mTORC1 stimulates nucleotide synthesis through both transcriptional and post-translational mechanisms
© Ben-Sahra et al; licensee BioMed Central Ltd. 2014
Published: 28 May 2014
Cellular growth signals stimulate anabolic processes. The mechanistic target of rapamycin (mTOR), as part of mTORC1, is a protein kinase that senses growth signals to regulate anabolic growth and proliferation. mTORC1 stimulates protein synthesis through effects on mRNA translation and ribosome biogenesis . mTORC1 signaling also promotes de novo lipid and sterol synthesis through the activation of the sterol-response element-binding protein (SREBP) transcription factors, which stimulate the expression of the enzymes driving this biosynthetic process .
Material and Methods
TSC2 +/+ MEFs, TSC2 -/- MEFs, MCF10A expressing pBabe-empty vector or PI3KCAH1047R, HeLa cells, U87MG cell line were used in this study. To determine the relative levels of intracellular metabolites, extracts were prepared and analyzed by LC/MS/MS . Regarding the U-14C-aspartate, U-14C-glycine incorporation into RNA and DNA, cells were serum starved for 15 hours and treated as indicated. Cells were harvested and RNA or DNA was isolated using Allprep DNA/RNA kits according to the manufacturer’s instructions and quantified using a spectrophotometer. For statistical analysis a two-tailed Student’s t-test was performed for all pairwise comparisons (n=3).
We find that activation of mTORC1 leads to the acute stimulation of metabolic flux through the de novo pyrimidine synthesis pathway . We recently found that mTORC1 stimulates the de novo purine synthesis pathway. In contrast with pyrimidine synthesis, the regulation of the purine synthesis by mTORC1 signaling occurs through long-term mechanism. Indeed, we found that mTORC1 regulates the de novo purine synthesis pathway through the transcription factor SREBP.
These findings demonstrate that growth signaling through mTORC1 promotes the production of new nucleotides to facilitate an increased demand for RNA and DNA. mTOR appears to be a central regulator of de novo nucleotide synthesis. Therefore, nucleotide synthesis joins protein and lipid synthesis as major anabolic processes stimulated by mTORC1 signaling.
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