Inhibitor of apoptosis (IAP) proteins, which bind to caspases via their baculoviral IAP repeat domains, also bear RING domains that enable them to promote ubiquitylation of themselves and other interacting proteins. Here we show that the RING domain of cIAP1 allows it to bind directly to the RING of X-linked IAP, causing its ubiquitylation and degradation by the proteasome, thus revealing a mechanism by which IAPs can regulate their abundance. Expression of a construct containing the RING of cellular IAP1 was able to deplete melanoma cells of endogenous X-linked IAP, promoted apoptosis, and also markedly reduced their clonogenicity when treated with cisplatin. Cross control of protein levels by RING domains may therefore enable their levels to be manipulated therapeutically.apoptosis ͉ ubiquitin ͉ homeostasis ͉ E3 ligase I nhibitor of apoptosis (IAP) proteins were initially identified in baculoviruses, where they prevent defensive apoptosis of the host cell (1), thereby increasing the time available for viral replication. Cellular IAP (cIAP) homologues, which all bear one to three baculoviral IAP repeat (BIR) domains, have been identified in yeasts and metazoans. Those that bear a RING domain in addition to BIR domains [X-linked IAP (XIAP), cIAP1, cIAP2, and ML-IAP͞Livin] appear to function as cell death inhibitors (reviewed in ref.2).The RING domains of IAPs can act as E3 ubiquitin ligases to promote the ubiquitylation of associated proteins such as TNF receptor-associated factors (TRAFs), Smac͞Diablo, and caspases (3-6). However, the importance of the RING domain for the antiapoptotic activity of the IAPs is unclear; on the one hand, a RING-less DIAP1 protein overexpressed in Drosophila had increased antiapoptotic activity (7); on the other hand, alleles of DIAP1 with mutations in the RING finger are null for Reaperinduced cell death, although more potent at blocking Hid-induced cell death (5,8,9).Our initial experiments showed that cIAP1 and XIAP can heterodimerize via a RING-RING interaction, but we also observed that expression of a stably integrated cIAP1 gene caused a specific reduction in the abundance of endogenous XIAP. Deletion studies revealed that the RING finger of cIAP1 was necessary and sufficient to cause loss of XIAP in a proteasome-dependent manner. cIAP1 RING-stimulated depletion of XIAP was seen in several cell types and associated with greatly increased sensitivity of several melanoma cells to cisplatin. Because several other E3 ligases such as BRCA1, BARD1, and RAG1 can interact via their RING fingers, it is possible that other RING-containing E3 ligases act to regulate the abundance of each other following heterodimerization. In this regard, it is striking that the E3 ligase activity of BRCA1 is greatly enhanced by heterodimerization with BARD1 (10, 11), suggesting a possible mechanism for homeostatic control of protein levels by RING domains. Materials and MethodsTransfections and Constructs. The complete sequence of all constructs used can be obtained upon request. Full length IAPs, RIN...

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