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.