Chemical Genetics Approach to Identify Peptide Ligands That Selectively Stimulate Dapk-1 Kinase Activity.
From: Cancer Research UK p53 Signal Transduction Group, University of Edinburgh Cancer Research Centre, Western General Hospital, Crewe Road South, Edinburgh EH4 2XR, UK.
Biochemistry
- Publish Date: Mar 2007
- ISSN: 0006-2960
- Volume: 46
- Issue: 10
- Pages: 2655-73
- Medium: Print
- Language: English
- Citation (JAMA): Fraser Jennifer A, Hupp Ted R, et al. Chemical Genetics Approach to Identify Peptide Ligands That Selectively Stimulate Dapk-1 Kinase Activity.. Biochemistry Mar 2007;46:2655-73
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.
Mesh Headings (Keywords): Animals, Apoptosis Regulatory Proteins, Calcium-Calmodulin-Dependent Protein Kinases, Cells, Cultured, Enzyme Activation, Humans, Insects, Ligands, Molecular Sequence Data, Peptides, Sequence Homology, Amino Acid
Check for Full Text / PubMed Unique Identifier (PMID): 17297916
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