Negative feedback in noncanonical NF-kappaB signaling modulates NIK stability through IKKalpha-mediated phosphorylation

Abstract

Canonical and noncanonical nuclear factor kappaB (NF-kappaB) signaling are the two basic pathways responsible for the release of NF-kappaB dimers from their inhibitors. Enhanced NF-kappaB signaling leads to inflammatory and proliferative diseases; thus, inhibitory pathways that limit its activity are critical. Whereas multiple negative feedback mechanisms control canonical NF-kappaB signaling, none has been identified for the noncanonical pathway. Here, we describe a mechanism of negative feedback control of noncanonical NF-kappaB signaling that attenuated the stabilization of NF-kappaB-inducing kinase (NIK), the central regulatory kinase of the noncanonical pathway, induced by B cell-activating factor receptor (BAFF-R) and lymphotoxin beta receptor (LTbetaR). Inhibitor of kappaB (IkappaB) kinase alpha (IKKalpha) was previously thought to lie downstream of NIK in the noncanonical NF-kappaB pathway; we showed that phosphorylation of NIK by IKKalpha destabilized NIK. In the absence of IKKalpha-mediated negative feedback, the abundance of NIK increased after receptor ligation. A form of NIK with mutations in the IKKalpha-targeted serine residues was more stable than wild-type NIK and resulted in increased noncanonical NF-kappaB signaling. Thus, in addition to the regulation of the basal abundance of NIK in unstimulated cells by a complex containing tumor necrosis factor receptor-associated factor (TRAF) and cellular inhibitor of apoptosis (cIAP) proteins, IKKalpha-dependent destabilization of NIK prevents the uncontrolled activity of the noncanonical NF-kappaB pathway after receptor ligation.

Fig. 1

IKKα inhibits NIK. (A and B) Long-term receptor ligation leads to the accumulation of a constant amount of NIK, in the absence of the resynthesis of TRAF3, as assayed by Western blotting in CH12.LX B cells treated with hBAFF (100 ng/ml) (A) or in murine fibroblasts treated with an agonistic antibody against LTβR (2 μg/ml) (B). (C) The abundance of NIK mRNA was assayed by quantitative polymerase chain reaction (PCR) assay in murine fibroblasts and did not decrease after treatment with agonistic antibody against LTβR (2 μg/ml). (D) NIK is basally stable in untreated fibroblasts from IKKα^−/− and NIK^aly/aly but not wild-type or p100^−/− fibroblasts as assessed by Western blotting. Values reported for the quantitation of bands here (in numbers beneath the blots) and in all other cases are relative arbitrary units as described in Materials and Methods. (E and F) Enhanced stability of NIK in freshly isolated splenic B cells (E) and primary bone marrow macrophages treated with recombinant mRANK (100 ng/ml) (F) from NIK^aly/aly compared to that in cells isolated from wild-type mice. (G) Enhanced basal stability of NIK from IKKα^−/− and NIK^aly/aly fibroblasts is comparable to that observed in TRAF2^−/− and TRAF3^−/− fibroblasts as assayed by Western blotting. (H) Enhanced basal stability of NIK observed in IKKα^−/− fibroblasts is dependent on the kinase activity of IKKα as evidenced by retroviral reconstitution of IKKα^−/− fibroblasts with pBABE-Empty Vector (−), pBABE-IKKα-WT (WT), or pBABE-IKKα-K44A (KD), the kinase-defective mutant of IKKα.

Fig. 2

TRAF-cIAP–mediated control of the stability of NIK is preserved in IKKα-deficient cells and aly cells. (A and B) Western blotting analysis revealed that the abundance of NIK continued to be responsive to LTβR stimulation with 4-hour treatment of IKKα^−/− fibroblasts (A) and NIK^aly/aly fibroblasts (B) with an agonistic antibody against LTβR. (C and D) Western blotting analysis showed that the abundance of NIK continued to be responsive to inhibition of cIAPs with 4-hour treatment of IKKα^−/− fibroblasts (C) and NIK^aly/aly fibroblasts (D) with LBW242 (1 μM), a small-molecule inhibitor of cIAP1 and cIAP2. (E) Knockdown of TRAF3 in both wild-type and NIK^aly/aly fibroblasts results in the enhanced stability of NIK as evidenced by retroviral reconstitution of wild-type, NIK^aly/aly, and TRAF2^−/− fibroblasts with control shRNA (−) or with shRNA specific for TRAF3 (+). (F) NIK in IKKα^−/− fibroblasts continues to interact with TRAF3 as evidenced by coimmunoprecipitation of endogenous TRAF3 with NIK with control immunoglobulin (Ig) or antibody against NIK (NIK) from extracts of wild-type or IKKα^−/− fibroblasts. The abundance of NIK in these cells was equilibrated with the proteasome inhibitor MG132.

