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关键作用， 细胞代谢和存活，通过磷酸化的核和细胞质的目标。在CD 4 T辅助细胞中 (Th)Akt和/或mTOR活性的分化抑制导致T调节（Treg）细胞的诱导， 其对于维持自身耐受性和预防自身免疫性是关键的。Treg诱导 通过将幼稚CD 4 T细胞暴露于低抗原（Ag）剂量， Akt/mTOR通路。Ag剂量对Th分化的影响已在几种体内模型中显示 使得低Ag剂量有利于Treg和Th 2分化，而高Ag剂量诱导炎性 Th 1细胞我们的初步数据表明，高强度与低强度的TCR信号导致定性的 不同的Akt磷酸化，导致Akt底物特异性的改变。定量质量 用抗磷酸化（Ser/Thr）Akt底物抗体对免疫沉淀物（IP）进行光谱分析 揭示了在用低或高浓度活化的T细胞中磷酸化的Akt底物之间的多重差异。 剂量Ag。有趣的是，我们观察到几种RNA加工因子被不同的磷酸化， Akt取决于Ag剂量。特别是，hnRNP L，它调节关键蛋白的选择性剪接， TCR信号通路的组分在用低，但不高， 剂量Ag。这导致TCR信号传导组分的可变剪接中的Akt依赖性变化。 这些结果表明，不同水平的TCR刺激启动定性不同的分化， Akt对选择性剪接的差异调节是这些程序的关键因素之一， 决定Th细胞的命运基于这些初步发现，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 效应细胞或调节细胞通过TCR信号传导组分的可变剪接的变化。 提出了三个具体目标：1）确定TCR信号强度如何控制Akt活性， 2）确定Akt磷酸化在控制选择性剪接中的作用， Teff和Treg细胞;和3）确定CD 247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.