title: Competitive and noncompetitive inhibition of the DNA-dependent protein kinase.
abstract: The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase that is involved in mammalian DNA double-strand break repair. The catalytic subunit of DNA-PK (DNA-PKcs) shares sequence homology in its kinase domain with phosphatidylinositol (PI) 3-kinase. Here, we provide a detailed kinetic analysis of DNA-PK inhibition by the PI 3-kinase inhibitor wortmannin and demonstrate this inhibition to be of a noncompetitive nature, with a Ki of 120 nM. Another inhibitor of PI 3-kinase. LY294002, its parent compound, quercetin, and other derivatives have also been studied. These chemicals are competitive inhibitors of DNA-PK, with LY294002 having a Ki of 6.0 microM. Using an antibody to wortmannin, we found that this compound binds covalently to the kinase domain of DNA-PKcs both in vitro and in vivo. Binding of wortmannin to the active site of DNA-PKcs is inhibited by ATP but not by a peptide substrate. Furthermore, wortmannin is able to bind to DNA-PKcs independently of Ku, and it is not stimulated by the presence of DNA. This suggests that the ATP binding site of DNA-PKcs is open constitutively and that DNA activation of the kinase is mediated via another mechanism.
authors.affiliation: Wellcome/CRC Institute, Cambridge, United Kingdom.
authors.forename: R A
authors.initials: RA
authors.lastname: Izzard
authors.forename: S P
authors.initials: SP
authors.lastname: Jackson
authors.forename: G C
authors.initials: GC
authors.lastname: Smith
mesh_terms: D000730:Androstadienes / Q000378:metabolism / Q000494:pharmacology*; D001667:Binding, Competitive; D002867:Chromones / Q000378:metabolism / Q000494:pharmacology*; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000378:metabolism / Q000494:pharmacology*; D018922:HL-60 Cells; D006801:Humans; D009025:Morpholines / Q000378:metabolism / Q000494:pharmacology*; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D011794:Quercetin / Q000031:analogs & derivatives / Q000494:pharmacology; D011838:Radiation-Sensitizing Agents / Q000378:metabolism / Q000494:pharmacology*; D012431:Rutin / Q000494:pharmacology; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D002867:Chromones; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D009025:Morpholines; D009687:Nuclear Proteins; D011838:Radiation-Sensitizing Agents; C012526:quercitrin; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D012431:Rutin; D011794:Quercetin; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D000077191:Wortmannin