DNA-dependent protein kinase catalytic subunit: a target for an ICE-like protease in apoptosis

EMBO J. 1996 Jul 1;15(13):3238-46.

Abstract

Radiosensitive cell lines derived from X-ray cross complementing group 5 (XRCC5), SCID mice and a human glioma cell line lack components of the DNA-dependent protein kinase, DNA-PK, suggesting that DNA-PK plays an important role in DNA double-strand break repair. Another enzyme implicated in DNA repair, poly(ADP-ribose) polymerase, is cleaved and inactivated during apoptosis, suggesting that some DNA repair proteins may be selectively targeted for destruction during apoptosis. Here we demonstrate that DNA-PKcs, the catalytic subunit of DNA-PK, is preferentially degraded after the exposure of different cell types to a variety of agents known to cause apoptosis. However, Ku, the DNA-binding component of the enzyme, remains intact. Degradation of DNA-PKcs was accompanied by loss of DNA-PK activity. One cell line resistant to etoposide-induced apoptosis failed to show degradation of DNA-PKcs. Protease inhibitor data implicated an ICE-like protease in the cleavage of DNA-PKcs, and it was subsequently shown that the cysteine protease CPP32, but not Mch2alpha, ICE or TX, cleaved purified DNA-PKcs into three fragments of comparable size with those observed in cells undergoing apoptosis. Cleavage sites in DNA-PKcs, determined by antibody mapping and microsequencing, were shown to be the same for CPP32 cleavage and for cleavage catalyzed by extracts from cells undergoing apoptosis. These observations suggest that DNA-PKcs is a critical target for proteolysis by an ICE-like protease during apoptosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies / chemistry
  • Apoptosis*
  • Base Sequence
  • Caspase 1
  • Catalysis
  • Cell Line
  • Cysteine Endopeptidases / metabolism*
  • DNA Primers
  • DNA-Activated Protein Kinase
  • DNA-Binding Proteins*
  • Etoposide / pharmacology
  • HeLa Cells
  • Humans
  • Hydrolysis
  • Mice
  • Mice, SCID
  • Molecular Sequence Data
  • Nuclear Proteins
  • Protein-Serine-Threonine Kinases / chemistry
  • Protein-Serine-Threonine Kinases / immunology
  • Protein-Serine-Threonine Kinases / metabolism*
  • Substrate Specificity
  • Tumor Cells, Cultured

Substances

  • Antibodies
  • DNA Primers
  • DNA-Binding Proteins
  • Nuclear Proteins
  • Etoposide
  • DNA-Activated Protein Kinase
  • PRKDC protein, human
  • Protein-Serine-Threonine Kinases
  • Cysteine Endopeptidases
  • Caspase 1