Reversible protein phosphorylation cascades are the dominant means by which cells process extracellular information and convert these signals into complex phenotypic responses such as growth, differentiation, adhesion and motility. The proper spatial and temporal control of intracellular phosphorylation events by protein kinases is essential for normal cellular function. Indeed, mis-regulation of protein kinase activity is a cause or consequence of a number of diseases, including cancer, diabetes and chronic inflammation. For this reason, protein kinases are one of the most highly pursued targets for the development of new therapeutics by the pharmaceutical industry. Despite widespread interest in the determination of kinase cellular function and in the development of kinase-targeted pharmacological agents, most members of this diverse enzyme family remain poorly characterized. A major reason for this shortcoming is that there remains a paucity of techniques for the global analysis of protein kinases in complex biological mixtures. The overall goal of the research in this proposal is to develop a general set of molecular tools that will allow the functional state of protein kinases to be profiled in cell lysates and living cells. The Specific Aims of the proposed research are: (1) To develop a panel of active site-directed, photo-affinity probes that allow global profiling of the kinase superfamily. (2) To develop a general set of assays for the analysis of probes developed in Specific Aim 1 and to use these assays to determine the selectivity of several clinically relevant kinase inhibitors. (3) To develop a general set of molecular probes that selectively bind to either the active or inactive conformation of protein kinases. If successful, these tools will provide a general method for determining the cellular targets of kinase inhibitors in physiologically relevant environments and for identifying aberrant kinase activities in human disease. PUBLIC HEALTH RELEVANCE: The mis-regulation of protein kinase activity is a cause or consequence of a number of diseases, including cancer, diabetes and chronic inflammation, which has made members of this diverse enzyme family targets for the development of new therapeutics. The goal of our proposed research is to develop new chemical tools for the molecular analysis of protein kinases in cellular lysates and in living cells. These reagents should allow us to gain a greater understanding of kinase function and aid in the discovery of new targets for the development of therapeutics.