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. ? ?
