Abstract
Cell growth can be suppressed by stressful environments, but the role of stress pathways in this process is largely unknown. Here we show that a cascade of p38β mitogen-activated protein kinase (MAPK) and p38-regulated/activated kinase (PRAK) plays a role in energy-starvation-induced suppression of mammalian target of rapamycin (mTOR), and that energy starvation activates the p38β–PRAK cascade. Depletion of p38β or PRAK diminishes the suppression of mTOR complex 1 (mTORC1) and reduction of cell size induced by energy starvation. We show that p38β–PRAK operates independently of the known mTORC1 inactivation pathways—phosphorylation of tuberous sclerosis protein 2 (TSC2) and Raptor by AMP-activated protein kinase (AMPK)—and surprisingly, that PRAK directly regulates Ras homologue enriched in brain (Rheb), a key component of the mTORC1 pathway, by phosphorylation. Phosphorylation of Rheb at Ser 130 by PRAK impairs the nucleotide-binding ability of Rheb and inhibits Rheb-mediated mTORC1 activation. The direct regulation of Rheb by PRAK integrates a stress pathway with the mTORC1 pathway in response to energy depletion.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Cuenda, A. & Rousseau, S. p38 MAP-kinases pathway regulation, function and role in human diseases. Biochim. Biophys. Acta 1773, 1358–1375 (2007).
English, J. M. & Cobb, M. H. Pharmacological inhibitors of MAPK pathways. Trends Pharmacol. Sci. 23, 40–45 (2002).
Nebreda, A. R. & Porras, A. p38 MAP kinases: beyond the stress response. Trends Biochem. Sci. 25, 257–260 (2000).
Ono, K. & Han, J. The p38 signal transduction pathway: activation and function. Cell Signal. 12, 1–13 (2000).
Han, J., Lee, J. D., Bibbs, L. & Ulevitch, R. J. A MAP kinase targeted by endotoxin and hyperosmolarity in mammalian cells. Science 265, 808–811 (1994).
Jiang, Y. et al. Characterization of the structure and function of a new mitogen-activated protein kinase (p38β). J. Biol. Chem. 271, 17920–17926 (1996).
Li, Z., Jiang, Y., Ulevitch, R. J. & Han, J. The primary structure of p38γ: a new member of p38 group of MAP kinases. Biochem. Biophys. Res. Commun. 228, 334–340 (1996).
Jiang, Y. et al. Characterization of the structure and function of the fourth member of p38 group mitogen-activated protein kinases, p38δ. J. Biol. Chem. 272, 30122–30128 (1997).
Zarubin, T. & Han, J. Activation and signaling of the p38 MAP kinase pathway. Cell Res. 15, 11–18 (2005).
Rincon, M. & Davis, R. J. Regulation of the immune response by stress-activated protein kinases. Immunol. Rev. 228, 212–224 (2009).
Reiling, J. H. & Sabatini, D. M. Stress and mTORture signaling. Oncogene 25, 6373–6383 (2006).
Wullschleger, S., Loewith, R. & Hall, M. N. TOR signaling in growth and metabolism. Cell 124, 471–484 (2006).
Sengupta, S., Peterson, T. R. & Sabatini, D. M. Regulation of the mTOR complex 1 pathway by nutrients, growth factors, and stress. Mol. Cell 40, 310–322 (2010).
Sabatini, D. M. mTOR and cancer: insights into a complex relationship. Nat. Rev. Cancer 6, 729–734 (2006).
Sabatini, D. M., Erdjument-Bromage, H., Lui, M., Tempst, P. & Snyder, S. H. RAFT1: a mammalian protein that binds to FKBP12 in a rapamycin-dependent fashion and is homologous to yeast TORs. Cell 78, 35–43 (1994).
Jacinto, E. et al. Mammalian TOR complex 2 controls the actin cytoskeleton and is rapamycin insensitive. Nat. Cell Biol. 6, 1122–1128 (2004).
Loewith, R. et al. Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Mol. Cell 10, 457–468 (2002).
Sarbassov, D. D., Guertin, D. A., Ali, S. M. & Sabatini, D. M. Phosphorylation and regulation of Akt/PKB by the rictor-mTOR complex. Science 307, 1098–1101 (2005).
Kim, D. H. et al. mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110, 163–175 (2002).
