The RING domain of cIAP1 mediates the degradation of RING-bearing inhibitor of apoptosis proteins by distinct pathways

Abstract

The Inhibitor of Apoptosis proteins (IAPs) are key repressors of apoptosis. Several IAP proteins contain a RING domain that functions as an E3 ubiquitin ligase involved in the ubiquitin-proteasome pathway. Here we investigated the interplay of ubiquitin-proteasome pathway and RING-mediated IAP turnover. We found that the CARD-RING domain of cIAP1 (cIAP1-CR) is capable of down-regulating protein levels of RING-bearing IAPs such as cIAP1, cIAP2, XIAP, and Livin, while sparing NAIP and Survivin, which do not possess a RING domain. To determine whether polyubiquitination was required, we tested the ability of cIAP1-CR to degrade IAPs under conditions that impair ubiquitination modifications. Remarkably, although the ablation of E1 ubiquitin-activating enzyme prevented cIAP1-CR-mediated down-regulation of cIAP1 and cIAP2, there was no impact on degradation of XIAP and Livin. XIAP mutants that were not ubiquitinated in vivo were readily down-regulated by cIAP1-CR. Moreover, XIAP degradation in response to cisplatin and doxorubicin was largely prevented in cIAP1-silenced cells, despite cIAP2 up-regulation. The knockdown of cIAP1 and cIAP2 partially blunted Fas ligand-mediated down-regulation of XIAP and protected cells from cell death. Together, these results show that the E3 ligase RING domain of cIAP1 targets RING-bearing IAPs for proteasomal degradation by ubiquitin-dependent and -independent pathways.

Figure 1.

cIAP1-CARD-RING mediates proteasomal-dependent down-regulation of RING-bearing IAPs. (A) HeLa cells were transfected with 6myc fusion proteins of different IAPs and with cIAP1-CR or cIAP1-CR-H588A. After 24 h of transfection, proteins were extracted, separated by SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblotting with anti-myc for the detection of 6myc-IAP, anti-FLAG for the detection of cIAP1-CR, and anti-GAPDH. (B–E) HeLa cells were transfected with plasmids encoding 6myc-cIAP1 (B), cIAP2 (C), XIAP (D), or Livin (E) along with cIAP1-CR or cIAP1-CR-H588A. After 19 h of transfection, cells were treated with 2 μM proteasomal inhibitor MG132 or vehicle for an additional 5 h. The inclusion of MG132 protected IAPs from cIAP1-CR–mediated degradation.

Figure 2.

cIAP1-mediated down-regulation of IAPs is independent of the E3 ligase function of the target. TREx-HeLa cells were transfected with plasmids encoding 6myc-cIAP1 or cIAP1-H588A (A), cIAP2 or cIAP2-H574A (B), XIAP or XIAP-H467A (C), or Livin or Livin-H269A and cIAP1-CR (D). Tetracycline was added to induce the expression of cIAP1-CR for the final 2, 4, 8, and 24 h of a total transfection time of 48 h. Cells were collected at the end of the transfection period and subjected to Western immunoblot analysis.

Figure 3.

cIAP1-CARD-RING mediated degradation of XIAP and Livin, but not cIAP1 and cIAP2, occurs independently of E1. (A) HeLa cells were transfected with nontargeting luciferase siRNA (NT) or ubiquitin-activating enzyme-specific siRNA (E1) for the indicated times. Protein extracts were subjected to Western immunoblot analysis with antibodies against ubiquitin, E1, and GAPDH. (B–E) After 80 h of siRNA transfection, HeLa cells were transfected with 6myc-cIAP1 (B), cIAP2 (C), XIAP (D), or Livin (E) in the presence or absence of LacZ, cIAP1-CR, cIAP1-CR-H588A, and Xenopus E1 for an additional 24 h. Protein extracts were collected and subjected to Western immunoblot analysis. 6myc-proteins, cIAP1-CR, and Xenopus E1 were detected with anti-myc, anti-FLAG, and anti-V5 antibodies, respectively.

Figure 4.

Mutation of XIAP ubiquitination sites does not affect cIAP1-CR–mediated degradation. (A) HeLa cells were transfected with the various 6myc fusion proteins of XIAP as well as with pCMV-Ub and pCMV-Ub-HA. Extracts were immunoprecipitated with an anti-myc antibody, followed by immunodetection with anti-HA for HA-ubiquitin or anti-XIAP for determining the relative pulldown of XIAP. (B) Relative expression of 6myc-XIAP in inputs was immunodetected with an anti-myc antibody. (C) HeLa cells were transfected with 6myc fusion proteins of XIAP wild type or mutants and with cIAP1-CR or cIAP1-CR-H588A. After 24 h of transfection, proteins were extracted and subjected to immunoblotting with anti-myc for the detection of 6myc-XIAP, anti-FLAG for the detection of cIAP1-CR and anti-GAPDH.

Figure 5.

cIAP1-CR mediates XIAP down-regulation in the presence of dominant negative ubiquitin. (A) HeLa cells were transfected with LacZ, cIAP1-CR, or cIAP1-CR-H588A in the presence of WT-Ub, Ub-K48R, or Ub-4K7R. Cells were collected 24 h after transfection and subjected to Western immunoblot analysis. (B and C) TREx-HeLa cells were transfected with 6myc-XIAP (B) or 6myc-cIAP1, cIAP1-CR (C) and in the presence of WT-Ub, Ub-K48R, Ub-3KR, or Ub-4K7R. Tetracycline was added to induce the expression of cIAP1-CR for the final 4, 8, 18, and 24 h of a total transfection time of 48 h. Cells were collected at the end of the transfection period and subjected to Western immunoblot analysis.

Figure 6.

Degradation of XIAP during apoptosis is cIAP1-dependent. (A) HeLa cells were treated simultaneously with cisplatin and ALLN, each at 20 μM, for 24 h. (B) After 16 h of 10 μM doxorubicin exposure, HeLa cells were treated with 10 μM lactacystin for an additional 8 h. (C–F) HeLa cells were transfected with the nontargeting luciferase (NT) and cIAP1-specific and cIAP2-specific (duplexes “2” and “9”) siRNA for 48 h, and then transfected cells were exposed to 20 μM cisplatin (C and D) or 10 μM doxorubicin (E and F) for 24 h. (G) HeLa cells were transfected with the nontargeting luciferase (NT), E1-specific or dually with E1- and cIAP1-specific siRNA for 80 h, and then transfected cells were exposed to 20 μM cisplatin for 24 h. Proteins were extracted and subjected to Western immunoblot analysis. cIAP1 and cIAP2 were recognized by an anti-RIAP1 antibody that cross-reacts with both proteins.

Figure 7.

Loss of cIAP1 protects against Fas-mediated cell death when mitochondrial pathway is blocked by BCL2. (A) SF295 cells were transfected with the nontargeting luciferase (NT) or both cIAP1-specific and cIAP2-specific (duplexes 9) siRNA. After 24 h of siRNA transfection, SF295 cells were transduced with an adenovirus that encodes for BCL2. After 24 h of induction, SF295 cells were treated with 0.1 μg/ml fas for an additional 18 h. Proteins were extracted and subjected to Western immunoblot analysis. (B) SF295 cells treated as in A were subjected to WST-1 viability assay. Bars represent the average ± SEM of four experiments. *p < 0.05; significantly different from vehicle-NT and fas-cIAP1/cIAP2, Student's t test.