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Science 339 (6125): 1323-1328

Copyright © 2013 by the American Association for the Advancement of Science

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

Issam Ben-Sahra1,*, Jessica J. Howell1,*, John M. Asara2, and Brendan D. Manning1,{dagger}

1 Department of Genetics and Complex Diseases, Harvard School of Public Health, Boston, MA 02115, USA.
2 Division of Signal Transduction, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, MA 02115, USA.


Figure 1
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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).

 

Figure 2
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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.

 

Figure 3
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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).

 

Figure 4
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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.

 


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