Chemical genetic methods for studying protein kinases

Our laboratory focuses on the development of novel chemically based tools to decipher signal transduction pathways on a genome‐wide scale. We have developed a method for producing small molecules that are specific for any protein kinase of interest in a signaling cascade by combining protein design with chemical synthesis. These highly specific inhibitors of individual kinases have revealed a number of new principles of signal transduction that have complemented genetic and biochemical studies of cell signaling. Examples where new pathways and new functions can be revealed by small molecule inhibitors of protein kinases will be highlighted. A second area of interest in our laboratory is the tracing of direct kinase substrates. We have designed and synthesized unnatural ATP analogs, which are substrates of our engineered kinases but are poorly accepted as substrates of wild‐type kinases. This specific nucleotide substrate of any kinase of interest allows for the radiolabeling of the direct substrates of a wide variety of protein kinases including both serine/threonine and tyrosine kinases. New methods for the isolation and identification of low abundance substrates of kinases from cells will be discussed. Once a phosphoprotein substrate of a kinase is identified, the specific phosphorylation site is often difficult to identify using traditional tryptic peptide phosphorylation site mapping. Using a novel strategy based on the design of tailor made proteases which specifically cleave proteins after sites of phosphorylation, we have developed a rapid means to map protein phosphorylation patterns. Finally, a potential link between the unnatural ligands of engineered kinases and a set of plant hormones, the cytokinins, will be discussed in the context of a custom designed database created for the genome wide analysis of protein kinase catalytic domains.