Stable isotope-labelling analysis of the impact of inhibition of the mammalian target of rapamycin on protein synthesis

The mammalian target-of-rapamycin complex 1 (mTORC1[1]) regulates diverse cell functions. mTORC1 controls the phosphorylation of several proteins involved in mRNA translation and the translation of specific mRNAs, including those containing 5'-terminal oligopyrimidines (5'-TOP). To date, most of the proteins encoded by known 5'-TOP mRNAs are proteins involved in mRNA translation, such as ribosomal proteins and elongation factors. Rapamycin inhibits some mTORC1 functions, whereas mTOR kinase inhibitors (mTOR-KIs) interfere with all of them. mTOR-KIs inhibit overall protein synthesis more strongly than rapamycin. To study the effects of rapamycin or mTOR-KIs on synthesis of specific proteins, we applied a pulsed stable isotope-labelling method (pSILAC). Our data reveal, firstly, that mTOR-KIs and rapamycin differentially affect the synthesis of many proteins. Second, mTOR-KIs inhibit the synthesis of proteins encoded by 5'-TOP mRNAs much more strongly than rapamycin does, revealing that these mRNAs are controlled by rapamycin-insensitive outputs from mTOR. Thirdly, the synthesis of certain other proteins shows a similar pattern of inhibition. Some of them appear to be encoded by 'novel' 5'-TOP mRNAs; they include proteins which, like known 5'-TOP mRNA-encoded proteins, are involved in protein synthesis, while others are enzymes involved in intermediary or anabolic metabolism. These data indicate that mTOR signalling may promote diverse biosynthetic processes through the translational upregulation of specific mRNAs. Lastly, a SILAC-based approach revealed that, although rapamycin and mTOR-KIs have little effect on general protein stability, they stabilise proteins encoded by 5'-TOP mRNAs