Chemical genetics approach to identify peptide ligands that selectively stimulate DAPK-1 kinase activity

Research output: Contribution to journalArticlepeer-review

Abstract / Description of output

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.
Original languageEnglish
Pages (from-to)2655-73
Number of pages19
Issue number10
Publication statusPublished - 13 Mar 2007

Keywords / Materials (for Non-textual outputs)

  • Animals
  • Apoptosis Regulatory Proteins
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Cells, Cultured
  • Death-Associated Protein Kinases
  • Enzyme Activation
  • Humans
  • Insects
  • Ligands
  • Molecular Sequence Data
  • Peptides
  • Sequence Homology, Amino Acid


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