Guertin, D. A. et al. Ablation in mice of the mTORC components raptor, rictor, or mLST8 reveals that mTORC2 is required for signaling to Akt-FOXO and PKCα, but not S6K1. Dev. Cell 11, 859–871 (2006).
Vander, H. E., Lee, S. I., Bandhakavi, S., Griffin, T. J. & Kim, D. H. Insulin signalling to mTOR mediated by the Akt/PKB substrate PRAS40. Nat. Cell Biol. 9, 316–323 (2007).
Peterson, T. R. et al. DEPTOR is an mTOR inhibitor frequently overexpressed in multiple myeloma cells and required for their survival. Cell 137, 873–886 (2009).
Hardie, D. G. Role of AMP-activated protein kinase in the metabolic syndrome and in heart disease. FEBS Lett. 582, 81–89 (2008).
Shackelford, D. B. & Shaw, R. J. The LKB1-AMPK pathway: metabolism and growth control in tumour suppression. Nat. Rev. Cancer 9, 563–575 (2009).
Inoki, K., Zhu, T. & Guan, K. L. TSC2 mediates cellular energy response to control cell growth and survival. Cell 115, 577–590 (2003).
Garami, A. et al. Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Mol. Cell 11, 1457–1466 (2003).
Aspuria, P. J. & Tamanoi, F. The Rheb family of GTP-binding proteins. Cell Signal. 16, 1105–1112 (2004).
Long, X., Lin, Y., Ortiz-Vega, S., Yonezawa, K. & Avruch, J. Rheb binds and regulates the mTOR kinase. Curr. Biol. 15, 702–713 (2005).
Zhang, Y. et al. Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins. Nat. Cell Biol. 5, 578–581 (2003).
Inoki, K., Li, Y., Xu, T. & Guan, K. L. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes Dev. 17, 1829–1834 (2003).
Gwinn, D. M. et al. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Mol. Cell 30, 214–226 (2008).
Um, S. H., D’Alessio, D. & Thomas, G. Nutrient overload, insulin resistance, and ribosomal protein S6 kinase 1, S6K1. Cell Metab. 3, 393–402 (2006).
Li, Y., Inoki, K., Vacratsis, P. & Guan, K. L. The p38 and MK2 kinase cascade phosphorylates tuberin, the tuberous sclerosis 2 gene product, and enhances its interaction with 14-3-3. J. Biol. Chem. 278, 13663–13671 (2003).
Cully, M. et al. A role for p38 stress-activated protein kinase in regulation of cell growth via TORC1. Mol. Cell. Biol. 30, 481–495 (2010).
Fingar, D. C., Salama, S., Tsou, C., Harlow, E. & Blenis, J. Mammalian cell size is controlled by mTOR and its downstream targets S6K1 and 4EBP1/eIF4E. Genes Dev. 16, 1472–1487 (2002).
Potter, C. J. & Xu, T. Mechanisms of size control. Curr. Opin. Genet. Dev. 11, 279–286 (2001).
New, L. et al. PRAK, a novel protein kinase regulated by the p38 MAP kinase. EMBO J. 17, 3372–3384 (1998).
Freshney, N. W. et al. Interleukin-1 activates a novel protein kinase cascade that results in the phosphorylation of Hsp27. Cell 78, 1039–1049 (1994).
Rouse, J. et al. A novel kinase cascade triggered by stress and heat shock that stimulates MAPKAP kinase-2 and phosphorylation of the small heat shock proteins. Cell 78, 1027–1037 (1994).
Li, Q. et al. Determinants that control the distinct subcellular localization of p38α-PRAK and p38β-PRAK complexes. J. Biol. Chem. 283, 11014–11023 (2008).
New, L., Jiang, Y. & Han, J. Regulation of PRAK subcellular location by p38 MAP kinases. Mol. Biol. Cell 14, 2603–2616 (2003).
Hardie, D. G., Carling, D. & Carlson, M. The AMP-activated/SNF1 protein kinase subfamily: metabolic sensors of the eukaryotic cell? Annu. Rev. Biochem. 67, 821–855 (1998).
Kalender, A. et al. Metformin, independent of AMPK, inhibits mTORC1 in a rag GTPase-dependent manner. Cell Metab. 11, 390–401 (2010).
Sancak, Y. et al. Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 141, 290–303 (2010).
Shaw, R. J. et al. The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress. Proc. Natl Acad. Sci. USA 101, 3329–3335 (2004).
