A protein kinase associated with apoptosis and tumor suppression: structure, activity, and discovery of peptide substrates.

Velentza AV, Schumacher AM, Weiss C, Egli M, Watterson DM
J Biol Chem. 2001 276 (42): 38956-65

PMID: 11483604 · DOI:10.1074/jbc.M104273200

Death-associated protein kinase (DAPK) has been implicated in apoptosis and tumor suppression, depending on cellular conditions, and associated with mechanisms 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 peptide substrate, and synthesized positional scanning substrate libraries in order to discover peptide substrates with K(m) values in the desired 10 microm range and to obtain knowledge about the preferences of DAPK for phosphorylation site sequences. Mutagenesis of DAPK catalytic domain at amino acids conserved among protein kinases or unique to DAPK provided a link between structure and activity. An enzyme assay for DAPK was developed and used to measure activity in adult brain and monitor protein purification based on the physical and chemical properties of the open reading frame of the DAPK cDNA. The results allow insight into substrate preferences and regulation of DAPK, provide a foundation for proteomic investigations and inhibitor discovery, and demonstrate the utility of the experimental approach, which can be extended potentially to kinase open reading frames identified by genome sequencing projects or functional genetics screens and lacking a known substrate.

MeSH Terms (30)

Amino Acid Sequence Animals Apoptosis Apoptosis Regulatory Proteins Binding Sites Blotting, Western Brain Calcium-Calmodulin-Dependent Protein Kinases Catalysis Catalytic Domain Cattle Death-Associated Protein Kinases DNA, Complementary Electrophoresis, Polyacrylamide Gel Humans Kinetics Models, Molecular Molecular Sequence Data Mutagenesis Mutagenesis, Site-Directed Open Reading Frames Peptide Library Peptides Phosphorylation Protein Binding Protein Structure, Tertiary Recombinant Proteins Sequence Homology, Amino Acid Substrate Specificity Tumor Suppressor Proteins

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