Executioner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis.
Apoptosis is orchestrated by a family of cysteine proteases known as the caspases. Fourteen mammalian caspases have been identified, three of which (caspase-3, -6, and -7) are thought to coordinate the execution phase of apoptosis by cleaving multiple structural and repair proteins. However, the relative contributions that the "executioner" caspases make to the demolition of the cell remains speculative. Here we have used cell-free extracts immuno-depleted of either caspase-3, -6, or -7 to examine the caspase requirements for apoptosis-associated proteolysis of 14 caspase substrates as well as nuclear condensation, chromatin margination, and DNA fragmentation. We show that caspase-3 is the primary executioner caspase in this system, necessary for cytochrome c/dATP-inducible cleavage of fodrin, gelsolin, U1 small nuclear ribonucleoprotein, DNA fragmentation factor 45 (DFF45)/inhibitor of caspase-activated DNase (ICAD), receptor-interacting protein (RIP), X-linked inhibitor of apoptosis protein (X-IAP), signal transducer and activator of transcription-1 (STAT1), topoisomerase I, vimentin, Rb, and lamin B but not for cleavage of poly(ADP-ribose) polymerase (PARP) or lamin A. In addition, caspase-3 was also essential for apoptosis-associated chromatin margination, DNA fragmentation, and nuclear collapse in this system. Surprisingly, although caspase-6 and -7 are considered to be important downstream effector caspases, depletion of either caspase had minimal impact on any of the parameters investigated, calling into question their precise role during the execution phase of apoptosis.
Slee
E A
EA
Molecular Cell Biology Laboratory, Department of Genetics, The Smurfit Institute, Trinity College, Dublin 2, Ireland.
Adrain
C
C
Martin
S J
SJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins / Q000378:metabolism; D053148:Caspase 3; D053178:Caspase 6; D053179:Caspase 7; D020169:Caspases / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D002843:Chromatin / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D053938:DNA Fragmentation; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D018260:Gelsolin / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D034904:Lamin Type A; D034921:Lamin Type B; D034882:Lamins; D008840:Microfilament Proteins / Q000378:metabolism; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D016160:Retinoblastoma Protein / Q000378:metabolism; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D050794:STAT1 Transcription Factor; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D014746:Vimentin / Q000378:metabolism; D051636:X-Linked Inhibitor of Apoptosis Protein; D013049:Spectrin
D000255:Adenosine Triphosphate; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; D053148:Caspase 3; D053178:Caspase 6; D053179:Caspase 7; D020169:Caspases; D002843:Chromatin; D003574:Cytochrome c Group; D004264:DNA Topoisomerases, Type I; D004268:DNA-Binding Proteins; D018260:Gelsolin; D034904:Lamin Type A; D034921:Lamin Type B; D034882:Lamins; D008840:Microfilament Proteins; D009687:Nuclear Proteins; D011506:Proteins; D053422:Receptor-Interacting Protein Serine-Threonine Kinases; D016160:Retinoblastoma Protein; D017412:Ribonucleoprotein, U1 Small Nuclear; D050794:STAT1 Transcription Factor; D015534:Trans-Activators; D014746:Vimentin; D051636:X-Linked Inhibitor of Apoptosis Protein; D013049:Spectrin; C494079:STAT1 protein, human; C496866:XIAP protein, human; C109664:caspase-activated DNase inhibitor; C506249:RIPK1 protein, human; C505407:CASP3 protein, human; C505514:CASP6 protein, human; C505520:CASP7 protein, human