New Insights into the Function of IAP Proteins: Modulation of the MYC/MAX/MAD Network

SUMMARY

A growing number of studies have revealed that the IAP (inhibitor of apoptosis) proteins play a variety of cellular roles in addition to suppression of apoptosis. A recent study in Molecular Cell demonstrates that one IAP member, c-IAP1, functions to potentiate the activity of Myc by triggering the ubiquitination and proteasomal degradation of the Myc inhibitory protein, Mad1.

When the iap (inhibitor of apoptosis) genes were first revealed in the genomes of higher eukaryotes and the term was coined, the function implied by the name frequently led to the mistaken assumption that all IAPs act to suppress apoptotic cell death. Certainly the prototype IAP, which was discovered in the genomes of insect viruses through an elegant screening approach designed to identify viral gene products capable of enhancing the survival of the infected cell (Crook et al., 1993), lives up to the name, but the proteins encoded by the related cellular genes actually participate in a wide range of cellular activities. The recent report by Xu and coworkers (Xu et al., 2007) aptly demonstrates this point.

Two important domains were originally identified in the baculovirus IAPs. The BIR (baculovirus iap repeat) is a zinc finger-like structure, two tandem repeats of which are present in the baculovirus IAPs. Largely through homology searches, the genomes of many higher eukaryotic organisms ranging from yeasts to human have now been shown to encode a group of proteins containing between one and three BIRs, and these proteins are collectively referred to as IAPs (Vaux and Silke, 2005). The second domain, which is characteristically found at the extreme carboxy terminus of the protype baculovirus IAP, as well as several cellular IAPs, is the RING motif. RING-containing proteins have been shown to direct the ubiquitination and proteasome-mediated degradation of target proteins. Initially, the BIRs were the focus of much investigation, particularly in the mammalian XIAP (X-linked IAP) in which the BIRs and adjacent sequences have been shown to directly bind to certain caspases, proteolytic enzymes which are critical executors of the apoptotic cell death program. However, a growing body of work has demonstrated the functional significance of the IAP RINGs, although the substrates of their ubiquitin ligase activities have remained largely elusive.

In their study, Xu and colleagues (Xu et al., 2007) examine the ubiquitin ligase properties of c-IAP1, a broadly expressed mammalian IAP that contains three BIRs and a RING. An interesting screening approach was taken to identify substrates of the E3 activity of c-IAP1, specifically testing a collection of proteins with well-established roles in tumorigenesis. The study focuses on one ‘hit’ from this approach: the tumor suppressor, MAD1, and the data support the notion that physiologically, MAD1 levels are regulated by polyubiquitination and proteasomal degradation through a mechanism that involves the RING-dependent ubiquitin ligase activity of c-IAP1.

The identification of MAD1 as a target of c-IAP1 is intriguing for several reasons. A pioneering study identified c-IAP1 as a putative oncoprotein that can function cooperatively with MYC in an experimental murine model of hepatocellular carcinoma (Zender et al., 2006). MAD1 is best known as a repressor of MYC through its ability to preferentially heterodimerize with the MYC binding partner, MAX (Grandori et al., 2000), and so by identifying MAD1 as a target of c-IAP1, the study by Xu and colleagues provides a potential explanation for the apparently synergistic tumor-enhancing effects of combining MYC and c-IAP1. Indeed, the study lends support to such a model, using transformation assays in which endogenous levels of c-IAP1, MAD1 and MYC were modulated by overexpression and RNA interference approaches, and the data suggest not only that c-IAP1 cooperates with MYC in tumorigenesis, but that this cooperativity is mediated by the antagonism of MAD1. Importantly, the RING-mediated ubiquitin ligase activity was shown to be required for these effects.

Another critically important implication of this study is that c-IAP1 was shown to function in oncogenesis through a mechanism which does not appear to involve direct apoptotic inhibition or direct interaction of the IAP with caspases. It is possible that while c-IAP1 might not function as a caspase inhibitor in the classical, biochemical sense, it might be able to target caspases for polyubiquitination, which would ultimately achieve the same effect. Additionally, recent reports have shown that c-IAP1 can ubiquitinate other IAPs, and can also function as a modulator of the signaling pathways controlling the NF-κB transcription factor pathways by directing the ubiquitination of key signaling intermediates such as NIK (Vince et al., 2007; Varfolomeev et al., 2007), RIP (Petersen et al., 2007) and NEMO/IKKγ (Tang et al., 2003), and so collectively these studies underscore the biological importance of IAPs in diverse cellular processes.

While these and other reports are beginning to shed light on the types of substrates that can be recognized and targeted by the RING-containing IAPs, we still know very little about how and when the ligase interacts with the substrate. The key finding over a decade ago that c-IAP1 is recruited to the type 2 tumor necrosis factor through its interaction with TRAF proteins (Rothe et al., 1995), strongly suggests a predominantly cytosolic role as a signal transduction intermediate, yet the study by Xu and coworkers reveals both a nuclear and cytosolic distribution of c-IAP1 and suggests that the nuclear pool is likely responsible for the targeting of MAD1. So we have much to learn about how the IAPs traffic intracellularly, how they meet their targets and how their ubiquitin ligase activities are triggered. Nevertheless, the identification of the targets of these E3 ligases is an essential first step in understanding the regulatory roles of the IAPs normally, as well as in disease states in which the activities of these enigmatic proteins are deregulated.