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Stimulation of de Novo Pyrimidine Synthesis by Growth Signaling Through mTOR and S6K1

Science, 15 March 2013
Vol. 339, Issue 6125, p. 1323-1328
DOI: 10.1126/science.1228792

Stimulation of de Novo Pyrimidine Synthesis by Growth Signaling Through mTOR and S6K1

  1. Issam Ben-Sahra1*,
  2. Jessica J. Howell1*,
  3. John M. Asara2,
  4. Brendan D. Manning1,
  1. 1Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA 02115, USA.
  2. 2Division of Signal Transduction, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.
  1. To whom correspondence should be addressed. E-mail: bmanning{at}hsph.harvard.edu
  1. * These authors contributed equally to this work.

  1. Fig. 1

    Influence of mTORC1 on the abundance of N-carbamoyl-aspartate. (A to C) Steady-state metabolite profiles from Tsc2+/+ and Tsc2−/− MEFs grown in the absence of serum for 15 hours and treated with either vehicle [dimethyl sulfoxide (DMSO)] or rapamycin (20 nM). Intracellular metabolites from three independent samples per condition were profiled by means of liquid chromatography (LC)/MS/MS, and those (A) significantly increased in Tsc2−/− relative to Tsc2+/+ cells or decreased in Tsc2−/− cells by either (B) 15 hours or (C) 1 hour of Rapamycin treatment are shown as row-normalized heat maps ranked according to P value. The complete metabolite profiles from these samples are provided in table S1. (D) Schematic of the de novo pyrimidine synthesis pathway and the source of carbon and nitrogen incorporated into the pyrimidine ring (bottom). (E and F) The effects of mTORC1 inhibition on the steady-state levels of N-carbamoyl-aspartate, measured via LC/MS/MS, in (E) Tsc2−/− MEFs or (F) MCF10A cells stably expressing K-RasG12V or PI3KCAH1047R after 15 hours of serum starvation and 1 hour of treatment with rapamycin (20 nM) or DMSO. (G) N-carbamoyl-aspartate levels were measured, as above, in U87MG cells stably expressing a doxycycline-inducible PTEN after 15 hours of serum starvation and treatment with doxycycline (1μg/mL) or rapamycin (20 nM) for the final 8 hours. In (E) to (G), data are shown as the mean ± SEM from triplicate samples, with immunoblots below. MetaboAnalyst (Jianguo Xia, University of Alberta, Edmonton, Canada) and GENE-E (Joshua Gould, Broad Institute, Cambridge, Massachusetts, USA) software were used to assist metabolite data analyses. All P values for pairwise comparisons were calculated by using a two-tailed Student's t test (n = 3 samples/condition).

  2. Fig. 2

    Effects of genetic or insulin-stimulated activation of mTORC1 on metabolic flux through the de novo pyrimidine synthesis pathway. (A) Normalized peak areas of 15N-labeled metabolites, measured by means of LC/MS/MS, extracted from Tsc2+/+ and Tsc2−/− MEFs grown in the absence of serum for 15 hours, with vehicle (DMSO) or rapamycin (20 nM) treatment over the last 1 hour and a 15-min pulse label of 15N-glutamine. (B) Normalized peak areas of 15N-labeled metabolites from wild-type MEFs treated as above, but stimulated with insulin (100 nM) for 1 hour where indicated. (C) Normalized peak areas of 13C-labeled metabolites from cells treated as in (A), but with a 15-min pulse label of [4-13C]-aspartate before metabolite extraction. (D) Normalized peak areas of singly 13C-labeled metabolites from cells treated as in (A) but with rapamycin treatment for either 1 hour or 15 hours and 15-min pulse label with [1,2-13C]-glucose before metabolite extraction. In (A) to (D), all data are presented as mean ± SEM over three independent samples per condition. *P < 0.05 for pairwise comparisons calculated by using a two-tailed Student's t test (n = 3 samples/condition), with all P values provided in table S3.

