Non-receptor protein tyrosine kinases (PTK's), Abl, Csk, Hck, and other Src family members, are central regulators of signal transduction and involved in multiple disease states, including many cancers and autoimmune syndromes. They share three highly homologous domains: an SH3 (Src homology 3) domain, an SH2 domain and a catalytic kinase domain. SH3 domains are involved in regulation of kinase activity in vitro and vivo. We propose structural characterization of the molecular mechanisms of SH3 domain control of the kinase activity using heteronuclear NMR coupled with protein segmental isotopic labeling. We will use chemical shift mapping and direct NMR structure determination to identify the intramolecular interaction surface between SH3 and catalytic domains of Csk which is likely to involve a novel mechanism of SH3/ligand recognition. Segmental isotopic labeling of SH3 domain within Abl SH(3,2,kinase) construct will be used to map the contacts of the SH3 with other parts of the molecule and assess structural modifications and changes in the presence of enzymatic and SH-related activators and inhibitors. We will conduct a broad survey of the solubility and association properties of Lek and Hck and other Src family members to establish the most likely practical candidate for future NMR studies. Based on the results of the survey, structural characterization of the SH3 autoinhibitory mechanisms in the active form of the Src-family of non-receptor tyrosine kinases will be conducted. The structural information derived from these studies will be important for understanding intramolecular mechanisms of kinase regulation and may represent a first step in designing highly specific therapeutic agents for regulating cellular PTK's activity.