Fig. 3

IKKα phosphorylates NIK. (A) NIK from IKKα^−/− fibroblasts migrates further on SDS-PAGE gels than does NIK from IKKα^+/+ fibroblasts regardless of the stimulus used to trigger its accumulation. Fibroblasts were treated for 4 hours with the indicated stimuli and the abundance of NIK was roughly equilibrated by loading different amounts of extract to facilitate migration analysis. (B) Differential migration of NIK from IKKα^+/+ and IKKα^−/− fibroblasts is due to phosphorylation. The accumulation of NIK was triggered with the proteasome inhibitor MG132, after which NIK was immunopurified and subjected to treatment with CIP. (C) IKKα leads to the phosphorylation of NIK in transfected HEK 293T cells. HEK 293T cells were cotransfected with plasmid encoding FLAG-tagged NIK alone or with plasmid encoding IKKα. Extracts were subjected to immunoprecipitation with agarose conjugated to antibody against FLAG. Samples were split, and half were subjected to treatment with CIP. (D) IKKα-dependent phosphorylation of NIK is retained in the C-terminal 247 amino acid residues of NIK. An additional thrombin cleavage site at residue 700 of NIK was generated by means of a T702G/D703R mutation of the protein. HEK 293T cells were cotransfected with plasmid encoding NIK, FLAG-tagged at the C terminus, with or without plasmid encoding IKKα. FLAG-NIK was immunopurified, subjected to partial digestion with thrombin, and analyzed by Western blotting with polyclonal rabbit antibody against FLAG. Potential thrombin cleavage products are shown on the right. (E) IKKα phosphorylates the C terminus of NIK. In vitro kinase assay (KA) of the GST-cNIK(700–942) substrate with increasing amounts of immunopurified IKKα from HEK 293T cells. (F) Addition of NIK substantially enhances the IKKα-dependent phosphorylation of the C terminus of NIK. HEK 293T cells were transfected as indicated; lysates were immunopurified with M2-conjugated agarose and subjected to kinase assay (KA). (G) Endogenous IKKα phosphorylates the C terminus of NIK in a signal-dependent manner. IKKα^+/+ or IKKα^−/− fibroblasts were treated for 3 hours with an agonistic antibody against LTβR (2 μg/ml). Lysates were immunoprecipitated with an antibody against IKKα and subjected to KA.

Fig. 4

IKKα phosphorylates NIK on Ser⁸⁰⁹, Ser⁸¹², and Ser⁸¹⁵. (A) Truncation analysis implicates amino acid residues 742 to 817 of NIK as encompassing the region that contains IKKα target residues. HEK 293T cells were cotransfected with plasmids encoding FLAG-tagged truncations of NIK spanning the C-terminal 200 residues (c200, residues 742 to 942) or 125 residues (c125, residues 817 to 942) with or without plasmid encoding IKKα. In all cases, NIK was included to stimulate the kinase activity of IKKα. (B) MS/MS spectrum of 981.1 (3+). Matching band y-ions, and one immonium ion have been marked. “P” indicates peaks resulting from neutral loss of phosphoric acid. The backbone fragmentation necessary to generate the b- (“\”) and y-ions (“/”) have been marked in the peptide sequence spanning residues 810 to 827 of NIK. (C) Ser→Ala mutations at residues Ser⁸⁰⁹, Ser⁸¹², or Ser⁸¹⁵ lead to enhanced stability of NIK. NIK^−/− fibroblasts were retrovirally reconstituted with pBABE-empty (−), Ser→Ala point mutations of NIK at given residues (8××), or combined mutation of all six serines (all). (D) Diagram of NIK indicating its TRAF3-binding motif (TRAF3-BM), kinase domain (KD), and expansion of the area harboring IKKα target serine residues (highlighted). (E) Combined Ser→Ala mutation of NIK at residues Ser⁸⁰⁹, Ser⁸¹², and Ser⁸¹⁵ generates a mutant protein, henceforth referred to as NIK-3SA, that interacts with IKKα. HEK 293T cells were transfected with plasmid encoding IKKα in combination with either empty vector (−), FLAG-tagged wild-type NIK (NIK-WT), NIK-aly, or NIK-3SA. Lysates subjected to immunoprecipitation with antibody against FLAG were analyzed by Western blotting for IKKα. (F) The NIK-3SA mutant retains kinase activity as assessed by transfecting HEK 293T cells with a 2xκB-luciferase reporter, CMV-βgal, and either pCDNA3-emtpy (−), NIK-WT (WT), the NIK KK430/431AA kinase-defective mutant (KD), or NIK-3SA (3SA). Mean raw light units (RLU) normalized to lacZ activity are shown for triplicate samples. Abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; G, Gly; H, His; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; and W, Trp.

Fig. 5

Negative feedback controls the stability of NIK and regulates noncanonical NF-κB signaling. (A) Mutation of Ser⁸⁰⁹, Ser⁸¹², and Ser⁸¹⁵ to alanine leads to substantially enhanced NIK stability, comparable to that of the NIK aly mutant. Western blotting of untreated NIK^−/− fibroblasts retrovirally reconstituted with pBABE empty (−), NIK-WT, NIK-aly, or the NIK-3SA mutant. (B) Differential stability of NIK mutants seen in (A) was not due to differences in gene expression because the abundance of NIK was equalized upon treatment of cells for 4 hours with the proteasome inhibitor MG132. (C) Differential stability of NIK mutants seen in (A) was dependent on IKKα feedback as evidenced by their equivalent abundance when retrovirally reconstituted into IKKα^−/− fibroblasts. (D) Elimination of IKKα-dependent feedback results in enhanced noncanonical NF-κB signaling. NIK^−/− fibroblasts were retrovirally reconstituted with pBABE-empty (−), NIK-WT, or NIK-3SA (3SA). Extracts were analyzed by Western blotting with an antibody against pIKKα and with an antibody against the N terminus of p100 that recognizes both p100 and its processed form, p52. (E and F) Elimination of IKKα-dependent feedback results in unabated accumulation of NIK during long-term stimulation of receptor. Western blotting analysis of IKKα^+/+ and IKKα^−/− fibroblasts (E) or NIK^+/+ and NIK^aly/aly fibroblasts (F) treated for the indicated times with an agonistic antibody against LTβR (2 μg/ml). (G) A refined model of noncanonical NF-κB signaling that includes the role of IKKα-dependent negative feedback on the stability of NIK. As shown, NIK stability is inhibited by the previously studied TRAF-cIAP complex under unstimulated conditions, whereas IKKα-dependent phosphorylation, described here, controls the abundance of NIK after receptor stimulation.