Abstract
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.
Publication types
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Comparative Study
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Research Support, Non-U.S. Gov't
MeSH terms
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Adenoviridae / metabolism*
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Adenoviridae Infections / metabolism*
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Adenoviridae Infections / physiopathology
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Apoptosis / drug effects
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Apoptosis / physiology*
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Calcium-Binding Proteins / metabolism
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Calpain / genetics
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Calpain / metabolism
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Caspase Inhibitors
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Caspases / genetics
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Caspases / metabolism
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Cell Line, Transformed
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Cell Membrane / drug effects
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Cell Membrane / metabolism
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Cell Membrane / ultrastructure
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Cytoplasm / drug effects
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Cytoplasm / metabolism
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Cytoplasm / ultrastructure
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DNA Fragmentation / drug effects
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DNA Fragmentation / physiology
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Enzyme Inhibitors / pharmacology
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Eukaryotic Cells / cytology
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Eukaryotic Cells / drug effects
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Eukaryotic Cells / metabolism*
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Green Fluorescent Proteins
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Humans
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Indicators and Reagents
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Luminescent Proteins
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Models, Biological
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Proto-Oncogene Proteins / genetics
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Proto-Oncogene Proteins / metabolism
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Proto-Oncogene Proteins c-bcl-2 / genetics
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Proto-Oncogene Proteins c-bcl-2 / metabolism
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Signal Transduction / drug effects
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Signal Transduction / physiology*
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Viral Proteins / genetics
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Viral Proteins / metabolism*
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bcl-2-Associated X Protein
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src-Family Kinases / drug effects
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src-Family Kinases / metabolism
Substances
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Calcium-Binding Proteins
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Caspase Inhibitors
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E4orf4 protein, adenovirus
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Enzyme Inhibitors
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Indicators and Reagents
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Luminescent Proteins
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Proto-Oncogene Proteins
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Proto-Oncogene Proteins c-bcl-2
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Viral Proteins
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bcl-2-Associated X Protein
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Green Fluorescent Proteins
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calpastatin
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src-Family Kinases
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Calpain
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Caspases