UC San Diego

Organisms rely on complex forms of chemical, physical and structural sensors that wire their communication networks at the cellular and molecular levels. These signal transduction pathways were classically treated as binary switches that relay information along specific routes from origins to destinations; however, this view is insufficient to describe the scale and diversity of responses offered by a limited number of molecular actors and pathways. Therefore, a more nuanced examination of structural components is needed for deeper understanding of high specificity and diversity of generated signals in the cellular context. The studies presented herein aim to explore the special molecular features of a major signaling component, the C-terminal Src kinase (Csk). It is a master regulator of the Src family of tyrosine kinases (SFKs) whose members participate in almost all aspects of cellular regulation and functions. We first studied the importance of a site-specific divergence within Csk's regulatory Src Homology 2 (SH2) domain and examined its effects on kinase behavior and functions using extended biophysical and activity techniques coupled to molecular dynamics simulations. Induced flexibility in the distal loop position was sensed throughout the protein as it modulated kinase functional motions and activity; revealing intramolecular routes of long-range communications in Csk. Along with insights on intrinsic functions, the results may provide a general framework for identifying potential effector sites for specific targeting by design. And since the distal motif is within a common adaptor domain, we characterized the changes in Csk's interaction with its physiological, membrane- recruiting activator, the Csk Binding Protein (CBP). Our enzymatic activation assays and Nuclear Magnetic Resonance binding analysis indicate that while the magnitude of maximal p-CBP activation of the kinase is largely unaffected, the overall catalytic efficiency is hampered by the non-contacting loop and allows for simultaneous, tunable control of localization and intrinsic activity. Lastly, unlike many modular scaffolds that could routinely be captured in multiple functional states, Csk's intrinsically dynamic character precludes extensive analysis through traditional structural techniques. Therefore, we used computational tools to study full- length Csk aiming to predict molecular transitions and large-scale coupled motions. The theoretical results were corroborated experimentally using our established kinetic, mutational, and structural-dynamic techniques; and construct a clearer model of dynamic intramolecular regulation. The data suggest that previously undetected, directional-global motions of the modular domains about the kinase core are inherently linked to Csk's enzymatic tasks of binding its substrates and catalyzing the phosphotransferase reaction