AbstractThe serine-threonine kinase mammalian target of rapamycin (mTOR) is an important integrator of nutrient, cytokine and growth factor sensing in T cells and controls transcriptional programs that determine CD8+ cytotoxic T cell fate and trafficking. mTORC1 is inhibited by the drug rapamycin which is a powerful immunosuppressant used in the clinic in the context of organ transplantation. However, not much is known about the full extent of the role of mTOR signalling in CTL.
We thus utilised high resolution quantitative mass spectrometry to define the mTOR regulated CTL proteome and map the abundance and isoform expression of more than 6700 proteins in CTL. The data provide unbiased analysis of how mTORC1 reprograms the transcriptional and proteome landscape of T cells. The results show that mTORC1 controls expression of approximately 700 proteins with equal numbers of up and down regulated proteins. This illustrates that mTORC1 inhibition does not lead to a general decreases in protein levels but initiates a diverse reprogramming in gene expression and in particular drives selective decreases and increases in the expression of key metabolic regulators, effector molecules, adhesion molecules and adapter proteins. The proteomic approach also allowed us to detect effects caused by mTORC1 inhibition that were not caused by changes in the corresponding transcripts but solely due to posttranscriptional mechanisms.
One striking result was the dominance of mTORC1 negative feedback control of the serine/threonine kinase PKB. This prompted detailed analysis of the role of mTOR in the regulation of the phosphatidylinositol (3,4,5)-trisphosphate (PIP3) signalling effector PKB in CTL. A striking observation was that mTOR inhibitors allow T cells to accumulate very high levels of PIP3 and cause T cells to hyperactivate the serine/threonine kinase PKB and moreover reprogram PKB activation. PKB activity was thus uncoupled from mTORC2 activity and explained the similar phenotype of selective mTORC1 vs combined mTORC1/2 inhibition.
Collectively these experiments highlight the power of high resolution analysis of proteomes to uncover critical signalling checkpoints that control T cell differentiation and give new insights about how mTORC1 inhibitors control T cell function.
|Date of Award
|Doreen Cantrell (Supervisor)