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和参与综合有机体功能的能力取决于蛋白激酶的活性。特别是，非受体酪氨酸蛋白激酶在将细胞外信息通过质膜传递到特定的细胞内蛋白靶点方面起着至关重要的作用。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。在本提案中，重点将放在了解Src家族非受体蛋白酪氨酸激酶如何通过Csk （COOH末端Src激酶）进行调节。Csk磷酸化c末端并下调Src酶家族的所有成员。这使得Csk处于许多对生物功能至关重要的信号级联的顶端。本研究的广泛、长期目标将是(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调节Csk功能中的作用，以及(c)确定支架/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，将创新地结合广泛的研究工具，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，快速猝灭流混合和停流荧光光谱。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，是一个可行的治疗靶点。

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.