Regulation of Nonreceptor Tyrosine Kinases through Csk.

描述(申请人提供)：真核细胞对外界刺激的适应能力和参与综合组织功能的能力取决于蛋白激酶的活性。特别是，非受体类的蛋白酪氨酸激酶在通过质膜将细胞外信息传递到特定的细胞内蛋白质靶点方面发挥着至关重要的作用。这是一个高度调控的过程，涉及磷酸化、亚细胞定位、结构域和蛋白质-蛋白质相互作用。在这项提案中，注意力将集中在了解Src家族非受体蛋白酪氨酸激酶是如何通过CSK(COOH末端Src激酶)来调节的。CSK使C末端磷酸化，下调Src酶家族的所有成员。这使CSK处于许多生物功能所必需的信号级联的顶端。这项研究的长期目标是：(A)确定CSK如何识别和磷酸化Src家族中的蛋白质底物；(B)研究丝氨酸磷酸化在体外和体内水平上调节CSK功能的作用；以及(C)确定支架/效应蛋白如何影响结构域通讯和催化功能。为了实现这些目标，将创新性地结合广泛的研究工具，包括氢-重离子交换、蛋白酶足迹、质谱学、体内激酶活性报告、质量标记、快速熄灭流动混合和停流荧光光谱分析。该项目将提供影响所有真核细胞的关键生物分子相互作用的基本信息，是一个可行的治疗目标。

DESCRIPTION (provided by applicant): The ability of the eucaryotic cell to adapt to external stimuli and participate in integrated organismal function depends on the activity of protein kinases. In particular, the nonreceptor class of protein tyrosine kinases plays essential roles in transferring extracellular information across the plasma membrane to specific intracellular protein targets. This is a highly regulated process that involves phosphorylation, subcellular localization and domain-domain and protein-protein interactions. In this proposal, attention will be focused on understanding how Src family nonreceptor protein tyrosine kinases are regulated through Csk (the COOH terminal Src kinase). Csk phosphorylates the C-termini and down-regulates all members of the Src enzyme family. This places Csk at the top of many signaling cascades essential for biological function. The broad, long-term objectives of this investigation will be (a) to determine how Csk recognizes and phosphorylates protein substrates in the Src family, (b) to study the role of serine phosphorylation in regulating Csk function at both in vitro and in vivo levels, and (c) to establish how scaffolding/effector proteins influence domain-domain communication and catalytic function. To accomplish these goals, a wide range of research tools will be innovatively coupled including hydrogen-deuterium exchange, protease footprinting, mass spectrometry, in vivo kinase activity reporters, mass tagging, rapid quench flow mixing, and stopped-flow fluorescence spectroscopy. This project will provide essential information on a critical biomolecular interaction which influences all eucaryotic cells and is a viable therapeutic target.