Dissecting the role of Janus Kinases in Common Cytokine Receptor y-chain signaling in T-cells using chemical genetics

使用化学遗传学剖析 Janus 激酶在 T 细胞常见细胞因子受体 y 链信号传导中的作用

基本信息

批准号：
9105153
负责人：
Geoffrey Alexander Smith
金额：
$ 3.59万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-07-01 至 2019-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): γ-chain cytokines, such as IL-2, signal through a receptor complex that contains both the Jak1 and Jak3 kinases. Whether these two kinases play distinct or redundant roles remains unclear, with recent work suggesting that while Jak1 kinase activity is essential, Jak3 acts primarily as a scaffold to bring together other parts of th signaling complex. To address the catalytic role of Jak3, a highly selective and potent Jak3 inhibitor was developed by targeting a cysteine found in Jak3, but not in family members Jak1, Jak2 or Tyk2. In murine CD4+ T-cell blasts, the Jak3 inhibitor potently blocked IL-2 driven proliferation and up-regulation of CD25. Unlike the Jak1/Jak2 inhibitor ruxolitinib or the pan-Jak inhibitor tofacitinib, this Jak3 inhibitor was not nearly as potent at blocking STAT5 phosphorylation after acute IL-2 stimulation. Most research on IL-2 signaling has focused on phosphorylation changes that occur in the first hour. However, preliminary data suggests that there are two "waves" of STAT5 phosphorylation following IL-2 stimulation: one that peaks 15 minutes after stimulation and reduces nearly to baseline by an hour and a second, weaker wave that peaks around 6 hours. Interestingly, although Jak1 blockade blocks phosphorylation in both of these waves, the Jak3 inhibitor potently blocks only this second wave of phosphorylation, suggesting a non-redundant role for Jak3 in sustaining signaling. The proposed work aims to explore the consequences and generality of this signaling pattern. Aim 1 will use mRNA-seq to determine whether a Jak3 inhibitor blocking just this second wave of signaling blocks all IL-2 driven transcriptional changes or a specific subset of those changes. Preliminary studies suggest that CD8+ T-cell blasts do not display these two waves of STAT5 phosphorylation but rather a single sustained peak of STAT5 phosphorylation. Aim 2 will exploit this difference to elucidate the mechanism and consequences of these distinct signaling patterns. The Jak3 inhibitor and the Jak1/2 inhibitor ruxolitinib will be used to probe the temporal roles of each kinase, and the expression of negative signaling regulators, such as SOCS proteins, will be compared to identify potential mechanisms. Aim 3 will assess the in vivo consequences of this signaling pattern in the SKG model of rheumatoid arthritis. This model is known to respond to tofacitinib, which blocks nearly all cytokine signaling, but it is unknown whether selectively blockingγ-chain cytokines is sufficient. To address this question, a high dose of the Jak3 inhibitor, sufficient to block all STAT5 phosphorylation, will be given and disease score monitored. In parallel, a low dose of the inhibitor will be used to assess whether blocking only sustained signaling is sufficient for a therapeutic effect. Completion of this project will both further our basic understanding of cytokine signaling and have immediate translational implications for the treatment of rheumatoid arthritis.

描述（由申请人提供）：γ链细胞因子，例如IL-2，信号，虽然受体竞争对手复合物，jak1 anases jak3 jak3 jak3这两个激酶是否扮演着独特的或冗余的角色，最近的工作表明JAK1激酶活动表明这是必不可少的，JAK3主要是将JAK3的其他型号组合在一起的脚手架。 Tyk2中的CD4+ T-Cell爆炸与JAK1/JAK2抑制剂ruxolitinib或Pan-Jak抑制剂Tofacitinib不同数据表明，在2个刺激之后，有两个“波浪”的磷酸化t峰值大约6小时，Althooth Jak1阻断了这两个波中的磷酸化，JAK3抑制剂可正常地阻止第二波的磷酸化。该信号传导模式。AIM 1将使用mRNA-SEQ来确定JAK3抑制剂是否仅阻止第二波信号传导阻塞所有IL-2的初步研究，而CD8+ T-Cell Blasts则不显示这两个STAT5磷酸化的波将利用这些限制性模式的机制和后果。所有的细胞因子信号传导是否有选择性地块</s> </s> OCK，所有STAT5 PHOSS STAT5都将被给予疾病评分。治疗。