Protein tyrosine kinases (PTK) are one of the largest enzyme families in a human cell. Many PTKs play central roles in the complex signal transduction pathways that control cellular functions at the most basic level, i.e. metabolism, development, cell division, differentiation, and apoptosis. Due to their key regulatory roles, many PTKs are also important targets for anti-cancer drug discovery. While all PTKs phosphorylate proteins on tyrosine (Tyr) residues, each PTK phosphorylates specific Tyr residues on only one or a small number of specific proteins. Thus, PTK substrate specificity determines the specificity and fidelity of signal transduction. At present, although the crystal structures of about a dozen PTKs are known, the structural and mechanistic basis for PTK substrate specificity has not been defined. This lack of information represents a major gap in our understanding of how PTKs function, which impedes our understanding of the mechanisms and pathways of signal transduction, and our ability to design specific PTK inhibitors to manipulate or interfere with signal transduction. This application focuses on elucidating the mechanisms for PTK substrate recognition and specificity, using Csk (C-terminal Src kinase) as a model PTK. Csk specifically phosphorylates Src family kinases on a C-terminal Tyr residue (Tyr527). However, Csk recognition of Src is not solely based on interactions with the Src tail containing the phosphorylation site. ? ? Our central hypothesis is that remote docking interactions between Csk and Src are largely responsible for this specific recognition. Preliminary studies have established the molecular system and methodology for elucidating the docking interactions, defined the structural parameters of the docking interactions, and identified part of the Csk substrate-docking site. These preliminary studies clearly validated the docking based recognition model. Building on this foundation, we now propose to map the full substrate-docking surface on Csk, identify the docking determinants on Src recognized by Csk, examine the implications of substrate docking on catalysis, and explore the potential of developing docking site-based PTK inhibitors. This work will not only reveal detailed information as to how Csk recognizes Src, but may also provide the basis for a general model of PTK substrate specificity and a new paradigm for PTK inhibitor design. ? ?