  3. Fig. 3

    CAD as a direct substrate of S6K1. (A) Effects of insulin and rapamycin on CAD phosphorylation sites. FLAG–hemagglutinin (HA)–CAD was immunopurified from serum-starved (16 hours) human embryonic kidney (HEK)–293E cells, treated for 1 hour with DMSO or rapamycin (20 nM), before stimulation with insulin (3 hours, 50 nM). The ratios of phosphorylated to total peptide levels, measured as total ion current (TIC) by means of LC/MS/MS, of the indicated sites on CAD under the different conditions are graphed. ND, phospho-peptide not detected. (B) HEK-293E cells expressing empty vector (EV) or wild-type (WT), S1859A, or S1900A versions of FLAG-HA-CAD were serum-starved (16 hours) and stimulated with insulin (1 hour, 100 nM). FLAG immunoprecipitates were immunoblotted with a phospho-14-3-3–binding motif antibody (P-Ser motif). (C) Cells were treated as in (B) but pretreated for 1 hour with rapamycin (20 nM) or the S6K1 inhibitor PF-4708671 (10 μM, S6K1i) before insulin stimulation. (D) Cells were treated as in (C) but were also transfected with siRNAs targeting S6K1, S6K2, or both, or nontargeting controls (siCtl). (E) In vitro kinase assays were performed with FLAG-HA-CAD substrate (WT or S1859A) immunoprecipitated from serum-starved, rapamycin-treated HEK-293E cells and HA-S6K1 [WT or kinase dead (KD)] immunoprecipitated from insulin-stimulated HEK-293E cells. (F) HeLa cells were serum-starved (16 hours) and pretreated for 1 hour with rapamycin, S6K1i, or the MEK inhibitor U0126 (10 μM) before 1 hour of stimulation with insulin (100 nM) or EGF (20 ng/mL).

  4. Fig. 4

    Requirement of S6K1 and S1859 on CAD for the mTORC1-dependent stimulation of the de novo pyrimidine synthesis pathway. (A) Normalized peak areas of 15N-labeled metabolites, measured by means of LC/MS/MS, extracted from WT MEFs serum starved (15 hours) and insulin-stimulated (1 hour, 100 nM) in the presence of DMSO, rapamycin (20 nM), or PF-4708671 (10 μM, S6Ki), with a 15-min pulse label of 15N-glutamine. (B) Normalized peak areas of 15N-labeled metabolites from WT MEFs transfected with siRNAs targeting S6K1, S6K2, or both, or nontargeting controls (siCtl) were treated 48 hours after transfection as in (A). (C) The relative incorporation of radiolabel from 14C-aspartate, 3H-uridine, or 3H-thymidine into RNA and DNA from WT MEFs transfected with siRNAs as in (B), serum starved (15 hours), and stimulated with insulin (6 hours, 100 nM), during which cells were radiolabeled. (D) The relative incorporation of radiolabel from 14C-aspartate or 3H-uridine into rRNA from WT MEFs treated as in (C). (Right) The purified rRNA was also assessed on an agarose gel. (E) Normalized peak areas of 15N-labeled metabolites in G9C cells expressing CAD WT or a S1859A mutant treated as in (A). ND, metabolite not detected. In (A) to (E), all data are presented as mean ± SEM over three independent samples per condition. *P < 0.05 for pairwise comparisons calculated by using a two-tailed Student's t test (n = 3 samples/condition), with all P values provided in table S4.

Citation:

I. Ben-Sahra, J. J. Howell, J. M. Asara, and B. D. Manning, Stimulation of de Novo Pyrimidine Synthesis by Growth Signaling Through mTOR and S6K1. Science 339, 1323-1328 (2013).

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B. G. Ng, L. A. Wolfe, M. Ichikawa, T. Markello, M. He, C. J. Tifft, W. A. Gahl, and H. H. Freeze
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A. P. Oliveira, C. Ludwig, M. Zampieri, H. Weisser, R. Aebersold, and U. Sauer
Sci Signal 8, rs4-rs4 (28 April 2015)

mTOR Signaling in Epilepsy: Insights from Malformations of Cortical Development
P. B. Crino, C. Ludwig, M. Zampieri, H. Weisser, R. Aebersold, and U. Sauer
Cold Spring Harb Perspect Med 5, a022442-a022442 (1 April 2015)

Signal Transduction in Cancer
R. Sever, and J. S. Brugge
Cold Spring Harb Perspect Med 5, a006098-a006098 (1 April 2015)

The expanding role of mTOR in cancer cell growth and proliferation
M. Cargnello, J. Tcherkezian, and P. P. Roux
Mutagenesis 30, 169-176 (1 March 2015)

Memory Retrieval Requires Ongoing Protein Synthesis and NMDA Receptor Activity-Mediated AMPA Receptor Trafficking
J. Lopez, K. Gamache, R. Schneider, and K. Nader
J. Neurosci. 35, 2465-2475 (11 February 2015)

Rheb Protein Binds CAD (Carbamoyl-phosphate Synthetase 2, Aspartate Transcarbamoylase, and Dihydroorotase) Protein in a GTP- and Effector Domain-dependent Manner and Influences Its Cellular Localization and Carbamoyl-phosphate Synthetase (CPSase) Activity
T. Sato, H. Akasu, W. Shimono, C. Matsu, Y. Fujiwara, Y. Shibagaki, J. J. Heard, F. Tamanoi, and S. Hattori
J Biol Chem 290, 1096-1105 (9 January 2015)

Huntingtin promotes mTORC1 signaling in the pathogenesis of Huntington's disease
W. M. Pryor, M. Biagioli, N. Shahani, S. Swarnkar, W.-C. Huang, D. T. Page, M. E. MacDonald, and S. Subramaniam
Sci Signal 7, ra103-ra103 (28 October 2014)

