The Serine/Threonine kinase Akt plays a critical role in multiple cellular processes including proliferation, cell metabolism and survival, through phosphorylation of nuclear and cytoplasmic targets. In CD4 T helper (Th) differentiation inhibition of Akt and/or mTOR activity results in the induction of T regulatory (Treg) cells, which are critical for the maintenance of self-tolerance and the prevention of autoimmunity. Treg induction through exposure of nave CD4 T cells to low antigen (Ag) doses is negatively correlated with activity of the Akt/mTOR pathway. The effect of Ag dose on Th differentiation has been shown in several in vivo models such that low Ag dose favors Treg and Th2 differentiation whereas high Ag doses induces inflammatory Th1 cells. Our preliminary data show that TCR signals of high vs. low strength result in qualitatively different Akt phosphorylation, resulting in a change in the substrate specificity of Akt. Quantitative mass spectrometry analysis of immunoprecipitates (IPs) with an anti-phospho-(Ser/Thr) Akt substrate antibody revealed multiple differences between Akt substrates phosphorylated in T cells activated with low or high dose Ag. Intriguingly, we observed that several RNA processing factors are differentially phosphorylated by Akt depending on Ag dose. In particular, hnRNP L, which regulates the alternative splicing of key components of the TCR signaling pathway is phosphorylated in T cells stimulated with low, but not high, dose Ag. This results in Akt-dependent changes in the alternative splicing of TCR signaling components. These results suggest that different levels of TCR stimulation initiate qualitatively distinct differentiation programs and that differential regulation of alternative splicing by Akt is one of the key elements determining Th cell fate decisions. Based on these preliminary findings, we hypothesize Akt-mediated phosphorylation of RNA processing factors induces the differentiation of nave Th cells to either effector or regulatory cells through changes in alternative splicing of TCR signaling components.
Three specific aims are proposed; 1) To determine how TCR signal strength controls Akt activity and function; 2) To determine the role of Akt phosphorylation in controlling alternative splicing in developing Teff and Treg cells; and 3) To determine the role of alternative splicing of CD247 in Th cell fate.
/Relevance Regulatory T (Treg) cells prevent autoimmunity by suppressing the activation and function of self-reactive T cells. Our overall goal with this research is to understand the mechanisms by which activity of a critical enzyme is altered by TCR signal strength and how this differential activity controls T cell differentiation into regulatory or effector cells. A deeper understanding of the mechanisms by which TCR signal strength controls Treg differentiation will lead to more effective therapies for a number of immune-mediated diseases, including cancer and autoimmunity.