17]. Indeed, inactivation of S6K1 by mTORC1 inactivation induced a mobility shift of Grb10, indicating dephosphorylation of this protein. Consequently, phosphorylation of Akt at Thr308, a PDK1 phosphorylation site [32], was elevated, probably due to activation of PI3K. Although the reason remains unknown, the Grb10 expression level was slightly decreased in p18Rev cells compared with WT cells. However, the phosphorylation level of Akt Thr308 in these cells was almost comparable with that in WT cells, suggesting that perturbation of Grb10 expression had no significant effect on the Akt pathway. Activation of Akt was further confirmed by the elevated phosphorylation of an Akt substrate, TSC2. These results suggest that inactivation of mTORC1 strongly activated Akt via activation of mTORC2 and abrogation of the negative-feedback inhibition mediated by Grb10. Activated Akt phosphorylates FoxO transcription factors and suppresses their nuclear function [26]. In p18KO cells, the levels of FoxO3a protein and mRNA were significantly upregulated (Figures 3A and B). Although the level of FoxO1 protein, a close relative of FoxO3a, appeared unchanged (Figure 3A), the FoxO1 mRNA level was significantly upregulated (Figure 3B). Furthermore, both FoxO3a and FoxO1 proteins exhibited apparent gel-mobility shifts (Figure 3A), as discussed further below. These findings suggest that the expression and modification of FoxO transcription factors are regulated via the p18-mTORC1 pathway. Based on the observations that FoxO3a protein is more markedly upregulated in p18KO cells than FoxO1 and that FoxO3a is widely expressed in various tissues [33], we hereafter focused on the regulatory mechanism for FoxO3a function.

Bottom Line: Although Akt was activated by mTORC1 inactivation, FoxO3a was upregulated via an epigenetic mechanism and hypophosphorylated at Ser314, which resulted in its nuclear accumulation.Consistently, mTORC1 inactivation induced downregulation of serum- and glucocorticoid-inducible kinase 1 (SGK1), the kinase responsible for Ser314 phosphorylation.SGK1 overexpression suppressed CDKI expression in p18-deficient cells, whereas SGK1 knockdown induced CDKI expression in wild-type cells, resulting in the suppression of cell proliferation.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka, Japan.

ABSTRACT
The mechanistic target of rapamycin (mTOR) functions as a component of two large complexes, mTORC1 and mTORC2, which play crucial roles in regulating cell growth and homeostasis. However, the molecular mechanisms by which mTOR controls cell proliferation remain elusive. Here we show that the FoxO3a transcription factor is coordinately regulated by mTORC1 and mTORC2, and plays a crucial role in controlling cell proliferation. To dissect mTOR signaling, mTORC1 was specifically inactivated by depleting p18, an essential anchor of mTORC1 on lysosomes. mTORC1 inactivation caused a marked retardation of cell proliferation, which was associated with upregulation of cyclin-dependent kinase inhibitors (CDKIs). Although Akt was activated by mTORC1 inactivation, FoxO3a was upregulated via an epigenetic mechanism and hypophosphorylated at Ser314, which resulted in its nuclear accumulation. Consistently, mTORC1 inactivation induced downregulation of serum- and glucocorticoid-inducible kinase 1 (SGK1), the kinase responsible for Ser314 phosphorylation. Expression of FoxO3a mutated at Ser314 suppressed cell proliferation by inducing CDKI expression. SGK1 overexpression suppressed CDKI expression in p18-deficient cells, whereas SGK1 knockdown induced CDKI expression in wild-type cells, resulting in the suppression of cell proliferation. These results suggest that mTORC1, in coordination with mTORC2, controls cell proliferation by regulating FoxO3a gene expression and SGK1-mediated phosphorylation of FoxO3a at Ser314.

Show MeSH
Related in: MedlinePlus