Through the use of sequence analyses and structural prediction algorithms the authors found that IAPs containing three BIRs and a RING finger also contain a UBA domain. This domain, which is located between the third BIR and the RING finger, enables interaction with Ub and is found in proteins that recognize Ub-modified proteins and activate a variety of signalling pathways. Further experiments established that cIAP1 (also known as BIRC2) and Drosophila melanogaster IAP2 interact with both Lys48 and Lys63 poly(Ub) chains, whereas cIAP2 (BIRC3) and XIAP (BIRC4) interact only with Lys63 poly(Ub) chains. None of the IAPs tested interacted with monoubiquitylated residues. Protein domain deletion experiments indicate that these IAPs need to oligomerize before they can interact with poly(Ub) chains and that RING finger-mediated dimerization, but not E3 ligase activity, is required for this interaction.
So how does the interaction with poly(Ub) affect the function of IAPs? Deletion of cIAP1 results in sensitivity to tumour necrosis factor-mediated apoptosis. Re-expression of wild-type cIAP1 (cIAP1WT) in mouse embryonic fibroblasts (MEFs) that were null for the cIAP1 gene rescued this phenotype, whereas expression of cIAP1 with a mutated UBA domain (cIAP1MF/AA) did not. Amplification of 11q22, which contains the gene that encodes cIAP1, is often seen in human tumours, and overexpression of cIAP1 cooperates with either loss of p53 or deregulated expression of MYC during tumorigenesis. Unlike cells expressing MYC and cIAP1WT, co-expression of MYC and cIAP1MF/AA in liver cells failed to induce subcutaneous tumours when injected into immunocompromised mice.
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