Systematic elucidation of calcineurin phosphatase signaling in humans

项目摘要 所有真核生物都使用基于磷酸化的信号网络，由蛋白激酶和 磷酸酶来调节细胞过程。虽然关于激酶信号传导的全球信息 爆炸，磷酸酶的知识已经落后。缺乏系统、公正的方法 分析磷酸酶信号留下了一个重大差距，在我们的理解细胞调控 网络. 25年来，我对钙调神经磷酸酶的研究，保守的Ca 2 +/钙调素调节， 磷酸酶，在S.酿酒酵母和人类已经直接解决了这个问题。钙调神经磷酸酶普遍存在于 在心血管、神经和免疫系统中表达并发挥重要的调节作用（1）; 然而，目前只有约70种蛋白质被确定为钙调磷酸酶底物（2）。钙调 使活化T细胞核因子（NFAT）转录因子去磷酸化，以活化 适应性免疫反应（3）和钙调磷酸酶抑制剂，FK 506和环孢菌素A，通常是 作为免疫抑制剂（4）。然而，这些药物会引起不良反应，包括 高血压、糖尿病和癫痫发作通过抑制非免疫组织中的钙调神经磷酸酶（5），突出了 需要系统地绘制人类钙调神经磷酸酶信号通路。我的工作阐明了钙调神经磷酸酶 通过基于发现短线性基序（SLiM）的新方法进行信号传导：短 在决定特异性、低- 对于信号传导至关重要的亲和相互作用（6）。使用实验和基于SLiM的计算机模拟 方法，我们最近定义了人类钙调磷酸酶信号网络（2）。这项工作揭示了 钙调神经磷酸酶对核孔复合物的保守调节，并显示出意想不到的钙调神经磷酸酶 接近中心体（2）。未来的努力将阐明这些细胞中的Ca 2+和钙调神经磷酸酶信号传导。 使用包括磷酸化蛋白质组学和邻近标记的方法的组合， 用更快标记的生物素连接酶（miniTurbo（33），Split TurboID）。荧光传感器将用于 探针Ca 2+信号在这些位置在体内。钙调神经磷酸酶在膜上的功能将通过 重点关注CNb 1，这是一种很少研究的亚型，其独特的定位和调节， 脂化C-末端序列（10）。通过动态棕榈酰化调节CNb 1活性将是 检查，CNb 1特异性信号通路，包括PI 4P合成的调节，将被 建立了一个全面的和机械的理解这种酶。最后 新技术，MRBLE-PEP（微球比例条形码镧系元素编码耦合到 PEPtides）（11）将被开发用于SLiM的定量分析和发现。总的来说，这项研究 旨在系统地绘制人类钙调神经磷酸酶信号通路， 基于SLiM的信令更广泛。

Project Summary All eukaryotes use phosphorylation-based signaling networks, composed of protein kinases and phosphatases, to regulate cellular processes. While global information about kinase signaling has exploded, knowledge of phosphatases has lagged behind. The lack of systematic, unbiased approaches to analyze phosphatase signaling leaves a major gap in our understanding of cellular regulatory networks. For >25 years, my research on calcineurin, the conserved Ca2+/calmodulin-regulated phosphatase, in S. cerevisiae and humans has directly addressed this issue. Calcineurin is ubiquitously expressed and plays critical regulatory roles in the cardiovascular, nervous and immune systems (1); however, only ~70 proteins are currently established as calcineurin substrates (2). Calcineurin dephosphorylates the Nuclear Factor of Activated T-cells (NFAT) transcription factors to activate the adaptive immune response (3), and calcineurin inhibitors, FK506 and cyclosporin A, are commonly prescribed as immunosuppressants (4). However, these drugs cause unwanted effects, including hypertension, diabetes, and seizures by inhibiting calcineurin in non-immune tissues (5), highlighting the need to map human calcineurin signaling pathways systematically. My work elucidates calcineurin signaling through novel approaches based on the discovery of short linear motifs (SLiMs): short degenerate peptide sequences found within regions of intrinsic disorder that determine specific, low- affinity interactions that are essential for signaling (6). Using experimental and in silico SLiM-based methods, we recently defined the human calcineurin signaling network (2). This work uncovered conserved regulation of nuclear pore complexes by calcineurin and showed unexpected calcineurin proximity to centrosomes (2). Future efforts will elucidate Ca2+ and calcineurin signaling at these compartments using a combination of methods that include phosphoproteomics and proximity labeling with faster-labeling biotin ligases (miniTurbo (33), Split TurboID). Fluorescent sensors will be used to probe Ca2+ signals at these locations in vivo. Calcineurin functions at membranes will be analyzed by focusing on CNb1, a little-studied isoform with distinct localization and regulation conferred by its unique, lipidated C-terminal sequences (10). Regulation of CNb1 activity via dynamic palmitoylation will be examined, and CNb1-specific signaling pathways, including regulation of PI4P synthesis, will be established to achieve a comprehensive and mechanistic understanding of this enzyme. Finally, the novel technology, MRBLE-PEP (Microspheres Ratiometric Barcode Lanthenide Encoding coupled to PEPtides) (11), will be developed for quantitative analysis and discovery of SLiMs. Overall this research aims to map human calcineurin signaling pathways systematically and to advance our understanding of SLiM-based signaling more broadly.