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Science 339 (6125): 1320-1323

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

Quantitative Phosphoproteomics Reveal mTORC1 Activates de Novo Pyrimidine Synthesis

Aaron M. Robitaille1, Stefan Christen2, Mitsugu Shimobayashi1, Marion Cornu1, Luca L. Fava1,*, Suzette Moes1, Cristina Prescianotto-Baschong1, Uwe Sauer2, Paul Jenoe1, and Michael N. Hall1,{dagger}

1 Biozentrum, University of Basel, 4056 Basel, Switzerland.
2 Institute of Molecular Systems Biology, Eidgenössische Technische Hochschule (ETH) Zürich, 8093 Zürich, Switzerland.


Figure 1
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Fig. 1. CAD as a target of mTORC1. (A) Diagram of CAD protein, including CPS (carbamoyl-phosphate synthetase), ATC (aspartate transcarbamylase), and DHO (dihydroorotase) enzymatic domains. (B) HeLa cells were deprived of serum for 16 hours in Dulbecco's minimum essential medium (DMEM) and incubated for 15 min without amino acids (AA) in 1x phosphate-buffered saline. Cells were then stimulated in DMEM with 10% dialyzed fetal calf serum (D-FCS) and 2x AA for 1 hour with or without rapamycin. (C) iRapKO and iRicKO MEFs were treated as in fig. S1B. The asterisk indicates a nonspecific band. (D) Mouse liver extract from wild-type (WT) (TSC1-fl/fl) or knockout (KO) (TSC1-fl/fl; Albumin-Cre) mice. Twelve-week-old littermates were starved overnight and treated with rapamycin or sham (0.9% NaCl) for 6 hours before killing. (E) WT (control) and DKO (S6K1-S6K2 double knockout) MEFs were deprived of serum for 2 hours and then stimulated for 1 hour in modified Hank's buffered salt solution plus 10% D-FCS, glucose, vitamins, and 2x AA, with or without rapamycin or PF-4708671.

 

Figure 2
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Fig. 2. Activation of de novo pyrimidine synthesis by mTORC1. (A) Diagram of de novo pyrimidine synthesis pathway. CAP, carbamoyl phosphate; CAA, carbamoyl aspartic acid; OMP, orotidine monophosphate. (B) HeLa cells were metabolically labeled with 4 mM 15N-amide glutamine. Metabolites were measured with targeted ultra high-performance liquid chromatography–tandem mass spectrometry. Values are expressed as mean ± SD. Asterisks indicate a statistical difference between stimulated and rapamycin treatment: *P < 0.05, **P < 0.01, ***P < 0.001 (Student's t test) n = 3 to 6. (C) Inhibition of growth factor–stimulated increase in DHOA, OA, and UTP cellular concentrations by rapamcyin. (D) Rapamycin did not inhibit growth factor–stimulated increase in GDP or GTP cellular concentrations.

 

Figure 3
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Fig. 3. Effects of mTORC1 on CAD localization and oligomerization. (A) HeLa cells were treated as in Fig. 1B. Endogenous CAD was visualized by immunocytochemistry. (B) G9c cells were transfected with plasmid-encoding, wild-type or mutant CAD. CAD oligomers were detected by sedimentation in 10 to 35% glycerol gradients. Fractions 2 (*) and 10 (**) correspond to 250-kD and 1.5-MD size standards, respectively. IB, immunoblot. (C) HeLa cells were treated as in Fig. 1B, G9c cells were treated as in (B), and iRapKO cells were treated as in fig. S1B. Oligomer is fraction 10 (**).

 

Figure 4
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Fig. 4. Promotion of S phase progression by mTORC1. (A) HeLa cells were synchronized in early S phase by using a double-thymidine block. Cells were then released in DMEM (starved) (red); DMEM, 10% D-FCS, and 2x AA (stimulated) (blue); or DMEM, 10% D-FCS, 2x AA, and 100 nM rapamycin (rapamycin) (green); in the absence (top) or presence (bottom) of 30 μM uridine. DNA content was analyzed by flow cytometry. (B and C) G9c cells were transfected with equal amounts of plasmid-encoding green fluorescent protein (GFP), WT, or mutant CAD. Cells were visualized with crystal violet 5 days after transfection (B), or proliferation was assayed via soft agar colony formation (C). Colonies were visualized after 8 days growth in the absence of uridine.

 


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