Ubiquitin Protein Ligase Activity of IAPs and Their Degradation in Proteasomes in Response to Apoptotic Stimuli

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Science  05 May 2000:
Vol. 288, Issue 5467, pp. 874-877
DOI: 10.1126/science.288.5467.874


To determine why proteasome inhibitors prevent thymocyte death, we examined whether proteasomes degrade anti-apoptotic molecules in cells induced to undergo apoptosis. The c-IAP1 and XIAP inhibitors of apoptosis were selectively lost in glucocorticoid- or etoposide-treated thymocytes in a proteasome-dependent manner before death. IAPs catalyzed their own ubiquitination in vitro, an activity requiring the RING domain. Overexpressed wild-type c-IAP1, but not a RING domain mutant, was spontaneously ubiquitinated and degraded, and stably expressed XIAP lacking the RING domain was relatively resistant to apoptosis-induced degradation and, correspondingly, more effective at preventing apoptosis than wild-type XIAP. Autoubiquitination and degradation of IAPs may be a key event in the apoptotic program.

Thymocytes undergo apoptosis in response to many stimuli, including glucocorticoids, etoposide, γ-radiation, and engagement of their receptors for antigen (1). Reagents that inhibit proteasomes, multicatalytic protease complexes responsible for the degradation of ubiquitinated cellular proteins (2), block cell death induced by many of these stimuli (3). One possible explanation is that proteasome-mediated degradation of anti-apoptotic proteins might be required for cell death to occur. Candidate anti-apoptotic molecules expressed in immature thymocytes include the Bcl-2 family member Bcl-xL and members of the IAP (inhibitors of apoptosis) family (4). To explore this idea, we induced thymocytes to die by treating them with dexamethasone (Dex). Under these conditions, amounts of both c-IAP1 (inhibitor of apoptosis–1) and XIAP (X-linked inhibitor of apoptosis) were substantially decreased (Fig. 1A) (5). No change occurred in amounts of Bcl-xL or β-actin. An increase in Dex-induced cell death was not observed until 8 hours of culture, and even at this time ∼90% of thymocytes were viable (Fig. 1B). Inhibition of proteasome activity greatly reduced apoptosis at 16 hours (Fig. 1B) (3). Amounts of c-IAP1 and XIAP began to decrease 4 to 6 hours after culture with Dex, and by 8 hours were decreased by 64 ± 1.7% for cIAP-1 (n = 3) and 73 ± 3.4% for XIAP (n = 5) (Fig. 1D) (6). Etoposide is a topoisomerase II inhibitor that, unlike glucocorticoids, induces apoptosis through a p53-dependent pathway (7). Etoposide induced a decrease in IAP levels that was first observed after 2 to 4 hours of culture, and by 6 hours the amounts of c-IAP1 and XIAP fell ∼87 ± 7.5% (n = 3) and 77 ± 8.5% (n = 5), respectively (Fig. 1E) (6). Cell death was first detected by trypan blue exclusion ∼8 hours after addition of etoposide and was largely prevented in cells treated with proteasome inhibitors (Fig. 1C). Therefore, induction of apoptosis by two different afferent pathways resulted in relatively early and specific loss of IAPs in thymocytes.

Figure 1

Glucocorticoids and etoposide induce the loss of XIAP and c-IAP1 but not Bcl-xL in thymocytes. (A) Immunoblot of c-IAP1, XIAP, Bcl-xL, and β-actin after treatment of thymocytes with 1 μM Dex for 8 hours. (B) Kinetics of induction of cell death by Dex. Thymocytes were cultured in the absence (□) or presence (○) of 1 μM Dex and cell viability was determined at the indicated times. The error bars represent the SEM of three independent experiments, each done in duplicate wells. (Inset) Percent specific apoptosis (±SEM) at 16 hours in the absence or presence of the proteasome inhibitorsN-acetyl-Leu-Leu-Leu-Nle-CHO (LLnL) (50 μM) or lactacystin (50 μM) (Calbiochem, San Diego, California). Specific apoptosis is defined as the amount of apoptosis in excess of the spontaneous apoptosis of control cultures (% experimental apoptosis − % apoptosis in medium)/(100 − % apoptosis in medium). Spontaneous apoptosis ranged from 4 to 12%. (C) Kinetics of induction of cell death by etoposide. Thymocytes were cultured in the absence (□) or presence (○) of etoposide (10 μg/ml), and cell viability was determined at the indicated times. (Inset) Percent specific apoptosis (±SEM) at 16 hours in the absence or presence of the indicated proteasome inhibitors. (D) Immunoblot analysis of the kinetics of XIAP and c-IAP1 decreases after treatment with 1 μM Dex. (E) Immunoblot analysis of the kinetics of XIAP and c-IAP1 decreases after treatment with etoposide (10 μg/ml). Immunoblots were incubated with 125I-labeled protein A and visualized with a Storm PhosphorImager.

