The Inhibitors of Apoptosis Protein (IAPs) are cellular guardians that are critical for controlling pro-death proteins such as caspases, Smac and HtrA2. IAPs contain both a RING ubiquitin ligase domain and characteristic baculoviral IAP repeat (BIR) domains that recognize substrates and promote their ubiquitination (Vaux and Silke, 2005
; Vucic et al., 2011
). Biochemical studies have shown that X chromosome-linked IAP (XIAP) can directly bind to and inhibit caspases, proteases that execute apoptotic cell death. Smac and HtrA2 are allies of caspases and antagonize XIAP using a characteristic N-terminal four residue IAP-binding motif (IBM) to prevent XIAP from binding caspases. However, these pro-apoptotic collaborators are also victims of XIAP as they become ubiquitinated and degraded in this mutually antagonistic relationship (Khan et al., 2008
; MacFarlane et al., 2002
). Cellular IAPs (mainly cIAP1 and cIAP2) have similar roles as XIAP in regulating caspases activity but also regulate NF-κB pathways by targeting TRAF2, RIP1 and NIK for ubiquitination (Vucic et al., 2011
). cIAP1 also mediates the ubiquitination and degradation of other IAP proteins, including XIAP and cIAP2, and thus fine tunes overall IAP levels in the cell (Silke et al., 2005
). Given the central importance of XIAP and cIAP1 in regulating cell death and inflammatory signaling, we sought an unbiased proteomics approach to search for additional substrates for the IAPs.
Identification of substrates for specific E3 ligases is extremely challenging because of the weak transient interaction between E3s and their substrates, the tremendous heterogeneity of modifications with over 5000 ubiquitinated proteins in cells (Kim et al., 2011
), and the rapid proteasomal degradation of ubiquitinated proteins. To facilitate E3 substrate identification we produced a catalytic tagging device, called the NEDDylator, that covalently marks E3 substrates with the rare and stable ubiquitin homolog, NEDD8 (Figure 1). NEDD8, like ubiquitin, is covalently conjugated to its substrate proteins through lysines. It uses an orthogonal system consisting of a NEDD8 E1 activating enzyme, E2 conjugating enzymes, and E3 ligases. NEDD8 is highly homologous to ubiquitin, but unlike ubiquitin, has a very limited number of endogenous protein substrates (Xirodimas, 2008
), mainly the cullins which are the scaffolding subunits of the cullin-RING ubiquitin ligase family (Deshaies et al., 2010
). NEDD8 modifications are known to regulate protein activity, and do not target proteins for proteasomal degradation.
Here, we generated a chimeric protein with a NEDD8 E2 (Ubc12) fused to the substrate binding domain of IAP ubiquitin ligases, termed the NEDDylator. This fusion protein permits robust, stable and simple-patterned NEDDylation of IAP substrate proteins that are readily purified and identified by mass spectrometry. These studies revealed >50 IAP substrate candidates. Almost all of these have classic IBM motifs of both cytosolic and mitochondrial origin. One of the strongest hits was PGAM5, a protein phosphatase located in the outer mitochondrial membrane. We show PGAM5 is cleaved by a non-caspase protease, and rapidly released from the mitochondria during apoptosis. The released form sensitizes cells to death, and cleavage uniquely generates a neo-IBM motif that can be bound to IAPs to mediate its ubiquitination. These studies both reveal important mutually antagonistic IAP substrates and a powerful approach for hunting substrates for E3 ligases or other protein-protein partners.