Phosphorylation of TSC2 (S1365) as a novel Regulator of mTORC1 Signaling in T Cells

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PROJECT SUMMARY mTOR plays a critical role in integrating signals from the immune microenvironment to regulate T cell activation, differentiation and function. We have been able to demonstrate that the Tuberous Sclerosis Complex 2 (TSC2) protein plays an important role in regulating mTORC1 activation in T cells. TSC2 is a RasGap protein that inhibits the activity of Rheb GTPase that in turn activates mTORC1. We have shown that genetic deletion of TSC2 in T cells leads to enhanced mTORC1 activity and a marked increase in CD8+ T cell effector function. However, while TSC2-/- T cells respond robustly to viruses and tumors, their persistent mTORC1 activity leads to a decrease in memory CD8+ T cell generation. Recently, the Kass lab has identified a novel phosphorylation site on TSC2 that regulates mTORC1 activity in cardiac myocytes. Phosphorylation of this site (S1365) leads to the inhibition of mTORC1 signaling. Mutating this site(SA) leads to an increase mTORC1 activity and the development of worse heart disease and mortality from pressure-overload (PO) stress. Alternatively, creating a phosphomimetic (SE) at this site mitigates mTORC1 activity and imparts protection from heart failure upon pressure overload. We hypothesized that the TSC2 (S1365) site might play an important role in regulating mTORC1 activity in T cells. Our preliminary studies demonstrate that upon TCR engagement this site is indeed phosphorylated. T cells harboring the SA mutation have unaltered mTORC1 activity in the non-stimulated condition (unlike TSC2-/- T cells), but show markedly increased activity upon TCR engagement. T cells with the SE mutation exhibit the opposite. Furthermore, phosphorylation of TSC2 (S1365) is markedly induced by hypoxia, low pH and reactive oxygen species suggesting that this pathway plays a critical role in integrating stress signals in order to regulate T cell differentiation and function. In this project we seek to define and understand a novel and selective mechanism of mTORC1 regulation in T cells. The overall goal of this proposal is to dissect the mechanisms by which phosphorylation of TSC2 at S1365 regulates mTORC1 activation in T cells, and consequently selectively regulates T cell activation, differentiation and function. Upon the completion of this proposal our findings will help elucidate novel and critical mTORC1 regulatory signaling mechanisms in T cells, and have implications for developing vaccines and engineering more robust T cells for Adoptive Cellular Therapy. This may in turn result in improved treatment strategies for preventing and treating infections as well as cancer.