c-IAP1 and c-IAP2 are critical mediators of tumor necrosis factor alpha (TNFalpha)-induced NF-kappaB activation

Abstract

The inhibitor of apoptosis (IAP) proteins are a family of anti-apoptotic regulators found in viruses and metazoans. c-IAP1 and c-IAP2 are recruited to tumor necrosis factor receptor 1 (TNFR1)-associated complexes where they can regulate receptor-mediated signaling. Both c-IAP1 and c-IAP2 have been implicated in TNFalpha-stimulated NF-kappaB activation. However, individual c-IAP1 and c-IAP2 gene knock-outs in mice did not reveal changes in TNF signaling pathways, and the phenotype of a combined deficiency of c-IAPs has yet to be reported. Here we investigate the role of c-IAP1 and c-IAP2 in TNFalpha-stimulated activation of NF-kappaB. We demonstrate that TNFalpha-induced NF-kappaB activation is severely diminished in the absence of both c-IAP proteins. In addition, combined absence of c-IAP1 and c-IAP2 rendered cells sensitive to TNFalpha-induced cell death. Using cells with genetic ablation of c-IAP1 or cells where the c-IAP proteins were eliminated using IAP antagonists, we show that TNFalpha-induced RIP1 ubiquitination is abrogated in the absence of c-IAPs. Furthermore, we reconstitute the ubiquitination process with purified components in vitro and demonstrate that c-IAP1, in collaboration with the ubiquitin conjugating enzyme (E2) enzyme UbcH5a, mediates polymerization of Lys-63-linked chains on RIP1. Therefore, c-IAP1 and c-IAP2 are required for TNFalpha-stimulated RIP1 ubiquitination and NF-kappaB activation.

FIGURE 1.

TNFα-stimulated NF-κB activation depends on c-IAP1 and c-IAP2. A, analysis of IκB protein levels in WT and in c-IAP1-, c-IAP2-, and XIAP-deficient MEFs. Cells were treated for the indicated periods of time with 20 ng/ml recombinant murine TNFα. The protein levels of IκB were analyzed by Western blotting. KO, knockout. B, c-IAP2-null MEFs were transfected with scrambled (Contr.) or c-IAP1-specific siRNA duplexes. 48 h later, cells were treated with recombinant murine TNFα (20 ng/ml), and protein levels were analyzed as in A. C, HT1080 cells were pretreated with vehicle (DMSO) or BV6 (5 μm) for 5 h followed by treatment with human recombinant TNFα (20 ng/ml) for the indicated time periods. The protein levels of IκB and c-IAP1 were analyzed by Western blotting. D, down-regulation of c-IAP1 and c-IAP2 expression reduces viability of TNFα-treated cells. Cells were transfected with scrambled, c-IAP1-specific, or c-IAP2-specific siRNA duplexes. 48 h later, cells were treated with TNFα as indicated. Cell viability was determined as described under “Experimental Procedures,” and protein levels were determined as described as in A. The error bars represent standard deviation from three independent experiments. The inset shows down-regulation of c-IAP2 in c-IAP1-deficient MEFs; down-regulation of c-IAP1 in c-IAP2-deficient MEFs is presented in panel B. Western blotting was performed with indicated antibodies.

FIGURE 2.

c-IAPs are critical for TNFα-induced RIP1 polyubiquitination. A, HT1080 cells were pretreated with DMSO or BV6 (5 μm) for 5 h followed by TNFα treatment as indicated. Cell lysates were immunoprecipitated (IP) with anti-TNFR1 antibodies, and protein levels in cellular lysates and in the TNFR1-associated complex were determined by Western blotting with the indicated antibodies. Ub, ubiquitin. B, WT or c-IAP1-null MEF cells were treated for the indicated periods of time with FLAG-tagged TNFα (1 mg/ml) followed by immunoprecipitation and Western blot analysis with the indicated antibodies. One-half of the immunoprecipitated protein complexes described above were dissociated by a 20-min incubation in 6 m urea. Collected supernatants were diluted 25-fold in the lysate buffer followed by immunoprecipitation and Western blot analysis with the indicated antibodies. KO, knock-out. C, RIP1 was immunoprecipitated from c-IAP1-null MEFs and incubated for 45 min in ubiquitination reactions with the indicated combination of recombinant c-IAP1, E2 enzymes, and ubiquitin proteins (WT, Lys-48 only, or Lys-63 only). RIP1 modifications were determined with anti-RIP1 antibodies. D, recombinant RIP1 was incubated for 45 min in ubiquitination reaction in the absence or presence of recombinant c-IAP1 or c-IAP1 RING mutant (Rm; H588A) and the indicated combination of E2 enzymes and ubiquitin proteins (WT, Lys-48 only, or Lys-63 only). RIP1 modifications were determined with anti-RIP1 antibodies.