A protein kinase associated with apoptosis and tumor suppression - Structure, activity, and discovery of peptide substrates

Death-associated protein kinase (DAPK) has been implicated in apoptosis and tumor suppression, depending on cellular conditions, and associated with m echanisms of disease. However, DAPK has not been characterized as an enzyme due to the lack of protein or peptide substrates. Therefore, we determined the structure of DAPK catalytic domain, used a homology model of docked pe ptide substrate, and synthesized positional scanning substrate libraries in order to discover peptide substrates with K-m values in the desired 10 muM range and to obtain knowledge about the preferences of DAPK for phosphoryl ation site sequences. Mutagenesis of DAPK catalytic domain at amino acids c onserved among protein kinases or unique to DAPK provided a link between st ructure and activity. An enzyme assay for DAPK was developed and used to me asure activity in adult brain and monitor protein purification based on the physical and chemical properties of the open reading frame of the DAPK cDN A. The results allow insight into substrate preferences and regulation of D APK, provide a foundation for proteomic investigations and inhibitor discov ery, and demonstrate the utility of the experimental approach, which can be extended potentially to kinase open reading frames identified by genome se quencing projects or functional genetics screens and lacking a known substr ate.