The relatively rapid decrease in the amount of IAPs in thymocytes stimulated to undergo apoptosis raised the possibility that these proteins were being targeted for degradation. Given that the proteasome is a major effector of intracellular protein degradation and that proteasome inhibitors block glucocorticoid- and etoposide-induced thymocyte apoptosis (3) (Fig. 1), thymocytes were cultured with Dex or etoposide in the absence or presence of proteasome inhibitors (Fig. 2). Both a peptide aldehyde proteasome inhibitor and the highly specific proteasome inhibitor lactacystin effectively blocked the decrease in IAP expression induced by the apoptotic stimuli.

Figure 2

Dex- and etoposide-induced decreases of XIAP and c-IAP1 can be prevented by proteasome inhibitors. (A) Thymocytes were cultured with 10−6 M Dex in the presence or absence of 50 μM LLnL or lactacystin proteasome inhibitors for 8 hours, and the levels of XIAP, c-IAP1, and β-actin were determined by immunoblot. (B) Thymocytes were treated with etoposide (10 μg/ml) for 6 hours in the presence or absence of 50 μM LLnL or lactacystin. The levels of XIAP, c-IAP1, and β-actin were determined by immunoblotting as in Fig. 1.

Protein ubiquitination involves the sequential action of ubiquitin activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin protein ligase (E3) (8). Ubiquitination by some known E3's and several proteins of previously unknown function is dependent on an intact RING finger (9, 10). IAPs contain a COOH-terminal RING domain, so we examined whether these molecules also have ubiquitin protein ligase activity. We used an in vitro ubiquitination assay that takes advantage of the fact that prokaryotic cells do not express the enzymes required for protein ubiquitination (9, 11). Glutathione S-transferase (GST) fusion proteins with full-length XIAP (GST-XIAP), c-IAP1 (GST-cIAP1), or GST alone were added to bacterial lysates containing recombinant E1 and E2 and 32P-labeled recombinant ubiquitin. After 90 min the reactions were stopped and proteins from the lysates resolved by SDS–polyacrylamide gel electrophoresis (PAGE) (Fig. 3A). In the presence of GST alone, little protein ubiquitination was detectable. In contrast, GST-XIAP and GST-cIAP1 caused a large increase in the number of ubiquitinated species. The pattern of ubiquitination for each IAP was distinct, with XIAP reproducibly yielding prominent (>180 kD) species of large molecular size and c-IAP1 generating species predominantly in the 80- to 130-kD range. We also examined GST fusions that contained a truncated form of XIAP lacking the RING domain (GST-XIAP1-351) or XIAP or c-IAP1 in which the metal-coordinating residues His467 or His588were replaced with Ala. Unlike the wild-type fusion proteins, GST-XIAP1-351, GST-XIAPH467A, and GST-cIAP1H588A did not increase the amount of ubiquitinated species in the ubiquitin protein ligase assay (Fig. 3, B and C). XIAP and c-IAP1 also mediated their own ubiquitination in vitro; in vitro–translated and metabolically labeled c-IAP1 and XIAP were ubiquitinated in an E2-dependent manner, which was increased when they were incubated with their corresponding GST fusion proteins; this activity was ablated by mutation of the RING domains (6).

Figure 3

(A to C) c-IAP1 and XIAP GST fusion proteins have ubiquitin protein ligase activity. The ubiquitination reaction was carried out with 2.5 pmol of bacterially expressed GST or the indicated GST fusion proteins bound to glutathione–Sepharose 4B beads added to the reaction buffer. After incubation, reaction mixtures were separated on 7.5% SDS-PAGE, and the 32P-ubiquitin-labeled species were visualized with a Storm PhosphorImager.

To determine if IAPs can mediate their own ubiquitination and degradation in vivo, Myc-tagged wild-type or RING-mutated c-IAP1 were transfected into 293 cells, immunoprecipitated with antibodies to Myc (anti-Myc), and blotted with anti-Myc or anti-ubiquitin. Both IAPs were expressed (Fig. 4A), and the RING mutant was reproducibly expressed in slightly higher amounts than the wild-type protein (Fig. 4B). Wild-type c-IAP1 but not the ubiquitination-defective form was heavily ubiquitinated (Fig. 4A). This ubiquitination appears to be functionally significant, because preventing proteasome-mediated degradation with lactacystin increased the abundance of the wild-type c-IAP1 protein but caused little change in the amount of the RING mutant (Fig. 4B). Therefore, overexpressed c-IAP1 undergoes ubiquitination and degradation in cells, phenomena that do not occur (or occur to a far lesser extent) when the IAP does not have endogenous ubiquitin protein ligase activity.

