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，调节细胞过程。而有关激酶信号转导的全球信息 在爆炸式增长的情况下，对磷酸酶的了解已经落后了。缺乏系统性、不偏不倚的方法 分析磷酸酶信号在我们对细胞调节的理解上留下了很大的空白 网络。25年来，我对钙调素调节的保守的钙调神经磷酸酶的研究 酿酒酵母和人类中的磷酸酶直接解决了这个问题。钙调神经磷酸酶无处不在 在心血管、神经和免疫系统中表达并发挥关键的调节作用(1)； 然而，目前只有70种蛋白质被确定为钙调神经磷酸酶底物(2)。钙调神经磷酸酶 使激活的T细胞核因子(NFAT)转录因子去磷酸化以激活 获得性免疫反应(3)和钙调神经磷酸酶抑制剂，FK506和环孢素A，是常见的 作为免疫抑制剂处方(4)。然而，这些药物会产生不良影响，包括 高血压、糖尿病和癫痫通过抑制非免疫组织中的钙调神经磷酸酶(5)，突出 需要系统地绘制人类钙调神经磷酸酶信号通路图。我的工作阐明了钙调神经磷酸酶 基于发现短线状模体(SLIMs)的新方法的信号传递 在内在紊乱区域内发现的简并多肽序列，决定了特定的、低水平的 对于信号传递至关重要的亲和力相互作用(6)。使用实验和基于硅胶的超薄技术 方法，我们最近定义了人钙调神经磷酸酶信号网络(2)。这部作品揭开了 钙调神经磷酸酶对核孔复合体的保守调节和意外的钙调神经磷酸酶 靠近中心体(2)。未来的努力将阐明钙离子和钙调神经磷酸酶信号在这些 使用包括磷酸蛋白质组学和邻近标记在内的组合方法的隔室 使用更快标记的生物素连接酶(mini Turbo(33)，Split TurboID)。荧光传感器将用于 在活体内探测这些位置的钙信号。钙调神经磷酸酶在膜上的功能将通过 关注CNb1，这是一个研究很少的异构体，具有独特的定位和调节作用， 脂化的C-末端序列(10)。通过动态棕榈酰化调节CNb1活性将是 研究，CNb1特异的信号通路，包括PI4P合成的调节，将被 建立了对这种酶的全面和机械性的理解。最后， 新技术MRBLE-PEP(微球比率条形码Lanthenide编码耦合到 多肽)(11)，将被开发用于定量分析和发现减肥。总体来说，这项研究 目的是系统地绘制人类钙调神经磷酸酶信号通路图，并增进我们对 更广泛的基于SLIM的信令。

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.