No abstract

Caspase 8/10-associated RING proteins (CARPs) are a recently described family of protein ubiquitin ligases that interact with and negatively regulate death receptor-mediated apoptosis. Because CARPs are overexpressed in cancer and their silencing reduces cell viability and sensitizes tumor cells to chemotherapeutic agents, we investigated their relationship to p53 tumor suppressor signaling. p53 is a major determinant of chemosensitivity, and its levels are increased following DNA damage through N-terminal phosphorylation and inhibition of degradation. Although p53 is well known to be negatively regulated by several ubiquitin ligases including MDM2, none are known to target phosphorylated p53 for degradation. CARPs physically interact with and ubiquitinate p53, targeting it for degradation in the absence of MDM2. Serine 20-phosphorylated p53 is also ubiquitinated by CARPs. CARP silencing stimulates p53 expression and promotes downstream effects, including transcriptional activation and tumor suppression.Caspase 8/10-associated RING domain proteins (CARP) 2 1/2 are a recently characterized protein family with a ubiquitin protein ligase encoded in their RING domain (1). CARP1 and CARP2 distinctively target apical death effector domain-containing caspases 8 and 10, but not 9, and are cleaved by active caspases 8 and 10 once the extrinsic cell death pathway is initiated and transduces a death signal (1). The CARP proteins share three highly conserved functional domains, FYVE (Fab1p, YOTB, Vac1P, and EEA1) (2), CID (Caspase-Interacting Domain), and RING (Really Interesting New Gene) (3) (Fig. 1a). A point mutation of a catalytically essential histidine to an alanine in the RING finger domain abolishes CARP1 (His-342) or CARP2 (His-333) E3 ligase activity (1) (Fig. 1a). The cysteine and histidine residues in the CARP RING domains completely align with the RING consensus sequence (4). The characteristic residues of RING domains are conserved in both CARP1 and CARP2 and to a large extent with the RING domains of MDM2 (5), c-Cbl (6), and tumor necrosis factor-R-associated factor 2 (7). CARP proteins, especially the functional domains, are also markedly conserved across different organisms from human, mouse, and frog to Drosophila (1). Despite their many similarities, we show here that CARP1 and CARP2 are also functionally distinct.Compared with the canonical IAPs (Inhibitor of Apoptosis Protein), which contain BIR (Baculovirus IAP Repeat), CARD (Caspase Activation and Recruitment Domain), and RING motifs and inhibit caspases 3, 7, and 9, CARPs represent a new family of apoptosis inhibitors specifically targeting death effector domain-containing caspases for degradation. In the cascade of the apical cell death pathway, how CARPs are involved in the regulation of caspases 8 and 10 remains elusive. One possible role for CARPs may be in purging excess pro-caspases 8 and 10 that could otherwise be activated to cause cell death, because CARPs do not appear to control active caspases (1).Because CARP silencing by RNA interfer...