Figure 4

The protein ubiquitin ligase activity of c-IAP1 regulates its ubiquitination and degradation in cells. The human embryonal kidney cell line 293 was transfected with the cDNA expression vector pcDNA3 alone, pcDNA3-myc-cIAP1, or cDNA3-myc-cIAP1H588A. (A) Lysate from transfected cells was immunoprecipitated with anti-Myc epitope (antibody 9E10). The antibody-coated beads were divided into two portions, and the immunoprecipitated material was immunoblotted with 9E10 or anti-ubiquitin, as indicated, and visualized with125I-labeled protein A as in Fig. 1. The transfected c-IAP1 is indicated above a minor nonspecific band. (B) Transfected 293 cells were treated with proteasome inhibitor lactacystin (50 μM) for 18 hours. Lysates were separated by SDS-PAGE and blotted with 9E10. Similar results were obtained in three independent experiments.

To investigate the functional relevance of the RING domain, 2B4.11 T hybridoma cells were stably transfected with full-length Flag-tagged XIAP or the RING-less XIAP1-351(12). Clones expressing equivalent amounts of the transfected gene products were chosen for further study. Treatment of untransfected 2B4.11 cells with Dex for 12 hours resulted in the loss of XIAP (Fig. 5A). As with thymocytes, amounts of β-actin did not change during this time. The amounts of transfected full-length XIAP (XFL.1 and XFL.2 cells) were also decreased after Dex treatment. In contrast, Dex caused only a small decrease in the amount of XIAP1-351 (XT cells). Expression of tagged full-length XIAP but not truncated XIAP appeared to increase the amount of endogenous XIAP in untreated cells, as well as the relative loss of this molecule in Dex-treated cells. The maintenance of expression of the various XIAPs was correlated with resistance to apoptosis (Fig. 5B).

Figure 5

Stability and efficacy of RING-less XIAP in protecting cells from glucocorticoid-induced apoptosis. 2B4.11 T hybridoma cells were stably transfected with full-length NH2-terminal Flag-tagged XIAP or XIAP1-351. (A) After treatment with 1 μM Dex for 10 hours, the indicated cells were analyzed for XIAP expression by immunoblotting with a polyclonal antibody to XIAP that recognizes both wild-type and truncated XIAP (AF822; R&D Systems, Minneapolis, MN). The blot was developed with horseradish peroxidase–labeled donkey anti-rabbit immunoglobulin G (Amersham Pharmacia Biotech, Piscataway, NJ) and SuperSignal chemiluminescent substrate (Pierce, Rockford, IL). The transfected full-length XIAP is slightly larger than the endogenous XIAP because of the Flag tag. The membrane was stripped, blotted with anti–β-actin, and developed with125I-labeled protein A. (B) Viability of the indicated cells was assessed by trypan blue exclusion 20 hours after treatment with 1 μM Dex. Specific cell death was calculated as inFig. 1. The error bars are the SEM of four independent experiments, each done in duplicate wells.

IAPs mediate their anti-apoptotic function, at least in part, by interactions between an ∼70–amino acid NH2-proximal domain containing three BIRs (baculovirus internal repeats) and particular caspases (4, 13). The role of the COOH-terminal RING domain in inhibition of apoptosis appears to vary depending on the IAP and the cell type, its mutation or absence having either a small negative effect or a positive effect on cell survival (14). Although our understanding of the function of RING domains is limited, this motif appears to play a role in protein ubiquitination, and a number of RING-containing proteins have ubiquitin protein ligase activity (9, 10). Our results demonstrate that IAPs themselves catalyze ubiquitination in a RING-dependent manner. Moreover, these anti-apoptotic proteins are capable of autoubiquitination, and when overexpressed or expressed in cells induced to die undergo RING-dependent proteasome-mediated degradation.

Because IAPs inhibit glucocorticoid- and etoposide-induced apoptosis (15) (as do proteasome inhibitors), their proteasome-mediated degradation may be an important regulatory step for cells that have been signaled to undergo apoptosis to actually progress to cell death. Furthermore, the finding that IAPs can catalyze their own ubiquitination provides the interesting possibility that their abundance is actively self-regulated. This is supported by the finding that RING-less XIAP is degraded to a lesser extent, and confers better protection from apoptosis, than wild-type protein. However, the RING-less XIAP was not completely resistant to Dex-induced and proteasome-mediated degradation, indicating that this protein can also serve as a target for other E3's, perhaps endogenous XIAP or other family members. The ability of IAPs to catalyze their own ubiquitination appears to be suppressed in resting thymocytes, because proteasome inhibitors had little effect on the amount of IAP in such cells. Whether IAPs can ubiquitinate other molecules and, if so, what might be their substrates, remains to be determined.

  • * To whom correspondence should be addressed. E-mail: jda{at}


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