Fas-induced DNA fragmentation and proteolysis of nuclear proteins.
BACKGROUND
Fas is a member of the tumour necrosis factor (TNF) receptor family. Activation of Fas by its ligand or an agonistic anti-Fas antibody causes apoptosis in Fas-bearing cells, by activating various members of the caspase family.


RESULTS
Specific fluorogenic substrates (MCA-DEVDAPK[dnp] and MCA-VEVDAPK[dnp]) for caspases 3 and 6 were prepared. Using these substrates, a gradual increase of the caspase 3-and 6-like proteases were detected during the Fas engagement in human Jurkat. This activation of caspases correlated well with the cleavage of poly(ADP-ribose) polymerase and lamin B1, as well as with DNA fragmentation. When the recombinant caspases were added to the extracts from Jurkat cells, caspase 3 produced active caspase 6-like protease, while caspase 6 activated the caspase 3 protease, suggesting that these proteases can activate each other. The caspase-treated cell extracts, as well as the extracts from the Fas-activated cells, caused the proteolysis of nuclear proteins and DNA degradation. The cleavage of nuclear proteins was inhibited by caspase inhibitors, while the same inhibitors had no effect on DNA degradation.


CONCLUSIONS
At one stage of the caspase cascade, caspases activate each other, and amplify the apoptotic signal. Caspases downstream of the cascade then cause the proteolysis of nuclear proteins and DNA degradation.
Department of Genetics, Osaka University Medical School, Suita, Japan.
A
A
Kawahara
M
M
Enari
R V
RV
Talanian
W W
WW
Wong
S
S
Nagata
D017209:Apoptosis / Q000502:physiology; D015153:Blotting, Western; D020170:Caspase 1; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases; D002457:Cell Extracts; D003546:Cysteine Endopeptidases / Q000502:physiology*; D053938:DNA Fragmentation / Q000502:physiology*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004789:Enzyme Activation; D005456:Fluorescent Dyes; D006801:Humans; D019169:Jurkat Cells; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; D011506:Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors; D019014:fas Receptor / Q000502:physiology*
D002457:Cell Extracts; D005456:Fluorescent Dyes; D034921:Lamin Type B; D034882:Lamins; D009687:Nuclear Proteins; D009842:Oligopeptides; D011506:Proteins; D019014:fas Receptor; C499320:PARP1 protein, human; D000071137:Poly (ADP-Ribose) Polymerase-1; D011065:Poly(ADP-ribose) Polymerases; C505407:CASP3 protein, human; C505514:CASP6 protein, human; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases; D003546:Cysteine Endopeptidases; D020170:Caspase 1; D004264:DNA Topoisomerases, Type I