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. 2008 Dec;9(12):1371-8.
doi: 10.1038/ni.1676. Epub 2008 Nov 9.

Noncanonical NF-kappaB activation requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2 and TRAF3 and the kinase NIK

Brian J Zarnegar  1 , Yaya Wang, Douglas J Mahoney, Paul W Dempsey, Herman H Cheung, Jeannie He, Travis Shiba, Xiaolu Yang, Wen-Chen Yeh, Tak W Mak, Robert G Korneluk, Genhong Cheng
Affiliations

Noncanonical NF-kappaB activation requires coordinated assembly of a regulatory complex of the adaptors cIAP1, cIAP2, TRAF2 and TRAF3 and the kinase NIK

Brian J Zarnegar et al. Nat Immunol. 2008 Dec.

Abstract

Recent studies suggest that nuclear factor kappaB-inducing kinase (NIK) is suppressed through constitutive proteasome-mediated degradation regulated by TRAF2, TRAF3 and cIAP1 or cIAP2. Here we demonstrated that the degradation of NIK occurs upon assembly of a regulatory complex through TRAF3 recruitment of NIK and TRAF2 recruitment of cIAP1 and cIAP2. In contrast to TRAF2 and TRAF3, cIAP1 and cIAP2 seem to play redundant roles in the degradation of NIK, as inhibition of both cIAPs was required for noncanonical NF-kappaB activation and increased survival and proliferation of primary B lymphocytes. Furthermore, the lethality of TRAF3 deficiency in mice could be rescued by a single NIK gene, highlighting the importance of tightly regulated NIK.

