It is estimated that approximately 5 percent of genes in the genome encode protein kinases. To understand how these protein kinases regulate various neuronal functions in the brain, it is essential to identify their direct physiological substrates. However, traditional biochemical methods are not adequate for unambiguous identification of kinase substrates because of the existence of large kinase superfamily and well-conserved ATP- binding and catalytic sites across the various kinases. These features make the interpretation of kinase inhibition studies difficult, thus complicating the cellular dissections of kinase functions. In the current application, we propose to employ a novel chemical genetic strategy to identify the direct substrate of brain-specific alpha-CaM kinase II, a key kinase involved in long-term potentiation and learning and memory. In our preliminary studies, we have created a functional mutant alphaCaMKII (F89G) in which its ATP binding pocket has been enlarged to fit the unnatural N6-substitute ATP analogs that are too big to be accepted by the wild-type alphaCaMKII by transferring 32P from the ATP analogs to the protein substrates. The identification and analysis of the labeled protein substrates via this novel chemical genetic approach will provide new insights into how the CaM kinase contributes to neuronal plasticity and memory formation.
