Imaging Protein Tyrosine Phosphorylation in vivo

描述（由申请人提供）：蛋白质和酶中酪氨酸残基的磷酸化和去磷酸化对健康和疾病中的许多细胞信号传导途径至关重要。在体内，酪氨酸磷酸化是动态的，并且受到蛋白酪氨酸磷酸酶（PTP）和蛋白酪氨酸激酶（PTK）的协同作用的严格控制。虽然酪氨酸磷酸化的重要性已经被认识了很多年，但探测重要信号蛋白中关键酪氨酸残基的磷酸化和去磷酸化所必需的工具一直发展缓慢。在这个探索性/开发性研究项目中，我们将创建一组新的酪氨酸磷酸化化学探针。将这些探针掺入活细胞中，并用于体内酪氨酸磷酸化和去磷酸化成像。该项目的长期目标是建立一种新的技术，用于实时分析体内酪氨酸磷酸化，并证明其在生理和病理条件下的细胞信号转导研究中的实用性。为此，我们提出了以下具体目标：（1）开发和验证一系列新的基于肽的PTP/PTK活性的体外探针，（2）阐明这些新的探针在体内动态酪氨酸磷酸化/去磷酸化过程中的成像的实用性。本建议书中描述的研究需要两位主要研究者在合成化学、生物化学和细胞生物学方面的专业知识的独特组合。通过EBRG R21机制为这个发展项目提供资金将有助于验证酪氨酸磷酸化成像的新技术，并为未来关于酪氨酸磷酸化在细胞信号传导过程中的重要性的假设驱动研究铺平道路。相关性：酪氨酸磷酸酶和酪氨酸激酶都是治疗许多人类疾病（包括糖尿病、自身免疫性疾病和癌症）的重要治疗靶标。在这个研究项目中，我们将开发一种新的技术来研究这些酶在活细胞中的活性和真实的时间。这项技术将帮助我们更多地了解这些酶在健康和疾病中的作用，并最终促进新疗法的设计。

DESCRIPTION (provided by applicant): The phosphorylation and dephosphorylation of tyrosine residues in proteins and enzymes is crucial to many cellular signaling pathways in both health and disease. In vivo, tyrosine phosphorylation is dynamic and tightly controlled by the concerted actions of protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs). Although the importance of tyrosine phosphorylation has been recognized for many years, the tools necessary to probe the phosphorylation and dephosphorylation of key tyrosine residues in important signaling proteins have been slow to develop. In this exploratory/developmental research project, we will create a new set of chemical probes of tyrosine phosphorylation. These probes will be incorporated into living cells and used to image tyrosine phosphorylation and dephosphorylation in vivo. The long-term objectives of this project are to establish a novel technology for real-time analysis of tyrosine phosphorylation in vivo and demonstrate its utility in the investigation of cell signaling under physiological and pathological conditions. To this end, we present the following SPECIFIC AIMS: (1) Develop and validate a series of novel peptide-based probes for PTP/PTK activity in vitro, (2) Illustrate the utility of these novel probes in imaging dynamic tyrosine phosphorylation/dephosphorylation processes in vivo. The research described in this proposal requires the unique combination of expertise in synthetic chemistry, biochemistry and cell biology embodied by the two Principal Investigators. Funding for this developmental project through the EBRG R21 mechanism will be instrumental in validating the new technology for imaging tyrosine phosphorylation and paving the way for future hypothesis-driven research on the importance of tyrosine phosphorylation in cellular signaling processes. RELEVANCE: Both tyrosine phosphatase and tyrosine kinase enzymes are important therapeutic targets for the treatment of many human diseases including diabetes, autoimmune disorders and cancer. In this research project we will develop a new technique for investigating the activity of these enzymes in live cells and in real time. This technique will help us to learn more about these enzymes in both health and disease, and ultimately facilitate the design of novel therapeutics.