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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 induces nuclear accumulation of FoxO3a.(A) Western-blot analyses to detect the indicated signaling molecules, using total cell lysates from WT, p18Rev, p18NΔ5-CAAX, and p18KO cells. The panels for FoxO3a and FoxO1 are the same panels used in Figure 3A. Mobility shifts of FoxO proteins are shown by bars. Locations of bands corresponding to long and short forms of SGK1 are indicated by arrows. (B) Schematic structure of FoxO3a. Sites of phosphorylation by the indicated kinases are shown. FH: Fork head domain. (C) HA-tagged FoxO3a constructs with point mutations at the indicated amino-acid positions were transiently expressed in WT cells, and their mobility shifts (indicated by bars) were analyzed by Western blotting. (D) Expression levels of mRNA encoding SGK1 in p18KO and WT cells were determined by quantitative real-time PCR. Means±SD were obtained from three independent assays. n.s.; not significant (Student's t-test). (E) Whole-cell lysates from p18KO and WT cells were separated into cytoplasmic and nuclear fractions, and the indicated proteins were detected by Western-blot analyses. β-tubulin and histone H3 represent control proteins for the cytoplasmic and nuclear fractions, respectively. (F) Immunofluorescence analysis for FoxO3a in p18KO and WT cells. Nuclei were visualized with propidium iodide (PI). Merged images are also shown. Scale bars: 10 µm. Upper graphs show the intensity of signals for FoxO3a obtained by scanning along the yellow dot lines. Right graph shows the statistic data of nuclear intensity of FoxO3a signals in p18KO and WT cells. Means±SD were obtained from 15 cells. ***P<0.001 (Student's t-test).
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pone-0088891-g005: Inactivation of mTORC1 induces nuclear accumulation of FoxO3a.(A) Western-blot analyses to detect the indicated signaling molecules, using total cell lysates from WT, p18Rev, p18NΔ5-CAAX, and p18KO cells. The panels for FoxO3a and FoxO1 are the same panels used in Figure 3A. Mobility shifts of FoxO proteins are shown by bars. Locations of bands corresponding to long and short forms of SGK1 are indicated by arrows. (B) Schematic structure of FoxO3a. Sites of phosphorylation by the indicated kinases are shown. FH: Fork head domain. (C) HA-tagged FoxO3a constructs with point mutations at the indicated amino-acid positions were transiently expressed in WT cells, and their mobility shifts (indicated by bars) were analyzed by Western blotting. (D) Expression levels of mRNA encoding SGK1 in p18KO and WT cells were determined by quantitative real-time PCR. Means±SD were obtained from three independent assays. n.s.; not significant (Student's t-test). (E) Whole-cell lysates from p18KO and WT cells were separated into cytoplasmic and nuclear fractions, and the indicated proteins were detected by Western-blot analyses. β-tubulin and histone H3 represent control proteins for the cytoplasmic and nuclear fractions, respectively. (F) Immunofluorescence analysis for FoxO3a in p18KO and WT cells. Nuclei were visualized with propidium iodide (PI). Merged images are also shown. Scale bars: 10 µm. Upper graphs show the intensity of signals for FoxO3a obtained by scanning along the yellow dot lines. Right graph shows the statistic data of nuclear intensity of FoxO3a signals in p18KO and WT cells. Means±SD were obtained from 15 cells. ***P<0.001 (Student's t-test).

Mentions: As mentioned above, we noticed that mTORC1 inactivation caused mobility shifts and hyperphosphorylation of Akt phosphorylation sites in FoxO3a and FoxO1 proteins (Figures 5A and 5B). Akt phosphorylates and inactivates FoxO proteins by causing them to translocate from the nucleus to the cytoplasm, which results in the promotion of cell proliferation via the suppression of the expression of CDKIs and pro-apoptotic molecules [25]. Surprisingly, however, expression of two CDKIs, p27Kip1 and p21Cip1, was increased by Akt-mediated FoxO phosphorylation (Figure 5A).


