title: Chemical genetics approach to identify peptide ligands that selectively stimulate DAPK-1 kinase activity.
abstract: Dissection of signal transduction pathways has been advanced by classic genetic approaches including targeted gene deletion and siRNA-based inhibition of gene product synthesis. Chemical genetics is a biochemical approach to develop small peptide-mimetic ligands to alter, post-translationally, how an enzyme functions. DAPK-1 was used as a model enzyme to develop selective peptide ligands that modulate its specific activity. The tumor modifier p21 has the most highly conserved elements of a DAPK consensus substrate, including a basic core followed by a hydrophobic core. Therefore, the p21 protein was synthesized in overlapping fragments to acquire a panel of peptide ligands for testing in DAPK binding and phosphorylation assays. Three distinct p21 derived peptide fragments were found to bind to DAPK; however, these had no stimulatory effect on its activity toward in vivo substrates, p21 and MLC. The p21 peptide ligands did, however, strikingly stimulate DAPK activity toward p53, a substrate that shows conservation in the hydrophobic part of its DAPK-1 consensus site. DAPK-1 stimulatory peptides attenuate tryptic cleavage of DAPK-1, suggesting that ligand binding can alter DAPK-1 conformation and lock the enzyme onto its substrate. We, therefore, generated an artificial p53, containing arginine residues N-terminal to the phospho-acceptor site, creating a better DAPK-1 peptide consensus and demonstrated that the Km for p531-66[ET-->RR] and ATP is elevated. The full-length p53E17T18-->R17R18 also functioned as a better Ser20 kinase substrate in vivo. These data suggest that DAPK-1 binding ligands can be generated to elevate its specific activity toward weak substrates and provide an approach to develop genetic assays to alter DAPK-1-specific activity in vivo.
authors.affiliation: Cancer Research UK p53 Signal Transduction Group, University of Edinburgh Cancer Research Centre, Western General Hospital, Crewe Road South, Edinburgh EH4 2XR, UK.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Fraser
authors.forename: Ted R
authors.initials: TR
authors.lastname: Hupp
mesh_terms: D000818:Animals; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D064550:Death-Associated Protein Kinases; D004789:Enzyme Activation / Q000187:drug effects*; D006801:Humans; D007313:Insecta / Q000166:cytology; D008024:Ligands; D008969:Molecular Sequence Data; D010455:Peptides / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid
chemical_list: D051017:Apoptosis Regulatory Proteins; D008024:Ligands; D010455:Peptides; C579096:DAPK1 protein, human; D064550:Death-Associated Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases