Revealing substrates and phosphoproteome level function of human STE20 kinases

揭示人类 STE20 激酶的底物和磷酸化蛋白质组水平功能

• 批准号: 10171453
• 负责人: Farren J. Isaacs
• 金额: $ 11.77万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-25 至 2021-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10171453
• 关键词: Address; Amino Acids; Apoptosis; Aspartate; Binding; Biological; Biological Process; Biotechnology; Cell Proliferation; Cells; Data; Development; Disease; Disease Pathway; Engineering; Escherichia coli; Event; Family; Functional disorder; Genomics; Glutamates; Human; Hypertension; Knowledge; Length; Malignant Neoplasms; Modification; Molecular; Organism; Outcome; Phosphoamino Acids; Phosphoproteins; Phosphorylation; Phosphorylation Site; Phosphoserine; Phosphotransferases; Physiological; Play; Post-Translational Protein Processing; Preparation; Process; Production; Protein Kinase; Proteins; Proteome; Proteomics; Recombinants; Regulation; Research; Role; Sense Codon; Signal Transduction; Site; Specificity; Structure; System; Technology; Terminator Codon; Translations; cell motility; drug candidate; human disease; improved; interest; new therapeutic target; novel; novel strategies; novel therapeutics; programs; protein protein interaction

Project summary Protein phosphorylation is one of the most common and critical post-translational modifications governing signaling cascades in humans. Phosphorylation of protein kinases governs their activity and regulation. The importance of regulation by phosphorylation is further emphasized by the fact that protein kinases comprise nearly 2% of the human proteome and numerous kinases have been implicated in processes that control cell proliferation, motility, and apoptosis in healthy and diseased human cells. While identification of phosphorylation sites within the human proteome has dramatically progressed in recent years, our understanding of phosphorylation cascades is limited due to a distinct lack of knowledge of which kinases are responsible for each phosphorylation event and the specific arrangement of phosphorylation sites leading to an active kinase that phosphorylates its target substrate. Establishing direct connections of all human kinases to the phosphoproteome and revealing a systems-level diagram of human signaling networks also remain defining challenges. Since phosphorylation plays a central role in protein-protein interactions through phospho- binding domains, new approaches that can address these questions in a comprehensive and unbiased fashion are needed. Studying protein phosphorylation has been limited by the inability to generate phosphoproteins with the specificity of natural systems. Genetically encoded non-standard amino acids (NSAAs) have recently enabled site-specific incorporation of phosphoserine into proteins. We showed that a genomically recoded organism (GRO), in which all TAG stop codons were converted to TAA and the deletion of RF-1, converted TAG to an open sense codon dedicated for incorporating phosphoamino acids. Importantly, this technological breakthrough enables site-specific expression of human phosphoproteins in an engineered bacterial system (i.e., GRO containing phosphoserine orthogonal translation system, OTS). Furthermore, it provides a platform technology to address questions probing the connectivity of the human kinome and the functional landscape of phospho-binding domains. Here, we aim to further develop and apply this technology to generate optimized platforms to address functional questions surrounding the phosphoserine component of the human phosphoproteome (Aim 1). These new, enhanced platforms will enable studies to identify STE20 kinase substrates that will directly inform future research into multiple human disease pathways as well as define a general strategy to elucidate human kinase substrates (Aim 2). Finally, we aim to identify phosphorylation sites that are drivers of protein-protein interactions in general, followed by, a systematic screen of the STE20 substrates in a coordinated effort to assign biological function to a portion of the human phosphoproteome (Aim 3).

项目摘要 蛋白质磷酸化是控制后最常见和最关键的翻译后修饰之一 在人类中发出信号级联。蛋白激酶的磷酸化控制其活性和调节。这 蛋白激酶包含以下事实，进一步强调了通过磷酸化调节的重要性 近2％的人蛋白质组和许多激酶与控制细胞的过程有关 健康和患病的人类细胞中的增殖，运动和凋亡。同时识别 近年来，人类蛋白质组内的磷酸化位点已取得了巨大进展，我们 了解磷酸化级联反应是有限的 负责每个磷酸化事件以及磷酸化位点的特定排列，导致 活性激酶可磷酸化其靶标底物。建立所有人类激酶与 磷蛋白体和揭示人类信号网络的系统级图也仍然保留 定义挑战。由于磷酸化在通过磷酸化的蛋白质 - 蛋白质相互作用中起着核心作用 具有约束力的领域，可以全面且公正的方式可以解决这些问题的新方法 需要。研究蛋白质磷酸化受到无法产生磷酸蛋白的限制 具有自然系统的特异性。遗传编码的非标准氨基酸（NSAA）最近具有 可以将磷酸磷酶特异性掺入蛋白质中。我们证明了一个基因组进行了重新编码 有机体（GRO），其中所有标签终止密码子都转换为TAA和RF-1的删除，转换为 标记用于用于掺入磷酸氨基酸的开放式密码子。重要的是，这种技术 突破使人类磷蛋白在工程细菌系统中的位点特异性表达 （即，含有磷serine的正交翻译系统的GRO）。此外，它提供了一个平台 解决探索人类群的连通性和功能格局的问题的技术 磷酸结合域。在这里，我们旨在进一步开发和应用这项技术以生成优化 解决人类磷serine成分的功能问题的平台 磷蛋白酶（AIM 1）。这些新的，增强的平台将使研究能够识别Ste20激酶 将直接告知未来对多种人类疾病途径的研究并定义A的底物 阐明人激酶底物的一般策略（AIM 2）。最后，我们旨在确定磷酸化 通常是蛋白质 - 蛋白质相互作用的驱动因素，然后是STE20的系统屏幕 在协调的努力中，将生物学功能分配给人类磷蛋白组的一部分的底物 （目标3）。