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Figures

Figure 1
Figure 1. Non-redundancy of TRAF2 and TRAF3 and redundancy of cIAP1 and cIAP2 in suppression of the noncanonical NF-κB pathway
(a) Immunoblot of lysates of wild-type (WT) and Traf3−/− (left panel), WT and Traf2−/− (right panel) MEFs reconstituted with empty pBABE-Flag vector (EV), pBABE-Flag-TRAF2 (T2) and pBABE-Flag-TRAF3 (T3). Noncanonical NF-κB activation was assessed by immunoblot analysis of NIK and p100 to p52 processing. (b) Immunoblot of WT, Birc2−/− and Birc3−/− MEFs transiently transfected with non-targeting (NT) siRNA or cIAP1-specific and/or cIAP2-specific siRNA as indicated. Activation of the noncanonical NF-κB pathway was then assessed by analysis of p100 to p52 processing 24 hr post-transfection. (c) Immunoblot of WT and Birc2−/− primary hepatocytes transiently transfected with NT, cIAP1-specific and/or cIAP2-specific siRNA as indicated. Activation of the noncanonical NF-κB pathway was then assessed by analysis of p100 to p52 processing 24 hr post-transfection. Results are representative of at least three independent experiments.
Figure 2
Figure 2. Assembly of the NIK regulatory complex by TRAF2 and TRAF3
(a) Immunoassays on lysates of primary murine B cells treated with the proteasomal inhibitor MG132 for 4 hr and then incubated with rabbit-IgG or anti-NIK. Protein complexes were then immunoprecipitated with Protein G beads followed by the indicated immunoblot analysis. (b) Immunoassays on lysates of Map3k14−/−, Traf3−/−, and Traf2−/− MEFs reconstituted with empty pBABE-Flag vector (EV) or pBABE-Flag-NIK (NIK) and complexes were immunoprecipitated with anti-Flag M2 beads followed by the indicated immunoblot. (c,d) Immunoassays on lysates from Map3k14−/− and Traf3−/− MEFs reconstituted with EV or NIK incubated with anti-Flag M2 beads and complexes were immunoprecipitated followed by the indicated immunoblot analysis. (e) Immunoassays on lysates from MG132 treated, BCR-Abl transformed WT and Traf3−/− B-cell lines incubated with control rabbit-IgG or anti-NIK followed by Protein G beads and complexes were immunoprecipitated followed by the indicated immunoblot analysis. (f) Immunoassays on lysates from WT and Traf2−/− 3T3s reconstituted with pBABE-Flag-cIAP1, -cIAP2 or empty vector (EV) control. Cell lysates were then immunoprecipitated with anti-Flag M2 beads and co-immunoprecipitation of TRAF2 and TRAF3 was detected by immunoblot. Data are representative of at least three experiments with similar results.
Figure 3
Figure 3. Differential affinities of TRAF2 and TRAF3 for cIAP1/2 and NIK
(a) Immunoblot of lysates of HEK 293T cells co-expressing Flag-NIK and titrations of Myc-TRAF2 and TRAF3. Lysates were incubated with anti-Flag M2 beads and NIK-TRAF complexes were detected by immunoblot analysis against Myc. (b) Immunoblot of lysates of HEK 293T cells co-expressing Myc-tagged cIAP1 and cIAP2 and Flag-TRAF2 and Flag-TRAF3. Lysates were incubated with anti-Flag M2 beads and cIAP-TRAF complexes were detected by immunoblot analysis against Myc. (c) Immunoassays on lysates from HEK 293T cell co-expressing NIK and Myc-cIAP1 or Myc-cIAP2 in the presence or absence of TRAF2, TRAF3 or both TRAF2 and TRAF3. NIK was then immunoprecipitated by NIK-specific antibody and NIK-cIAPs complexes were detected by immunoblot against Myc. All experiments were done at least three times with similar results.
Figure 4
Figure 4. Generation of a TRAF2-TRAF3 chimera
(a,b) Traf3−/− (a) and Traf2−/− (b) MEFs were reconstituted with pBABE-Flag-TRAF2 (T2), pBABE-Flag-TRAF3 (T3), pBABE-Flag-3TD, and pBABE-Flag-2TD. Rescue of constitutive noncanonical NF-κB activation was assessed by immunoblot analysis of p100 to p52 processing. Data are representative of at least three independent experiments.
Figure 5
Figure 5. The TRAF2-TRAF3 Chimera, 3TD
(a) Flag-NIK was co-expressed with Flag-tagged TRAF2, 3TD, TRAF3, and 2TD in HEK 293T cells. Lysates were incubated with NIK-specific antibody and NIK-TRAF complexes were detected by immunoblot analysis against Flag. (b) Myc-tagged cIAP1 and cIAP2 were co-expressed with Flag-tagged TRAF2, TRAF3, 3TD, and 2TD. Lysates were incubated with anti-Flag M2 beads and cIAP-TRAF complexes were detected by immunoblot analysis against Myc. (c,d) Myc-cIAP1 (c) or Myc-cIAP2 (d) together with Flag-NIK were co-expressed alone or in the presence of Flag-tagged TRAF2, 3TD, TRAF3, or 2TD in HEK 293T cells. Lysates were incubated with anti-NIK antibody and NIK-cIAPs complexes were detected by immunoblot analysis against Myc. All data are representative of at least three independent experiments.
Figure 6
Figure 6. Receptor-induced stabilization of NIK after TRAF2 and TRAF3 degradation
(a) A20 cells were treated with 5 µg/ml of agonistic anti-CD40 antibody (FgK-45) in media alone (left panel), and in the presence of the cIAP inhibitor, SM (100nM, middle panel) or the proteasome inhibitor MG132 (10 µM, right panel), for the indicated times. SDS Lysates were then subjected to immunoblot analysis of the indicated proteins. (b) Primary murine B-cells were treated with 5 µg/ml of FgK in media alone and in the presence SM (100nM). SDS Lysates were then subjected to immunoblot analysis of the indicated proteins. (c) A20 cells were treated with MG132 or FgK for 4 hr as indicated. Cell Lysates were then incubated with either rabbit-IgG or anti-NIK antibody followed by immunoprecipitation with anti-rabbit-IgG beads. Immunoprecipitates were then subjected to immunoblot detection of the indicated proteins. Data are representative of at least three independent experiments.
Figure 7
Figure 7. SMAC mimetic compounds mimic co-stimulatory properties of BAFF in B lymphocyte activation
(a) WT and Traf3−/− MEFs were cultured in media alone or a 5-fold dilution series (high dose 500 nM) of SM for 12 hr. Activation of the noncanonical NF-κB pathway was then assessed by immunoblot analysis of p100 to p52 processing. (b) WT and Map3k14−/− 3T3s were cultured in media alone, in the presence of 2 µg/ml agonistic αLTβR antibody, or 50 nM SM for 12 hr. Activation of the noncanonical NF-κB pathway was then assessed by immunoblot analysis of p100 to p52 processing. (c) Primary B-lymphocytes were cultured in media alone, in the presence of a 5-fold dilution series (high dose 500 nM) of SM, 100 ng/ml of BAFF, or 5 µg/ml of anti-CD40 for 12 hr. Activation of the noncanonical NF-κB pathway was then assessed by immunoblot analysis of p100 to p52 processing. (d) Primary B-lymphocytes were cultured in media alone or in the presence of 3 fold titrations of BAFF, SM, or LBW242 (high dose 300 ng/ml, 300 nM, 300 nM respectively) for 72 hr. Cell viability was then determined by propidium iodide exclusion assay and analyzed by FACS. Stimulations were performed in triplicate. (e) Primary B-lymphocytes were cultured in the presence of the indicated titrations of CpG alone, or in the presence of the indicated concentrations of BAFF or LBW242 for 72 hr. Cellular Proliferation was then assessed by measurement of incorporated [H3]-Thymidine. Stimulations were performed in triplicate with bars indicating ± s.e.m. *, P < 0.05, **, P < 0.01. Data are representative of at least three independent experiments.
Figure 8
Figure 8. Rescue of the TRAF3-null phenotype by deletion of a single allele of NIK
(a) Survival curve of mice of the indicated genotypes (n = 5 per strain). (b) Body Mass of mice of the indicated genotypes (n = 3 per strain), ***, P < 0.001. (c,d) Survival curve (n = 6 per strain) (c) and serum corticosterone levels (n = 3 per strain) (d) of mice of the indicated genotypes.
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References

    1. Baeuerle PA, Baltimore D. NF-κB: ten years after. Cell. 1996;87:13–20. - PubMed
    1. Iotsova V, et al. Osteopetrosis in mice lacking NF-κB1 and NF-κB2. Nat Med. 1997;3:1285–1289. - PubMed
    1. Kopp EB, Ghosh S. NF-κB and rel proteins in innate immunity. Adv Immunol. 1995;58:1–27. - PubMed
    1. Bonizzi G, Karin M. The two NF-κB activation pathways and their role in innate and adaptive immunity. Trends Immunol. 2004;25:280–288. - PubMed
    1. Zandi E, Karin M. Bridging the gap: composition, regulation, and physiological function of the IκB kinase complex. Mol Cell Biol. 1999;19:4547–4551. - PMC - PubMed

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