Distinct cell death pathways triggered by the adenovirus early region 4 ORF 4 protein.
In transformed cells, induction of apoptosis by adenovirus type 2 (Ad2) early region 4 ORF 4 (E4orf4) correlates with accumulation of E4orf4 in the cell membrane-cytoskeleton fraction. However, E4orf4 is largely expressed in nuclear regions before the onset of apoptosis. To determine the relative contribution of nuclear E4orf4 versus membrane-associated E4orf4 to cell death signaling, we engineered green fluorescent fusion proteins to target E4orf4 to specific cell compartments. The targeting of Ad2 E4orf4 to cell membranes through a CAAX-box or a myristylation consensus signal sufficed to mimic the fast Src-dependent apoptotic program induced by wild-type E4orf4. In marked contrast, the nuclear targeting of E4orf4 abolished the early induction of extranuclear apoptosis. However, nuclear E4orf4 still induced a delayed cell death response independent of Src-like activity and of E4orf4 tyrosine phosphorylation. The zVAD.fmk-inhibitable caspases were dispensable for execution of both cell death programs. Nevertheless, both pathways led to caspase activation in some cell types through the mitochondrial pathway. Finally, our data support a critical role for calpains upstream in the death effector pathway triggered by the Src-mediated cytoplasmic death signal. We conclude that Ad2 E4orf4 induces two distinct cell death responses, whose relative contributions to cell killing may be determined by the genetic background.
Robert
Amélie
A
Centre de recherche en cancérologie de l'Université Laval, L'Hôtel-Dieu de Québec, CHUQ, Québec G1R 2J6, Canada.
Miron
Marie-Joëlle
MJ
Champagne
Claudia
C
Gingras
Marie-Claude
MC
Branton
Philip E
PE
Lavoie
Josée N
JN
D000256:Adenoviridae / Q000378:metabolism*; D000257:Adenoviridae Infections / Q000378:metabolism* / Q000503:physiopathology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002154:Calpain / Q000235:genetics / Q000378:metabolism; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D002461:Cell Line, Transformed; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D053938:DNA Fragmentation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005057:Eukaryotic Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008954:Models, Biological; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D051028:bcl-2-Associated X Protein; D019061:src-Family Kinases / Q000187:drug effects / Q000378:metabolism
D002135:Calcium-Binding Proteins; D061945:Caspase Inhibitors; C121517:E4orf4 protein, adenovirus; D004791:Enzyme Inhibitors; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D014764:Viral Proteins; D051028:bcl-2-Associated X Protein; D049452:Green Fluorescent Proteins; C033668:calpastatin; D019061:src-Family Kinases; D002154:Calpain; D020169:Caspases