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Competitive and Noncompetitive Inhibition of the DNA-dependent Protein Kinase¹

Wellcome/CRC Institute, Cambridge, CB2 1QR; and Department of Zoology, University of Cambridge, Cambridge, CB2 3EJ, United Kingdom

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 K_i 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 K_i of 6.0 µM. 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.

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