Regulation of Nonreceptor Tyrosine Kinases through Csk.

描述（由申请人提供）：真核细胞适应外部刺激并参与整合生物体功能的能力取决于蛋白激酶的活性。特别是，非受体类蛋白酪氨酸激酶在跨质膜将细胞外信息传递到特定的细胞内蛋白靶点中起着重要作用。这是一个高度调控的过程，涉及磷酸化，亚细胞定位和结构域-结构域和蛋白质-蛋白质相互作用。在这个建议中，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）进行调节的。Csk磷酸化C-末端并下调Src酶家族的所有成员。这使得Csk处于许多生物功能所必需的信号级联的顶端。这项研究的广泛的，长期的目标将是（a）确定如何Csk识别和磷酸化蛋白质底物的Src家族，（B）研究丝氨酸磷酸化在调节Csk功能在体外和体内水平的作用，和（c）建立支架/效应蛋白如何影响域域通信和催化功能。为了实现这些目标，广泛的研究工具将创新地结合起来，包括氢氘交换，蛋白酶足迹，质谱，体内激酶活性报告，质量标记，快速淬灭流动混合和停流荧光光谱。该项目将提供一个关键的生物分子相互作用，影响所有真核细胞，是一个可行的治疗目标的基本信息。

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.