A Chemical Genetic Approach for the Identifi cation of Direct Substrates of Protein Kinases

Protein kinases form one of the largest superfamily of enzymes that play pivotal roles in controlling almost every signaling pathway (1). Deregulated kinase activity thus leads to multiple diseases, including various forms of cancer (2), inflammatory and autoimmune diseases (3), neurodegenerative diseases (4,5), diabetes (6), and HIV infection (7). Signaling networks regulated by kinases are complex and highly interconnected. Additionally, many kinases display overlapping substrate specificities in vitro and can functionally compensate for each other in single gene knockout experiments (8,9). Therefore, unraveling of these pathways to dissect the role of any particular kinase (normal or oncogenic) has remained one of the major challenges ever since the first kinase was purified. Ideally, if the substrate of each kinase could be identified in a cell, it would provide a baseline for understanding the complex cellular functions and consequently also provide a blueprint for novel targets for drug discovery.