Abstract
Apoptosis depends critically on regulated cytoskeletal reorganization events in a cell. We demonstrate that death effector domain containing DNA binding protein (DEDD), a highly conserved and ubiquitous death effector domain containing protein, exists predominantly as mono- or diubiquitinated, and that diubiquitinated DEDD interacts with both the K8/18 intermediate filament network and pro-caspase-3. Early in apoptosis, both cytosolic DEDD and its close homologue DEDD2 formed filaments that colocalized with and depended on K8/18 and active caspase-3. Subsequently, these filamentous structures collapsed into intracellular inclusions that migrated into cytoplasmic blebs and contained DEDD, DEDD2, active caspase-3, and caspase-3-cleaved K18 late in apoptosis. Biochemical studies further confirmed that DEDD coimmunoprecipitated with both K18 and pro-caspase-3, and kinetic analyses placed apoptotic DEDD staining prior to caspase-3 activation and K18 cleavage. In addition, both caspase-3 activation and K18 cleavage was inhibited by expression of DEDDDeltaNLS1-3, a cytosolic form of DEDD that cannot be ubiquitinated. Finally, siRNA mediated DEDD knockdown cells exhibited inhibition of staurosporine-induced DNA degradation. Our data suggest that DEDD represents a novel scaffold protein that directs the effector caspase-3 to certain substrates facilitating their ordered degradation during apoptosis.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Apoptosis Regulatory Proteins
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Apoptosis*
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Carrier Proteins / metabolism
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Caspase 3
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Caspases / metabolism
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DNA / metabolism
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DNA-Binding Proteins / physiology*
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Death Domain Receptor Signaling Adaptor Proteins
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Enzyme Inhibitors / pharmacology
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Enzyme Precursors / metabolism
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Female
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HeLa Cells
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Humans
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Inclusion Bodies / chemistry
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Inclusion Bodies / physiology
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Inclusion Bodies / ultrastructure
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Intermediate Filaments / metabolism*
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Intermediate Filaments / ultrastructure
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Intracellular Signaling Peptides and Proteins*
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Jurkat Cells
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Keratins / metabolism
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Kinetics
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Male
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Membrane Glycoproteins / metabolism
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Mice
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Mice, Inbred C3H
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Mice, Inbred C57BL
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Nuclear Proteins / metabolism
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RNA, Small Interfering
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RNA, Untranslated / metabolism
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Staurosporine / pharmacology
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TNF-Related Apoptosis-Inducing Ligand
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Tumor Cells, Cultured
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Tumor Necrosis Factor-alpha / metabolism
Substances
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Apoptosis Regulatory Proteins
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Carrier Proteins
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DEDD protein, human
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DEDD2 protein, human
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DNA-Binding Proteins
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Death Domain Receptor Signaling Adaptor Proteins
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Dedd protein, mouse
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Enzyme Inhibitors
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Enzyme Precursors
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Intracellular Signaling Peptides and Proteins
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Membrane Glycoproteins
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Nuclear Proteins
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RNA, Small Interfering
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RNA, Untranslated
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TNF-Related Apoptosis-Inducing Ligand
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TNFSF10 protein, human
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Tnfsf10 protein, mouse
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Tumor Necrosis Factor-alpha
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Keratins
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DNA
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CASP3 protein, human
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Casp3 protein, mouse
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Caspase 3
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Caspases
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Staurosporine