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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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Nuclear localization of FoxO3a suppresses cell proliferation.(A) Schematic structures of WT FoxO3a, FoxO3a S314A mutant (S314A), and FoxO3a T32/S252/S314A triple mutant (3A). (B) Immunofluorescence analyses to determine localizations of HA-tagged WT FoxO3a, S314A, and 3A cells to determine FoxO3a (HA) localization. Merged images with PI staining are shown. Scale bars: 10 µm. (C) Western-blot analyses for the indicated molecules in p18KO cells, mock-treated WT cells, and WT cells expressing FoxO3a, S314A, or 3A. (D) Cell proliferation of WT cells and WT cells expressing WT FoxO3a, S314A, or 3A was analyzed by the WST-1 growth assay over the indicated time course. Means±SD were obtained from three independent assays. **P<0.01 and *P<0.05 (Student's t-test). (E) Expression level of p27Kip1 mRNA in cells used in (C) was determined by quantitative real-time PCR. Means±SD were obtained from three independent assays. **P<0.01, (Student's t-test).
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pone-0088891-g006: Nuclear localization of FoxO3a suppresses cell proliferation.(A) Schematic structures of WT FoxO3a, FoxO3a S314A mutant (S314A), and FoxO3a T32/S252/S314A triple mutant (3A). (B) Immunofluorescence analyses to determine localizations of HA-tagged WT FoxO3a, S314A, and 3A cells to determine FoxO3a (HA) localization. Merged images with PI staining are shown. Scale bars: 10 µm. (C) Western-blot analyses for the indicated molecules in p18KO cells, mock-treated WT cells, and WT cells expressing FoxO3a, S314A, or 3A. (D) Cell proliferation of WT cells and WT cells expressing WT FoxO3a, S314A, or 3A was analyzed by the WST-1 growth assay over the indicated time course. Means±SD were obtained from three independent assays. **P<0.01 and *P<0.05 (Student's t-test). (E) Expression level of p27Kip1 mRNA in cells used in (C) was determined by quantitative real-time PCR. Means±SD were obtained from three independent assays. **P<0.01, (Student's t-test).

Mentions: To examine the role of Ser314 phosphorylation in the regulation of cell proliferation, we generated WT cell lines that stably overexpressed wild-type FoxO3a, the S314A mutant (S314A), or a mutant with triple alanine replacements at T32, S252, and S314 (3A) (Figure 6A). Immunofluorescence analysis revealed that whereas WT FoxO3a was mostly distributed in the cytoplasm, the S314A mutant was localized to both the cytoplasm and the nucleus (Figure 6B). This distribution pattern of the S314A mutant was consistent with that of FoxO3a in p18KO cells (Figure 5F), indicating that hypophosphorylation of Ser314 allows nuclear entry of FoxO3a even though other sites are hyperphosphorylated (Figure 6C). The 3A mutant was predominantly localized to the nucleus (Figure 6B), which supports the results of a previous observation showing that Akt-mediated phosphorylation contributes to the nuclear export of FoxO3a [33]. In both cases, the nuclear localization of FoxO3a mutants strongly suppressed cell proliferation (Figure 6D). Western-blot and real-time PCR analyses confirmed that expression of the S314A and 3A mutants resulted in elevated protein and mRNA levels of p27Kip1, and these levels were inversely correlated with cyclin D1 expression (Figures 6C and 6E). These findings suggest that the phosphorylation status of Ser314 is crucial for the regulation of the nuclear function of FoxO3a.


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)

Nuclear localization of FoxO3a suppresses cell proliferation.(A) Schematic structures of WT FoxO3a, FoxO3a S314A mutant (S314A), and FoxO3a T32/S252/S314A triple mutant (3A). (B) Immunofluorescence analyses to determine localizations of HA-tagged WT FoxO3a, S314A, and 3A cells to determine FoxO3a (HA) localization. Merged images with PI staining are shown. Scale bars: 10 µm. (C) Western-blot analyses for the indicated molecules in p18KO cells, mock-treated WT cells, and WT cells expressing FoxO3a, S314A, or 3A. (D) Cell proliferation of WT cells and WT cells expressing WT FoxO3a, S314A, or 3A was analyzed by the WST-1 growth assay over the indicated time course. Means±SD were obtained from three independent assays. **P<0.01 and *P<0.05 (Student's t-test). (E) Expression level of p27Kip1 mRNA in cells used in (C) was determined by quantitative real-time PCR. Means±SD were obtained from three independent assays. **P<0.01, (Student's t-test).
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Related In: Results  -  Collection

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pone-0088891-g006: Nuclear localization of FoxO3a suppresses cell proliferation.(A) Schematic structures of WT FoxO3a, FoxO3a S314A mutant (S314A), and FoxO3a T32/S252/S314A triple mutant (3A). (B) Immunofluorescence analyses to determine localizations of HA-tagged WT FoxO3a, S314A, and 3A cells to determine FoxO3a (HA) localization. Merged images with PI staining are shown. Scale bars: 10 µm. (C) Western-blot analyses for the indicated molecules in p18KO cells, mock-treated WT cells, and WT cells expressing FoxO3a, S314A, or 3A. (D) Cell proliferation of WT cells and WT cells expressing WT FoxO3a, S314A, or 3A was analyzed by the WST-1 growth assay over the indicated time course. Means±SD were obtained from three independent assays. **P<0.01 and *P<0.05 (Student's t-test). (E) Expression level of p27Kip1 mRNA in cells used in (C) was determined by quantitative real-time PCR. Means±SD were obtained from three independent assays. **P<0.01, (Student's t-test).
Mentions: To examine the role of Ser314 phosphorylation in the regulation of cell proliferation, we generated WT cell lines that stably overexpressed wild-type FoxO3a, the S314A mutant (S314A), or a mutant with triple alanine replacements at T32, S252, and S314 (3A) (Figure 6A). Immunofluorescence analysis revealed that whereas WT FoxO3a was mostly distributed in the cytoplasm, the S314A mutant was localized to both the cytoplasm and the nucleus (Figure 6B). This distribution pattern of the S314A mutant was consistent with that of FoxO3a in p18KO cells (Figure 5F), indicating that hypophosphorylation of Ser314 allows nuclear entry of FoxO3a even though other sites are hyperphosphorylated (Figure 6C). The 3A mutant was predominantly localized to the nucleus (Figure 6B), which supports the results of a previous observation showing that Akt-mediated phosphorylation contributes to the nuclear export of FoxO3a [33]. In both cases, the nuclear localization of FoxO3a mutants strongly suppressed cell proliferation (Figure 6D). Western-blot and real-time PCR analyses confirmed that expression of the S314A and 3A mutants resulted in elevated protein and mRNA levels of p27Kip1, and these levels were inversely correlated with cyclin D1 expression (Figures 6C and 6E). These findings suggest that the phosphorylation status of Ser314 is crucial for the regulation of the nuclear function of FoxO3a.

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