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We have employed a stable isotope- labelling approach to study the impact of rapamycin and mTOR kinase inhibitors (mTOR- Kls) on protein synthesis in Hela cells. As expected, rapamycin impaired the synthesis of proteins encoded by mRNAs containing S'- terminal oligopyrimidines (5'-TOPs), but, surpri singly, mTOR- Kls inhibited this much more strongly. Synthesis of several other proteins displayed the same pattern and, importantly, their mRNAs appear to contain a 5'- TOP, indicating they are novel members of this class of mRNAs. The mRNAs for translationally controlled tumour protein TCTP, an anti - apoptotic protein that is up- regulated by serum stimulation and in cancer, also contain a 5'-TOP. The seruminduced increase in TCTP levels is partially inhibited by rapamycin and completely by mTORKls. Polysome profile analysis combined with northern blotting revealed that these effects on TCTP and other novel 5' -TOP mRNA- encoded proteins are exerted at the translational level. Consistent with control by mTORCl, TCTP levels are not down- regulated by serumstarvation in mouse embryo fibroblasts lacking tuberous sclerosis complex 2 (TSC2). Although elF4E overexpression enhances the expression of TCTP and the polysomal association of other 5'-TOP mRNAs, it does not 'rescue ' their translation from the effects of mTOR inhibition, indicating that additional regulatory mechanisms account for th eir control by mTOR.

The mammalian target of rapamycin (mTOR), which controls diverse cellular processes, is regulated by the integration of many signals. Rapamycin strongly inhibits the proliferation of many cancer cell lines and there is a high level of interest in its potential use as an anti-cancer agent. However, some tumours and cancer cells are resistant to rapamycin. This has prompted the development of mTOR kinase inhibitors (mTOR-KIs), such as PP242 and AZD8055, which compete with ATP for binding to the kinase domain in mTOR. In this research, I have studied whether the effects of mTOR-KIs on cell signalling and protein synthesis differed in comparison to those of rapamycin. My data shows that mTOR-KIs have strikingly different effects on proteins (including formation of the eIF4F translation factor complex) that control mRNA translation. Furthermore, while rapamycin only has a very small inhibitory effect on the rate of protein synthesis, mTOR-KIs have a much bigger effect. A new mass spectrometric approach, ?pSILAC‘, was applied to explore the effects of rapamycin and mTOR-KIs on the synthesis of specific proteins. The data from pSILAC reveal (i) mTOR-KIs impair synthesis of many proteins; (ii) rapamycin always inhibits less than mTOR-KIs; (iii) their effects are strongest for proteins encoded by 5‘-TOP mRNAs, but mTOR-KIs again inhibit more strongly; (iv) synthesis of some other proteins which are not encoded by known 5‘-TOP mRNAs shows a similar pattern of inhibition to 5‘-TOP mRNAs. These data show that pSILAC is a valuable tool for studying the control of the synthesis of specific proteins. I have also investigated the effects of disruption of eukaryotic translation initiation factor 4E (eIF4E) phosphorylation on (i) its modification by SUMO-1 (ii) TNF? biosynthesis in macrophages and (iii) the interaction with specific mRNAs encoding protumourigenic factors.