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 induces nuclear accumulation of FoxO3a.(A) Western-blot analyses to detect the indicated signaling molecules, using total cell lysates from WT, p18Rev, p18NΔ5-CAAX, and p18KO cells. The panels for FoxO3a and FoxO1 are the same panels used in Figure 3A. Mobility shifts of FoxO proteins are shown by bars. Locations of bands corresponding to long and short forms of SGK1 are indicated by arrows. (B) Schematic structure of FoxO3a. Sites of phosphorylation by the indicated kinases are shown. FH: Fork head domain. (C) HA-tagged FoxO3a constructs with point mutations at the indicated amino-acid positions were transiently expressed in WT cells, and their mobility shifts (indicated by bars) were analyzed by Western blotting. (D) Expression levels of mRNA encoding SGK1 in p18KO and WT cells were determined by quantitative real-time PCR. Means±SD were obtained from three independent assays. n.s.; not significant (Student's t-test). (E) Whole-cell lysates from p18KO and WT cells were separated into cytoplasmic and nuclear fractions, and the indicated proteins were detected by Western-blot analyses. β-tubulin and histone H3 represent control proteins for the cytoplasmic and nuclear fractions, respectively. (F) Immunofluorescence analysis for FoxO3a in p18KO and WT cells. Nuclei were visualized with propidium iodide (PI). Merged images are also shown. Scale bars: 10 µm. Upper graphs show the intensity of signals for FoxO3a obtained by scanning along the yellow dot lines. Right graph shows the statistic data of nuclear intensity of FoxO3a signals in p18KO and WT cells. Means±SD were obtained from 15 cells. ***P<0.001 (Student's t-test).
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Related In: Results  -  Collection

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pone-0088891-g005: Inactivation of mTORC1 induces nuclear accumulation of FoxO3a.(A) Western-blot analyses to detect the indicated signaling molecules, using total cell lysates from WT, p18Rev, p18NΔ5-CAAX, and p18KO cells. The panels for FoxO3a and FoxO1 are the same panels used in Figure 3A. Mobility shifts of FoxO proteins are shown by bars. Locations of bands corresponding to long and short forms of SGK1 are indicated by arrows. (B) Schematic structure of FoxO3a. Sites of phosphorylation by the indicated kinases are shown. FH: Fork head domain. (C) HA-tagged FoxO3a constructs with point mutations at the indicated amino-acid positions were transiently expressed in WT cells, and their mobility shifts (indicated by bars) were analyzed by Western blotting. (D) Expression levels of mRNA encoding SGK1 in p18KO and WT cells were determined by quantitative real-time PCR. Means±SD were obtained from three independent assays. n.s.; not significant (Student's t-test). (E) Whole-cell lysates from p18KO and WT cells were separated into cytoplasmic and nuclear fractions, and the indicated proteins were detected by Western-blot analyses. β-tubulin and histone H3 represent control proteins for the cytoplasmic and nuclear fractions, respectively. (F) Immunofluorescence analysis for FoxO3a in p18KO and WT cells. Nuclei were visualized with propidium iodide (PI). Merged images are also shown. Scale bars: 10 µm. Upper graphs show the intensity of signals for FoxO3a obtained by scanning along the yellow dot lines. Right graph shows the statistic data of nuclear intensity of FoxO3a signals in p18KO and WT cells. Means±SD were obtained from 15 cells. ***P<0.001 (Student's t-test).
Mentions: As mentioned above, we noticed that mTORC1 inactivation caused mobility shifts and hyperphosphorylation of Akt phosphorylation sites in FoxO3a and FoxO1 proteins (Figures 5A and 5B). Akt phosphorylates and inactivates FoxO proteins by causing them to translocate from the nucleus to the cytoplasm, which results in the promotion of cell proliferation via the suppression of the expression of CDKIs and pro-apoptotic molecules [25]. Surprisingly, however, expression of two CDKIs, p27Kip1 and p21Cip1, was increased by Akt-mediated FoxO phosphorylation (Figure 5A).

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