title: Caspase-9 is activated in a cytochrome c-independent manner early during TNFalpha-induced apoptosis in murine cells.
abstract: FL5.12 pro-B lymphoma cells utilize the mitochondrial pathway to apoptosis in response to tumor necrosis factor (TNF) receptor occupation, yet high levels of the Bcl-2 family antiapoptotic protein, Bcl-x(L), fail to protect these cells against TNF-receptor-activated death. Bcl-x(L) expression delays, but does not totally block, the release of mitochondrial cytochrome c (cyt c) in these cells in response to TNFalpha-induced apoptosis and caspase-9 is processed prior to mitochondrial cyt c release under these circumstances. Early processing of caspase-9 also occurred in Apaf-1 knockout murine fibroblasts in response to TNF-receptor occupation. A caspase-9-specific inhibitor was more effective in delaying the progression of apoptosis in the FL5.12 Bcl-x(L) cells than was an inhibitor specific to caspase-3. Furthermore, downregulation of caspase-9 levels by RNA interference resulted in partial protection of these cells against TNF-receptor-activated apoptosis, indicating that caspase-9 activation contributed to early amplification of the caspase cascade. Consistent with this, proteolytic processing of caspase-9 was observed prior to processing by caspase-3, suggesting that caspase-3 was not responsible for early caspase-9 activation. We show that murine caspase-9 is efficiently processed by active caspase-8 at SEPD, the motif at which caspase-9 autoprocesses following its recruitment to the apoptosome. Our results suggest that, in addition to processing procaspase-3 and the BH3 protein Bid, active caspase-8 can cleave and activate procaspase-9 in response to TNF receptor crosslinking in murine cells.
authors.affiliation: Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis MN 55455, USA.
authors.forename: M A
authors.initials: MA
authors.lastname: McDonnell
authors.forename: D
authors.initials: D
authors.lastname: Wang
authors.forename: S M
authors.initials: SM
authors.lastname: Khan
authors.forename: M G
authors.initials: MG
authors.lastname: Vander Heiden
authors.forename: A
authors.initials: A
authors.lastname: Kelekar
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis; D053477:Apoptotic Protease-Activating Factor 1; D001402:B-Lymphocytes / Q000166:cytology; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D045744:Cell Line, Tumor; D045304:Cytochromes c / Q000378:metabolism*; D004789:Enzyme Activation; D007700:Kinetics; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D011506:Proteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000502:physiology; D034622:RNA Interference; D018124:Receptors, Tumor Necrosis Factor / Q000378:metabolism; D016415:Sequence Alignment; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000633:toxicity*; D051020:bcl-X Protein
chemical_list: C506593:Apaf1 protein, mouse; D053477:Apoptotic Protease-Activating Factor 1; C494812:Bcl2l1 protein, mouse; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D051020:bcl-X Protein; D045304:Cytochromes c; C505526:Casp8 protein, mouse; C506434:Casp9 protein, mouse; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases