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 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.
Velentza
A V
AV
Drug Discovery Program and Department of Molecular Pharmacology and Biological Chemistry, Northwestern University, Chicago, Illinois 60611, USA.
Schumacher
A M
AM
Weiss
C
C
Egli
M
M
Watterson
D M
DM
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D001665:Binding Sites; D015153:Blotting, Western; D001921:Brain / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D002417:Cattle; D018076:DNA, Complementary / Q000378:metabolism; D064550:Death-Associated Protein Kinases; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D025521:Tumor Suppressor Proteins / Q000737:chemistry*
D051017:Apoptosis Regulatory Proteins; D018076:DNA, Complementary; D019151:Peptide Library; D010455:Peptides; D011994:Recombinant Proteins; D025521:Tumor Suppressor Proteins; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases