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PDK1 regulation of mTOR and hypoxia-inducible factor 1 integrate metabolism and migration of CD8+ T cells.

Finlay DK, Rosenzweig E, Sinclair LV, Feijoo-Carnero C, Hukelmann JL, Rolf J, Panteleyev AA, Okkenhaug K, Cantrell DA - J. Exp. Med. (2012)

Bottom Line: We also show that PI3K- and Akt-independent pathways mediated by mTORC1 regulate the expression of HIF1 (hypoxia-inducible factor 1) transcription factor complex.This mTORC1-HIF1 pathway is required to sustain glucose metabolism and glycolysis in effector CTLs and strikingly functions to couple mTORC1 to a diverse transcriptional program that controls expression of glucose transporters, multiple rate-limiting glycolytic enzymes, cytolytic effector molecules, and essential chemokine and adhesion receptors that regulate T cell trafficking.These data reveal a fundamental mechanism linking nutrient and oxygen sensing to transcriptional control of CD8+ T cell differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland. finlayd@tcd.ie

ABSTRACT
mTORC1 (mammalian target of rapamycin complex 1) controls transcriptional programs that determine CD8+ cytolytic T cell (CTL) fate. In some cell systems, mTORC1 couples phosphatidylinositol-3 kinase (PI3K) and Akt to the control of glucose uptake and glycolysis. However, PI3K-Akt-independent mechanisms control glucose metabolism in CD8+ T cells, and the role of mTORC1 has not been explored. The present study now demonstrates that mTORC1 activity in CD8+ T cells is not dependent on PI3K or Akt but is critical to sustain glucose uptake and glycolysis in CD8+ T cells. We also show that PI3K- and Akt-independent pathways mediated by mTORC1 regulate the expression of HIF1 (hypoxia-inducible factor 1) transcription factor complex. This mTORC1-HIF1 pathway is required to sustain glucose metabolism and glycolysis in effector CTLs and strikingly functions to couple mTORC1 to a diverse transcriptional program that controls expression of glucose transporters, multiple rate-limiting glycolytic enzymes, cytolytic effector molecules, and essential chemokine and adhesion receptors that regulate T cell trafficking. These data reveal a fundamental mechanism linking nutrient and oxygen sensing to transcriptional control of CD8+ T cell differentiation.

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PI3K and Akt do not regulate mTORC1 activity. (A and B) CTLs were cultured in the presence or absence of Akti1/2, IC87114, rapamycin, or LY294002 for 60 min (A and B) or 24 h (A) and subjected to immunoblot analysis with the indicated antibodies. (C) CTLs generated from WT or p110δD910A mice were subjected to immunoblot analysis with or without rapamycin treatment (30 min). Data are representative of two experiments. (D) CTLs were cultured in the presence or absence of Akti1/2, IC87114, or rapamycin for 24 h and subjected to immunoblot analysis with the indicated antibodies. (E–G) CTLs generated from PDK1flox/flox TamoxCre (PDK1Flox) and PDK1WT/WT TamoxCre (WT) mice were treated ± 4′OHT for 3 d to delete PDK1 and subjected to immunoblot analysis. For, A, B, and D–G, data are representative of at least three experiments. Molecular mass is indicated in kilodaltons.
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fig7: PI3K and Akt do not regulate mTORC1 activity. (A and B) CTLs were cultured in the presence or absence of Akti1/2, IC87114, rapamycin, or LY294002 for 60 min (A and B) or 24 h (A) and subjected to immunoblot analysis with the indicated antibodies. (C) CTLs generated from WT or p110δD910A mice were subjected to immunoblot analysis with or without rapamycin treatment (30 min). Data are representative of two experiments. (D) CTLs were cultured in the presence or absence of Akti1/2, IC87114, or rapamycin for 24 h and subjected to immunoblot analysis with the indicated antibodies. (E–G) CTLs generated from PDK1flox/flox TamoxCre (PDK1Flox) and PDK1WT/WT TamoxCre (WT) mice were treated ± 4′OHT for 3 d to delete PDK1 and subjected to immunoblot analysis. For, A, B, and D–G, data are representative of at least three experiments. Molecular mass is indicated in kilodaltons.

