Tyrosine phosphorylation of both STAT5A and STAT5B is necessary for maximal IL-2 signaling and its mitogenic action

STAT5 proteins are vital for lymphocyte development and function. Cytokine activation of STAT5A and STAT5B involves the tyrosine phosphorylation of a C-terminal tyrosine in each protein; however, the importance of STAT5 tyrosine phosphorylation in vivo has not been assessed. Here we generated Stat5a and Stat5b tyrosine mutant knockin mice and found they had greatly reduced CD8+ T-cell numbers. Morever, these cells exhibited profoundly diminished IL-2-induced proliferation, correlating with reduced IL-2- mediated induction of Myc, pRB, a range of cyclins and CDKs, and a G1àS phase- transition block. The mutant CD8+ T cells also exhibited decreased IL-2-mediated activation of pERK and pAKT, which can in part be attributed to diminished IL-2-induced expression of IL-2Rb and IL-2Rg. Our findings demonstrate that tyrosine phosphorylation of both STAT5A and STAT5B is essential for maximal IL-2 signaling, and our transcriptomic and proteomic analyses elucidate the molecular basis for the mitogenic effects of IL-2 on CD8+ T cells. Overall design: ChIP-Seq and RNA-Seq analyses using freshly isolated mouse CD8+ T cells that were expanded with either no cytokine, or with IL-2 at various time points.

信号转导与转录激活因子5（STAT5）蛋白对于淋巴细胞的发育与功能至关重要。细胞因子介导的STAT5A与STAT5B激活，依赖于两种蛋白各自羧基末端酪氨酸位点的酪氨酸磷酸化修饰；然而，体内STAT5酪氨酸磷酸化的重要性尚未得到评估。本研究构建了Stat5a与Stat5b酪氨酸突变型敲入小鼠，发现其体内CD8阳性（CD8+）T细胞数量显著减少。此外，这些细胞的白细胞介素2（IL-2）诱导的增殖能力大幅受损，其与IL-2介导的原癌基因Myc、视网膜母细胞瘤蛋白（pRB）、多种细胞周期蛋白（cyclins）及细胞周期蛋白依赖性激酶（CDKs）的诱导表达下调相关，同时存在G1到S期的细胞周期阻滞。突变型CD8+ T细胞中，IL-2介导的磷酸化细胞外调节蛋白激酶（pERK）与磷酸化蛋白激酶B（pAKT）的激活水平也有所下降，这在一定程度上可归因于IL-2诱导的IL-2受体β链（IL-2Rβ）与IL-2受体γ链（IL-2Rγ）的表达下调。本研究结果证实，STAT5A与STAT5B的酪氨酸磷酸化对于实现最大化的IL-2信号转导至关重要；同时，我们的转录组学与蛋白质组学分析阐明了IL-2对CD8+ T细胞的促有丝分裂效应的分子机制。整体实验设计：使用新鲜分离的小鼠CD8+ T细胞进行染色质免疫沉淀测序（ChIP-Seq）与RNA测序（RNA-Seq）分析，这些细胞分别在无细胞因子或不同时间点的IL-2处理下进行扩增培养。