Targeting mTORC1-Mediated Metabolic Addiction Overcomes Fludarabine Resistance in Malignant B Cells
A. Sharma, A. J. Janocha, B. T. Hill, M. R. Smith, S. C. Erzurum, and A. Almasan
Mol Cancer Res 12, 1205-1215 (1 September 2014)

Targeting mTOR dependency in pancreatic cancer
D. C. Morran, J. Wu, N. B. Jamieson, A. Mrowinska, G. Kalna, S. A. Karim, A. Y. M. Au, C. J. Scarlett, D. K. Chang, M. Z. Pajak et al.
Gut 63, 1481-1489 (1 September 2014)

Hepatic mTORC1 controls locomotor activity, body temperature, and lipid metabolism through FGF21
M. Cornu, W. Oppliger, V. Albert, A. M. Robitaille, F. Trapani, L. Quagliata, T. Fuhrer, U. Sauer, L. Terracciano, M. N. Hall et al.
Proc. Natl. Acad. Sci. USA 111, 11592-11599 (12 August 2014)

Epidermal Growth Factor Receptor (EGFR) Signaling Regulates Global Metabolic Pathways in EGFR-mutated Lung Adenocarcinoma
H. Makinoshima, M. Takita, S. Matsumoto, A. Yagishita, S. Owada, H. Esumi, and K. Tsuchihara
J Biol Chem 289, 20813-20823 (25 July 2014)

PPAR{gamma} activation attenuates glucose intolerance induced by mTOR inhibition with rapamycin in rats
W. T. Festuccia, P.-G. Blanchard, T. Belchior, P. Chimin, V. A. Paschoal, J. Magdalon, S. M. Hirabara, D. Simoes, P. St-Pierre, A. Carpinelli et al.
Am. J. Physiol. Endocrinol. Metab. 306, E1046-E1054 (1 May 2014)

Chromosome 3p loss of heterozygosity is associated with a unique metabolic network in clear cell renal carcinoma
F. Gatto, I. Nookaew, and J. Nielsen
Proc. Natl. Acad. Sci. USA 111, E866-E875 (4 March 2014)

The Adaptor Protein p66Shc Inhibits mTOR-Dependent Anabolic Metabolism
M. A. Soliman, A. M. Abdel Rahman, D. W. Lamming, K. Birsoy, J. Pawling, M. E. Frigolet, H. Lu, I. G. Fantus, A. Pasculescu, Y. Zheng et al.
Sci Signal 7, ra17-ra17 (18 February 2014)

The Role of Target of Rapamycin Signaling Networks in Plant Growth and Metabolism
Y. Xiong, and J. Sheen
Plant Physiol. 164, 499-512 (1 February 2014)

Hepatic signaling by the mechanistic target of rapamycin complex 2 (mTORC2)
D. W. Lamming, G. Demirkan, J. M. Boylan, M. M. Mihaylova, T. Peng, J. Ferreira, N. Neretti, A. Salomon, D. M. Sabatini, P. A. Gruppuso et al.
FASEB J. 28, 300-315 (1 January 2014)

Where is mTOR and what is it doing there?
C. Betz, and M. N. Hall
JCB 203, 563-574 (25 November 2013)

Evolution and cell physiology. 2. The evolution of cell signaling: from mitochondria to Metazoa
N. W. Blackstone, and M. N. Hall
Am. J. Physiol. Cell Physiol. 305, C909-C915 (1 November 2013)

Metastatic Castration-Resistant Prostate Cancer Hungers for Leucine
A. R. Tee, and M. N. Hall
JNCI J Natl Cancer Inst 105, 1427-1428 (2 October 2013)

New Strategies in Endometrial Cancer: Targeting the PI3K/mTOR Pathway--The Devil Is in the Details
A. P. Myers, and M. N. Hall
Clin. Cancer Res. 19, 5264-5274 (1 October 2013)

Adaptation to chronic mTOR inhibition in cancer and in aging
R. Gilley, K. Balmanno, C. L. Cope, and S. J. Cook
Biochm. Soc. Trans. 41, 956-961 (1 August 2013)

A growing role for mTOR in promoting anabolic metabolism
J. J. Howell, S. J. H. Ricoult, I. Ben-Sahra, and B. D. Manning
Biochm. Soc. Trans. 41, 906-912 (1 August 2013)

Exploiting Tumor Vulnerabilities: Epigenetics, Cancer Metabolism and the mTOR Pathway in the Era of Personalized Medicine
C. Munoz-Pinedo, E. Gonzalez-Suarez, A. Portela, A. Gentilella, and M. Esteller
Cancer Res. 73, 4185-4189 (15 July 2013)

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