Mentions: What about the second question? Are PI3K and Akt activity required for mTORC1 activation and HIF1 expression in CD8+ T cells? To address this issue, we used complementary genetic and pharmacological strategies to block PI3K and Akt and then monitored the impact of these perturbations on mTORC1 activity by assessing the phosphorylation of mTORC1 substrate sequences on S6K1 (T389 and S421/424) and 4EBP1 (S35/47) and the phosphorylation of the S6K1 substrate S6 ribosomal protein (S235/236). The data show that IL-2–maintained CTLs contain high levels of active Akt phosphorylated on threonine 308 and high levels of mTORC1 signaling (Fig. 7 A). mTORC1 inhibition with rapamycin abolished the phosphorylation of S6K1 on T389 and S421/424 and blocked S6 phosphorylation. In CD8+ T cells, Akt is activated via a PI3K complex containing the p110δ catalytic subunit (Macintyre et al., 2011). The Akt inhibitor, Akti1/2, or a p110δ inhibitor, IC87114, potently inhibited Akt activity in CTLs as judged by the loss of phosphorylated Akt on T308 and S473 and inhibition of the phosphorylation of the Akt substrates, Foxo transcription factors (Fig. 7, A and B). However, neither Akti1/2 nor IC87114 prevented mTORC1 activity in CD8+ T cells as neither compound blocked the phosphorylation of mTORC1 substrates S6K1 and 4EBP1 or S6 phosphorylation in CTLs (Fig. 7 A). Moreover, CTLs that had WT PI3K p110δ catalytic subunits substituted with a catalytically inactive mutant (p110δD910A) did not activate Akt in response to IL-2 but showed normal rapamycin-sensitive phosphorylation of S6K1 and S6 (Fig. 7 C). Further evidence that mTORC1 activity is independent of PI3K and Akt is that the expression of HIF1α was not regulated by Akt and PI3K inhibitors (Fig. 7 D). These data reveal that PI3K–Akt activity is dispensable for mTORC1 activity and HIF1 expression in CTLs.


PDK1 regulation of mTOR and hypoxia-inducible factor 1 integrate metabolism and migration of CD8+ T cells.

Finlay DK, Rosenzweig E, Sinclair LV, Feijoo-Carnero C, Hukelmann JL, Rolf J, Panteleyev AA, Okkenhaug K, Cantrell DA - J. Exp. Med. (2012)

PI3K and Akt do not regulate mTORC1 activity. (A and B) CTLs were cultured in the presence or absence of Akti1/2, IC87114, rapamycin, or LY294002 for 60 min (A and B) or 24 h (A) and subjected to immunoblot analysis with the indicated antibodies. (C) CTLs generated from WT or p110δD910A mice were subjected to immunoblot analysis with or without rapamycin treatment (30 min). Data are representative of two experiments. (D) CTLs were cultured in the presence or absence of Akti1/2, IC87114, or rapamycin for 24 h and subjected to immunoblot analysis with the indicated antibodies. (E–G) CTLs generated from PDK1flox/flox TamoxCre (PDK1Flox) and PDK1WT/WT TamoxCre (WT) mice were treated ± 4′OHT for 3 d to delete PDK1 and subjected to immunoblot analysis. For, A, B, and D–G, data are representative of at least three experiments. Molecular mass is indicated in kilodaltons.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3526360&req=5