Yu, Y. et al. Structural basis for the unique biological function of small GTPase RHEB. J. Biol. Chem. 280, 17093–17100 (2005).
Yuan, J. et al. Identification and characterization of RHEBL1, a novel member of Ras family, which activates transcriptional activities of NF-kappa B. Mol. Biol. Rep. 32, 205–214 (2005).
Sun, P. et al. PRAK is essential for ras-induced senescence and tumor suppression. Cell 128, 295–308 (2007).
New, L. et al. PRAK, a novel protein kinase regulated by the p38 MAP kinase. EMBO J. 17, 3372–3384 (1998).
Inoki, K., Zhu, T. & Guan, K. L. TSC2 mediates cellular energy response to control cell growth and survival. Cell 115, 577–590 (2003).
Zhang, H. et al. Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J. Clin. Invest 112, 1223–1233 (2003).
Kwong, J. et al. p38α and p38γ mediate oncogenic ras-induced senescence through differential mechanisms. J. Biol. Chem. 284, 11237–11246 (2009).
Inoki, K., Li, Y., Xu, T. & Guan, K. L. Rheb GTPase is a direct target ofTSC2 GAP activity and regulates mTOR signaling. Genes Dev. 17, 1829–1834 (2003).
Kinoshita, E., Kinoshita-Kikuta, E., Takiyama, K. & Koike, T. Phosphate-binding tag, a new tool to visualize phosphorylated proteins. Mol. Cell Proteomics 5, 749–757 (2006).
Yates, J. R. III, Eng, J. K., McCormack, A. L. & Schieltz, D. Method to correlate tandem mass spectra of modified peptides to amino acid sequences in the protein database. Anal. Chem. 67, 1426–1436 (1995).
Tabb, D. L., McDonald, W. H. & Yates, J. R. III DTASelect and Contrast: tools for assembling and comparing protein identifications from shotgun proteomics. J. Proteome Res. 1, 21–26 (2002).
Anderson, J. C. & Peck, S. C. A simple and rapid technique for detecting protein phosphorylation using one-dimensional isoelectric focusing gels and immunoblot analysis. Plant J. 55, 881–885 (2008).
Garami, A. et al. Insulin activation of Rheb, a mediator of mTOR/S6K/4E-BP signaling, is inhibited by TSC1 and 2. Mol. Cell 11, 1457–1466 (2003).
Roccio, M., Bos, J. L. & Zwartkruis, F. J. Regulation of the small GTPase Rheb by amino acids. Oncogene 25, 657–664 (2006).
Acknowledgements
We would like to thank H. Jiang (Boehringer Ingelheim Pharmaceuticals) for providing p38 conditional knockout mice, B. Viollet (Universite Paris Descartes) and K. R. Laderoute (Stanford University School of Medicine) for A M P K α 1/α 2−/− cells. This work was supported by grants from NSF China 30830092, 30921005, 30828219, 973 program 2009CB22200, National Institutes of Health AI41637 and AI68896.
Author information
Authors and Affiliations
Contributions
M.Z., Y-H.W., X-N.W., S-Q.W. and J.H. designed and carried out the experiments. B-J.L. and M-Q.D. carried out mass spectrometry analysis. M.Z., H.Z., P.S., S-C.L., K-L.G. and J.H. participated in the interpretation of the data. M.Z. and J.H. wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Information
Supplementary Information (PDF 3550 kb)
Rights and permissions
About this article
Cite this article
Zheng, M., Wang, YH., Wu, XN. et al. Inactivation of Rheb by PRAK-mediated phosphorylation is essential for energy-depletion-induced suppression of mTORC1. Nat Cell Biol 13, 263–272 (2011). https://doi.org/10.1038/ncb2168
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ncb2168
This article is cited by
-
Polo-like kinase 1 promotes pulmonary hypertension
Respiratory Research (2023)
-
The essential role of PRAK in tumor metastasis and its therapeutic potential
Nature Communications (2021)
-
History and progress of hypotheses and clinical trials for Alzheimer’s disease
Signal Transduction and Targeted Therapy (2019)
-
Metformin potentiates the effect of arsenic trioxide suppressing intrahepatic cholangiocarcinoma: roles of p38 MAPK, ERK3, and mTORC1
Journal of Hematology & Oncology (2017)
-
The novel RAGE interactor PRAK is associated with autophagy signaling in Alzheimer’s disease pathogenesis
Molecular Neurodegeneration (2016)