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Chemical Genetics Approach to Identify Peptide Ligands that Selectively Stimulate DAPK-1 Kinase Activity

Fraser, Jennifer A.; Hupp, Ted R.


Jennifer A. Fraser

Ted R. Hupp


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.

Journal Article Type Article
Online Publication Date Feb 13, 2007
Publication Date 2007-03
Deposit Date Jul 26, 2016
Journal Biochemistry
Print ISSN 0006-2960
Electronic ISSN 1520-4995
Publisher American Chemical Society
Peer Reviewed Peer Reviewed
Volume 46
Issue 10
Pages 2655-2673
Keywords signal transduction pathways, Chemical genetics, genetic assays
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