Determinants of killer /DR5 signaling and death ligand sensitivity

Programmed cell death is carried out by a conserved protease family called caspases. Executioner caspases (-3, -6 and -7) produce the morphological changes associated with apoptosis but first require activation via initiator caspases (-2, -9, -8 and -10) that respond to the cellular insult. Cellular cues include death ligand binding to a cell surface receptor (the extrinsic pathway) or cellular stresses such as DNA damage (the intrinsic pathway). Activation of these pathways leads to adaptor-mediated initiator caspase recruitment and activation, triggering the caspase cascade. Death ligands such as TRAIL and their receptors belong to the tumor necrosis factor (TNF) and TNF Receptor (TNFR) superfamily, respectively. Classification as a death receptor requires a conserved cytoplasmic “death domain” (DD). TRAIL is the most complex ligand with two apoptotic death receptors, DR4 and KILLER/DR5, and three anti-apoptotic “decoy” receptors. Following death ligand exposure, death receptors bind a DD-containing adaptor molecule, FADD that recruits initiator caspases-8 and -10 via a death effector domain (DED) interaction between adaptor and caspase. In order to gain a better understanding of TRAIL signaling, we carried out alanine scanning mutagenesis within the KILLER/DR5 DD to identify residues critical for death. This study suggested that multiple charged residues cooperate for efficient adaptor binding and that maintenance of the hydrophobic core was paramount for death signaling. The severity of the mutations directly correlated with the ability to recruit FADD and caspase-8. To identify novel regulatory molecules, the DD of KILLER/DR5 and the DEDs of caspase-10 were used as bait in yeast two-hybrid screens. The KILLER/DR5 DD interacted with an uncharacterized protein (24g). Furthermore, another novel protein, CARP1 was identified as a caspase-10 interacting protein. CARP1 and a closely related protein, CARP2 represent a novel family of DED-caspase binding proteins with phospholipid-binding and ubiquitin ligase activities. Knockdown of CARP1 or CARP2 leads to sensitization of cells to death ligands and chemotherapeutic agents due to increased caspase-8 and -10 activity. CARPs represent DED-caspase inhibitors that are themselves targets of caspase-mediated cleavage. These studies reveal determinants of TRAIL signaling and identify a novel family of proteins that regulate apical caspases of the extrinsic pathway