Two distinct signalling cascades target the NF-kappaB regulatory factor c-IAP1 for degradation

Abstract

c-IAP1 (cellular inhibitor of apoptosis 1) has recently emerged as a negative regulator of the non-canonical NF-kappaB (nuclear factor kappaB) signalling cascade. Whereas synthetic IAP inhibitors have been shown to trigger the autoubiquitination and degradation of c-IAP1, less is known about the physiological mechanisms by which c-IAP1 stability is regulated. In the present paper, we describe two distinct cellular processes that lead to the targeted loss of c-IAP1. Recruitment of a TRAF2 (tumour necrosis factor receptor-associated factor 2)-c-IAP1 complex to the cytoplasmic domain of the Hodgkin's/anaplastic large-cell lymphoma-associated receptor, CD30, leads to the targeting and degradation of the TRAF2-c-IAP1 heterodimer through a mechanism requiring the RING (really interesting new gene) domain of TRAF2, but not c-IAP1. In contrast, the induced autoubiquitination of c-IAP1 by IAP antagonists causes the selective loss of c-IAP1, but not TRAF2, thereby releasing TRAF2. Thus c-IAP1 can be targeted for degradation by two distinct processes, revealing the critical importance of this molecule as a regulator of numerous intracellular signalling cascades.

Figure 1. CD30 activation induces the degradation of both c-IAP1 and TRAF2

(A) Karpas 299 cells were layered on to CHO cells, or CD30L⁺ CHO cells, for the indicated periods of time. Cells were recovered, lysed in RIPA buffer and immunoblotted for p100/p52, p105/p50, c-IAP1 and TRAF2 as indicated. (B) HEK-293 cells were transfected with the indicated combinations of c-IAP1, wild-type TRAF2 and DN-TRAF2, together with a control plasmid or a constitutively active form of CD30. At 24 h after transfection, cell lysates were prepared, and immunoblot analysis was performed for HA–c-IAP1 and TRAF2. (C) HEK-293 cells were transfected with plasmids encoding TRAF2, HA–c-IAP1 or HA–c-IAP1 H588A, together with a control plasmid or a constitutively active form of CD30, as indicated. Cells were harvested and lysates were analysed by immunoblot as described in (B). Equivalent protein loading was verified by immunoblotting for β-actin.

Figure 2. Cytosolic Smac triggers the autoubiquitination and degradation of c-IAP1, but not TRAF2

(A) HEK-293 cells were transiently transfected with HA-tagged c-IAP1 or the c-IAP1 H588A mutant expression plasmid along with plasmids encoding TRAF2 and mature Smac. At 24 h after transfection, cell lysates were prepared, and immunoblot analysis was performed for HA–c-IAP1/H588A mutant, Smac and TRAF2. (B) HEK-293 cells were transfected with combinations of Smac–FLAG, HA–c-IAP1, TRAF2 and His₆–ubiquitin (His-Ub). At 24 h after transfection, cell lysates were prepared in 8 M urea buffer and precipitated using Ni-NTA–agarose beads. The presence of HA–c-IAP1, TRAF2 and Smac–FLAG in the precipitate was determined by immunoblotting. Equivalent protein loading was verified in (A) and (B) by immunoblotting for β-actin.

Figure 3. c-IAP1 is an inhibitor of the non-canonical NF-κB pathway, and Smac neutralizes c-IAP1-mediated NF-κB inhibition

HEK-293 cells were transfected with mature Smac–FLAG, HA–c-IAP1 and TRAF2. At 24 h after transfection, cell lysates were prepared, and immunoblot analysis was performed for p100/p52, HA–c-IAP1, TRAF2 and Smac–FLAG. Equivalent protein loading was confirmed by immunoblotting for β-actin.

Figure 4. The IAP antagonist AEG40730 induces the degradation of c-IAP1, but not TRAF2

(A) HEK-293 cells were transfected with HA–c-IAP1 and TRAF2. At 24 h after transfection, cells were treated with 0, 1, 5, 10 or 25 nM AEG40730 or DMSO. At 24 h after treatment with AEG40730, cell lysates were prepared, and immunoblot analysis was performed for HA–c-IAP1 and TRAF2. Equivalent protein loading was confirmed by immunoblotting for β-actin. (B) HEK-293 cells were transfected with HA–c-IAP1 H588A. At 24 h after transfection, cells were treated with 0, 1, 5, 10 or 25 nM AEG40730 or DMSO. At 24 h after treatment with AEG40730, cell lysates were prepared and immunoblotted for HA–c-IAP1 H588A. As a control, c-IAP1 H588A was transfected with mature Smac–FLAG. Equivalent protein loading was verified by immunoblotting for β-actin.

Figure 5. Rapid degradation of c-IAP1, but not TRAF2, precedes activation of the non-canonical NF-κB pathway

The IAP antagonist AEG40730 triggers the processing of p100 to its p52 form. (A) HEK-293 cells or (B) Karpas 299 cells were treated with DMSO or 0, 1, 5, 10 or 25 nM AEG40730 and incubated for 24 h. (C) Karpas 299 cells were treated for 0–48 h with 25 nM AEG40730 or DMSO. Following treatment with AEG40730, cell lysates were prepared, and immunoblot analysis was performed for c-IAP1, TRAF2 and p52/p100. Equivalent protein loading was confirmed by immunoblotting for β-actin. n.s., non-specific.

Figure 6. Smac is a modulator of CD30-mediated signalling and activates transcription of endogenous NF-κB-dependent genes

(A) Karpas 299 cells were electroporated with an expression plasmid encoding mature Smac–FLAG, or an empty control plasmid, as indicated. At 24 h after transfection, cells were layered on to CD30L⁺ CHO cells or control CHO cells for 2 h. Total RNA was extracted and reverse-transcribed, and cDNA was analysed by qRT-PCR using Taqman probes for the indicated NF-κB-dependent genes. (B) Karpas 299 cells were treated with 25 nM AEG40730 or DMSO. At 24 h after following treatment, cells were layered on to CD30L⁺ CHO cells or control CHO cells for 2 h. Total RNA was extracted and reverse-transcribed, and cDNA was analysed by qRT-PCR using Taqman probes for the indicated NF-κB-dependent genes. (C) Karpas 299 cells were electroporated with double-stranded siRNA oligonucleotides targeting Smac (siSmac) or an irrelevant sequence (siGFP), as indicated. At 48 h after transfection, cells were layered on to CD30L⁺ CHO cells or control CHO cells, as indicated, for 2 h. Total RNA was extracted and reverse-transcribed, and cDNA was analysed by qRT-PCR using Taqman probes for the indicated NF-κB-responsive genes. ICAM, intercellular adhesion molecule; IκBα, inhibitory κB α.

Figure 7. Model of the two mechanisms by which the NF-κB regulatory factor c-IAP1 can be regulated

Smac and IAP antagonists induce the selective autoubiquitination and degradation of c-IAP1. In the presence of mature Smac, c-IAP1, but not TRAF2, is degraded. Smac promotes processing of p100 to p52 and augments non-canonical NF-κB signalling. CD30 activation promotes the degradation of c-IAP1 and TRAF2. Degradation of c-IAP1 and TRAF2 does not require the E3 ubiquitin ligase activity of c-IAP1, but does requires the RING domain in TRAF2. CD30 signalling results in the induction of NF-κB through processing of p100 to the active p52 subunit.