fig7: PI3K and Akt do not regulate mTORC1 activity. (A and B) CTLs were cultured in the presence or absence of Akti1/2, IC87114, rapamycin, or LY294002 for 60 min (A and B) or 24 h (A) and subjected to immunoblot analysis with the indicated antibodies. (C) CTLs generated from WT or p110δD910A mice were subjected to immunoblot analysis with or without rapamycin treatment (30 min). Data are representative of two experiments. (D) CTLs were cultured in the presence or absence of Akti1/2, IC87114, or rapamycin for 24 h and subjected to immunoblot analysis with the indicated antibodies. (E–G) CTLs generated from PDK1flox/flox TamoxCre (PDK1Flox) and PDK1WT/WT TamoxCre (WT) mice were treated ± 4′OHT for 3 d to delete PDK1 and subjected to immunoblot analysis. For, A, B, and D–G, data are representative of at least three experiments. Molecular mass is indicated in kilodaltons.
Mentions: What about the second question? Are PI3K and Akt activity required for mTORC1 activation and HIF1 expression in CD8+ T cells? To address this issue, we used complementary genetic and pharmacological strategies to block PI3K and Akt and then monitored the impact of these perturbations on mTORC1 activity by assessing the phosphorylation of mTORC1 substrate sequences on S6K1 (T389 and S421/424) and 4EBP1 (S35/47) and the phosphorylation of the S6K1 substrate S6 ribosomal protein (S235/236). The data show that IL-2–maintained CTLs contain high levels of active Akt phosphorylated on threonine 308 and high levels of mTORC1 signaling (Fig. 7 A). mTORC1 inhibition with rapamycin abolished the phosphorylation of S6K1 on T389 and S421/424 and blocked S6 phosphorylation. In CD8+ T cells, Akt is activated via a PI3K complex containing the p110δ catalytic subunit (Macintyre et al., 2011). The Akt inhibitor, Akti1/2, or a p110δ inhibitor, IC87114, potently inhibited Akt activity in CTLs as judged by the loss of phosphorylated Akt on T308 and S473 and inhibition of the phosphorylation of the Akt substrates, Foxo transcription factors (Fig. 7, A and B). However, neither Akti1/2 nor IC87114 prevented mTORC1 activity in CD8+ T cells as neither compound blocked the phosphorylation of mTORC1 substrates S6K1 and 4EBP1 or S6 phosphorylation in CTLs (Fig. 7 A). Moreover, CTLs that had WT PI3K p110δ catalytic subunits substituted with a catalytically inactive mutant (p110δD910A) did not activate Akt in response to IL-2 but showed normal rapamycin-sensitive phosphorylation of S6K1 and S6 (Fig. 7 C). Further evidence that mTORC1 activity is independent of PI3K and Akt is that the expression of HIF1α was not regulated by Akt and PI3K inhibitors (Fig. 7 D). These data reveal that PI3K–Akt activity is dispensable for mTORC1 activity and HIF1 expression in CTLs.

Bottom Line: We also show that PI3K- and Akt-independent pathways mediated by mTORC1 regulate the expression of HIF1 (hypoxia-inducible factor 1) transcription factor complex.This mTORC1-HIF1 pathway is required to sustain glucose metabolism and glycolysis in effector CTLs and strikingly functions to couple mTORC1 to a diverse transcriptional program that controls expression of glucose transporters, multiple rate-limiting glycolytic enzymes, cytolytic effector molecules, and essential chemokine and adhesion receptors that regulate T cell trafficking.These data reveal a fundamental mechanism linking nutrient and oxygen sensing to transcriptional control of CD8+ T cell differentiation.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biochemistry and Immunology, Trinity Biomedical Sciences Institute, Trinity College Dublin, Dublin 2, Ireland. finlayd@tcd.ie

ABSTRACT
mTORC1 (mammalian target of rapamycin complex 1) controls transcriptional programs that determine CD8+ cytolytic T cell (CTL) fate. In some cell systems, mTORC1 couples phosphatidylinositol-3 kinase (PI3K) and Akt to the control of glucose uptake and glycolysis. However, PI3K-Akt-independent mechanisms control glucose metabolism in CD8+ T cells, and the role of mTORC1 has not been explored. The present study now demonstrates that mTORC1 activity in CD8+ T cells is not dependent on PI3K or Akt but is critical to sustain glucose uptake and glycolysis in CD8+ T cells. We also show that PI3K- and Akt-independent pathways mediated by mTORC1 regulate the expression of HIF1 (hypoxia-inducible factor 1) transcription factor complex. This mTORC1-HIF1 pathway is required to sustain glucose metabolism and glycolysis in effector CTLs and strikingly functions to couple mTORC1 to a diverse transcriptional program that controls expression of glucose transporters, multiple rate-limiting glycolytic enzymes, cytolytic effector molecules, and essential chemokine and adhesion receptors that regulate T cell trafficking. These data reveal a fundamental mechanism linking nutrient and oxygen sensing to transcriptional control of CD8+ T cell differentiation.

Show MeSH
Related in: MedlinePlus