Interleukin-1 beta-converting enzyme-like protease cleaves DNA-dependent protein kinase in cytotoxic T cell killing.
Cytotoxic T cells (CTL) represent the major defense mechanism against the spread of virus infection. It is believed that the pore-forming protein, perforin, facilitates the entry of a series of serine proteases (particularly granzyme B) into the target cell which ultimately leads to DNA fragmentation and apoptosis. We demonstrate here that during CTL-mediated cytolysis the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs), an enzyme implicated in the repair of double strand breaks in DNA, is specifically cleaved by an interleukin (IL)-1 beta-converting enzyme (ICE)-like protease. A serine protease inhibitor, 3,4-dichloroisocoumarin (DCl), which is known to block granzyme B activity, inhibited CTL-induced apoptosis and prevented the degradation of DNA-PKcs in cells but failed to prevent the degradation of purified DNA-PKcs by CTL extracts. However, Tyr-Val-Ala-Asp-CH2Cl (YVAD-CMK) and other cysteine protease inhibitors prevented the degradation of purified DNA-PKcs by CTL extracts. Furthermore, incubation of DNA-PKcs with granzyme B did not produce the same cleavage pattern observed in cells undergoing apoptosis and when this substrate was incubated with either CTL extracts or the ICE-like protease, CPP32. Sequence analysis revealed that the cleavage site in DNA-PKcs during CTL killing was the same as that when this substrate was exposed to CPP32. This study demonstrates for the first time that the cleavage of DNA-PKcs in this intact cell system is exclusively due to an ICE-like protease.
Song
Q
Q
Queensland Cancer Fund Research Unit, Queensland Institute of Medical Research, Bancroft Centre, PO Royal Brisbane Hospital, Herston, Australia.
Burrows
S R
SR
Smith
G
G
Lees-Miller
S P
SP
Kumar
S
S
Chan
D W
DW
Trapani
J A
JA
Alnemri
E
E
Litwack
G
G
Lu
H
H
Moss
D J
DJ
Jackson
S
S
Lavin
M F
MF
D000595:Amino Acid Sequence; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D003602:Cytotoxicity, Immunologic; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D053804:Granzymes; D006801:Humans; D006868:Hydrolysis; D008666:Metalloendopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000378:metabolism; D010455:Peptides / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism; D015398:Signal Transduction; D013602:T-Lymphocytes, Cytotoxic / Q000201:enzymology*; D013997:Time Factors
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D010455:Peptides; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases; D003546:Cysteine Endopeptidases; D008666:Metalloendopeptidases; C089403:protease resembling ICE