Akt controls alternative splicing in T helper call fate decisions

摘要 丝氨酸/苏氨酸激酶Akt在多种细胞过程中起着关键作用，包括增殖、 通过胞核和胞浆靶标的磷酸化，细胞的新陈代谢和存活。在CD4T辅助对象中 (Th)Akt和/或mTOR活性的分化抑制导致T调节(Treg)细胞的诱导， 这对维持自身耐受性和预防自身免疫至关重要。Treg诱导 通过将幼稚的CD4T细胞暴露于低剂量的抗原(Ag)与其活性呈负相关 AKT/mTOR通路。在几种体内模型中，银剂量对Th分化的影响已经显示出来 低银剂量有利于Treg和Th2的分化，而高银剂量则诱导炎症 Th1细胞。我们的初步数据显示，高强度的TCR信号与低强度的TCR信号在质量上 不同的Akt磷酸化，导致Akt底物特异性的改变。定量质量 抗磷酸(丝氨酸/苏氨酸)Akt底物抗体对免疫沉淀物的光谱分析 低或高激活的T细胞中Akt底物磷酸化的多重差异 剂量Ag。有趣的是，我们观察到几个RNA加工因子被不同的 AKT依赖于Ag剂量。特别是调控KEY选择性剪接的hnRNP L 在低而不是高刺激的T细胞中，TCR信号通路的组成部分被磷酸化。 剂量Ag。这导致TCR信号组件的选择性剪接发生依赖于Akt的变化。 这些结果表明，不同水平的TCR刺激启动了性质上不同的分化 程序和Akt对选择性剪接的差异调控是关键因素之一 决定Th细胞命运的决定。基于这些初步发现，我们假设Akt介导的 RNA加工因子的磷酸化诱导幼稚Th细胞分化为 效应细胞或调控细胞通过改变TCR信号元件的选择性剪接。 提出了三个具体目标：1)确定TCR信号强度如何控制Akt活性和 功能；2)确定Akt磷酸化在发育过程中控制选择性剪接的作用 3)确定CD247的选择性剪接在Th细胞命运中的作用。

Abstract 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 naïve 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 naïve 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.