| This dissertation describes the development and characterization of molecules to study phosphorylation in the context of Src family kinases (SFKs). SFKs, with its nine members, play central roles in several cell signaling pathways, including cell adhesion, migration, proliferation and survival. Chapter 1 reviews current methodology in detecting and monitoring phosphorylation events.;Chapter 2 focuses on the development of tyrosine kinase reporters based on bipartite tetracysteine display. Using this strategy, we designed an encodable Src-family kinase sensor, E2. This sensor efficiently bound ReAsH upon phosphorylation, and the fluorescence increase was selective to phosphorylation at one of the two tyrosines in the substrate sequence. This observation suggests that E2 could be useful in visualizing position-dependent phosphorylation events. The fluorescence increase is comparable to existing FRET-based sensors of Src kinase activity. As an additional advantage, E2 displayed an increase in fluorescence when phosphorylated, whereas FRET-based Src kinase sensors typically show a signal decrease that can be difficult to differentiate from photobleaching. E2 represents the first generation scaffold for bipartite tetracysteine display-mediated visualization of kinase activity, and will be optimized further for rapid kinetics of ReAsH binding and kinase selectivity.;Chapter 3 discusses the development of ligands and activators of SFKs that recognize SH3 domains with high specificity. Using APP12 as a model ligand for the Src SH3 domain, a series of miniature protein ligands based on the aPP and PYY scaffolds were designed and characterized with a wide range of SH3 domains. Equilibrium dissociation constants of binding suggest that the grafting approach imparted a level of specificity in SH3 domain recognition, which APP12 did not have. Some of these molecules were even shown to activate Hck kinase in vitro with more potency than APP12. Preliminary work to test the activity of these molecules in live cells suggests that some of the miniature proteins may be able to increase lick kinase phosphorylation to levels comparable to Nef, a natural protein ligand for Hck. The section concludes with some isothermal titration calorimetry experiments to gain a better understanding of the thermodynamic relationship between the structure and function of the designed miniature proteins. |
