Functional Role of Nck and Crk Binding Interactions

Nck 和 Crk 结合相互作用的功能作用

• 批准号: 6827231
• 负责人: BRUCE J. MAYER
• 金额: $ 32.42万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6827231
• 关键词: RNA interference; affinity chromatography; binding proteins; binding sites; biological signal transduction; chemical aggregate; fluorescent dye /probe; green fluorescent proteins; mass spectrometry; neoplastic transformation; oncoproteins; phosphorylation; polymerization; protein localization; protein protein interaction; protein structure function; protein tyrosine kinase; protooncogene; transfection /expression vector; transport proteins

DESCRIPTION (provided by applicant): SH2/SH3 adaptors such as Nck and Crk function by recruiting effector proteins that bind their SH3 domains to sites of tyrosine phosphorylation bound by their SH2 domains. Some forms of Crk can induce cell transformation when highly expressed, and endogenous Crk is implicated in regulating motility and phagocytosis. Nck has been has been shown to mediate localized actin polymerization and plays a role in pathogen invasion and motility. Despite the importance of these proteins and the seeming simplicity of their mechanism of action, little is currently known about the interactions that are important for their biological activity. Because the SH2 and SH3 protein binding domains are relatively promiscuous, both Crk and Nck bind to a multitude of partners in the cell, and the functional importance of any specific interaction or binding partner is largely unknown. Progress is further hampered by the fact that the stimuli that generate phosphorylated binding sites for adaptor SH2 domains have many other effects in the cell, making it difficult to isolate the specific role of the adaptor. Furthermore, the binding partners and ultimate biological effects of the adaptors are highly dependent on their subcellular localization and local concentration. We will use a suite of new experimental tools and approaches developed in the laboratory to manipulate the binding interactions of the adaptors, and the local concentration of the adaptors and their binding partners in the cell. We will identity and determine the subcellular localization of tyrosine-phosphorylated SH2 binding sites for Crk and Nck, identify those Crk SH3- and SH2-binding proteins whose interaction plays a role in Crk transformation, elucidate normal physiological roles for Crk and Nck, and determine the functional relevance of specific protein-protein interactions to those normal roles.

描述（由申请人提供）：SH2/SH3适配器（例如NCK和CRK功能）通过募集效应子蛋白将其SH3结构域与酪氨酸磷酸化位点结合到受其SH2结构域绑定的位点。高度表达时，某些形式的CRK会诱导细胞转化，并且内源性CRK与调节运动性和吞噬作用有关。 NCK已被证明可以介导局部的肌动蛋白聚合，并在病原体侵袭和运动中起作用。尽管这些蛋白质的重要​​性以及其作用机理的看似简单性，但目前对它们的生物活性很重要的相互作用知之甚少。由于SH2和SH3蛋白结合结构域是相对滥交的，因此CRK和NCK都与细胞中的许多伴侣结合，并且任何特定相互作用或结合伴侣的功能重要性在很大程度上都是未知的。由于在细胞中产生磷酸化的结合位点的刺激在细胞中具有许多其他效果，因此很难隔离适配器的特定作用，这进一步阻碍了进步。此外，适配器的结合伙伴和最终生物学效应高度取决于其亚细胞定位和局部浓度。我们将使用在实验室中开发的一系列新的实验工具和方法来操纵适配器的结合相互作用，以及适配器的局部浓度及其在细胞中的结合伙伴。我们将认同并确定CRK和NCK的酪氨酸磷酸化SH2结合位点的亚细胞定位，识别那些CRK SH3-和SH2结合蛋白，其相互作用在CRK转化中起作用，阐明CRK和NCK的正常生理作用，并确定与这些正常蛋白质相互作用的功能相关性。