Identifying protein kinase substrates:: hunting for the organ-grinder's monkeys

Despite an extraordinary increase in our understanding of protein kinase biology, the discovery of novel substrates for protein serine/threonine kinases has become a rate-limiting step in advancing our knowledge of cell signalling. This is largely due to the lack of suitable methods for the identification and purification of these molecules. Traditional in vitro methods of finding protein serine/threonine kinase substrates are now being complemented by more contemporary methodologies designed to identify substrates in intact cells. In particular, both phospho-motif antibodies that can recognize a broad consensus signature sequence phosphorylated by a specific protein kinase, and phospho-motif-binding proteins that can recognize and enrich protein kinase substrates are increasing in use. Potential protein kinase substrates discovered by these new methods must be verified both in vitro and in the intact cell by testing them against several specified criteria. Major recent advances in substrate detection technology, including global phosphoproteome analysis and protein kinase chips, have the potential to transform our ability to identify new protein kinase targets. The current status and future development of this technology is discussed.