Signal processing by its coil zipper domain activates IKK gamma

Abstract

NF-kappaB activation occurs upon degradation of its inhibitor I-kappaB and requires prior phosphorylation of the inhibitor by I-kappaB kinase (IKK). Activity of IKK is governed by its noncatalytic subunit IKKgamma. Signaling defects due to missense mutations in IKKgamma have been correlated to its inability to either become ubiquitylated or bind ubiquitin noncovalently. Because the relative contribution of these events to signaling had remained unknown, we have studied mutations in the coil-zipper (CoZi) domain of IKKgamma that either impair signaling or cause constitutive NF-kappaB activity. Certain signaling-deficient alleles neither bound ubiquitin nor were they ubiquitylated by TRAF6. Introducing an activating mutation into those signaling-impaired alleles restored their ubiquitylation and created mutants constitutively activating NF-kappaB without repairing the ubiquitin-binding defect. Constitutive activity therefore arises downstream of ubiquitin binding but upstream of ubiquitylation. Such constitutive activity reveals a signal-processing function for IKKgamma beyond that of a mere ubiquitin-binding adaptor. We propose that this signal processing may involve homophilic CoZi interactions as suggested by the enhanced affinity of CoZi domains from constitutively active IKKgamma.

Fig. 1.

Defect in NF-κB signaling in F40, F29, and J77 cells due to mutations in IKKγ. (A) Cells were stimulated for 24 h with 1 μg/ml LPS before analysis for NF-κB-dependent GFP expression. (B) Nuclear extracts from cells stimulated with 10 μg/ml LPS and probed for RelA and PCNA. (C) Cells stimulated for 24 h with 1 μg/ml LPS, 10 μg/ml PGN, 250 nM CpG DNA, or 50 ng/ml PMA/1 μM Ionomycin analyzed for NF-κB-dependent GFP and Thy1 expression by flow cytometry. (D) Cell lysates were probed for IKKγ. (E) Secondary structure of IKKγ (CC1, coiled coil 1; CC2, coiled coil 2; LZ, leucine zipper; Zn, zinc finger). F40 carries a frame shift (T47fs). F29 and J77 contain point mutations (E308V and R331P, respectively). D304N occurred in a patient with EDA-ID. K270A is a designed mutation. (F) F40 cells complemented with the indicated IKKγ alleles. Cells stimulated for 24 h with 1 μg/ml LPS were analyzed for NF-κB-dependent GFP expression. Lysates were probed for AU1-tagged IKKγ.

Fig. 2.

IKKγ^K270A dominantly controls NF-κB activity. (A) Helical wheel representation of CC2. Beginning (position 253) and end (position 288) of CC2 and the occurrence of K270 in a “d” position are indicated. (B) NF-κB-dependent GFP induction measured 48 h after the transduction of GTPT3 cells with a control gene (bsd) or the indicated IKKγ alleles. Cells were stimulated with or without 1 μg/ml LPS for the final 24 h as indicated. An IRES-controlled CD8 was used to distinguish transduced from nontransduced cells. (C) F40 cells containing NF-κB-dependent luciferase were transduced with IKKγ, either wild type or mutant in position 270, and carrying additional mutations as indicated. Luciferase activity was measured 24 h later. Lysates were probed for AU1-tagged IKKγ.

Fig. 3.

IKKγ binds Ub via its CoZi domain. (A and B) Purified GST fusion proteins coupled to beads were incubated with lysates of 293 cells expressing luciferase IKKγ. The ratio between luciferase activity bound to beads and present in lysates is shown. GST fusion proteins were visualized by Coomassie blue staining. (C) Purified GST fusion proteins coupled to beads were incubated with lysate from bacteria expressing His-tagged CoZi. (Upper) Eluates from beads were blotted with anti-His antibody. (Lower) GST fusion proteins were visualized by Coomassie blue staining. (D) Purified GST fusion proteins coupled to beads were incubated with lysates of 293 cells expressing a luciferase IKKγ. The ratio between luciferase activity bound to beads and present in lysates is shown. GST fusion proteins were visualized by Coomassie blue staining. (E) The 293 cells were transfected with the indicated AU1-tagged IKKγ constructs. Lysates were incubated with purified GST-tetra-Ub (GST-4xUb) bound to beads. Lysates and eluates were blotted for AU1-IKKγ.

Fig. 4.

Differential ubiquitylation of IKKγ alleles. (A) The 293 cells were transfected with plasmids encoding AU1-tagged IKKγ, myc-tagged Ub, and HA-tagged TRAFs. Lysates were precipitated with an antibody against AU1. Precipitates and lysates were blotted for AU1-IKKγ, HA-TRAFs, and myc-Ub. (B) The 293 cells were transfected with plasmids encoding AU1-tagged IKKγ alleles (wild type, K270A, D304N, E308V, and R331P) and HA-tagged TRAF6 (wild type or C70A). Lysates were immunoprecipitated with an antibody against AU1. Precipitates and lysates were blotted for AU1-IKKγ and HA-TRAFs. (C) The 293 cells were transfected with plasmids encoding AU1-tagged IKKγ alleles containing either one mutation (K270A) or two mutations (K270A/D304N, K270A/E308V, and K270A/R331P) and HA-tagged TRAF6 (wild type or C70A). Lysates were immunoprecipitated with an antibody against AU1. Precipitates and lysates were blotted for AU1-IKKγ and HA-TRAFs. The asterisk indicates lanes that were removed electronically from the blot.

Fig. 5.

K270A causes high-affinity CoZi interactions. (A) GTPT3 cells were transduced with the indicated IKKγ alleles. AU1-tagged IKKγ was precipitated, and a kinase assay was performed with GST-IκBα (amino acids 1–100) as a substrate. The expression level of AU1-tagged IKKγ in lysates was analyzed. (B) NF-κB-dependent luciferase activity in 293 cells 48 h after transfection with the indicated combinations of plasmids. IKKβDN corresponds to IKKβ^K44A and IκBαDN indicates to IκBα^S32A+S36A. (C) (Left) CoZi^WT and CoZi^K270A were purified from E. coli. (Right) Mass-action-driven association was analyzed by analytical ultracentrifugation. Plots are from sedimentation equilibrium runs and indicate the formation of dimers. The K_d value for 30 ± 5 μM CoZi^WT and the monomer size as determined from fitting the raw data are indicated. The latter is in excellent agreement with the theoretical value (10.9 kDa). CoZi^K270A did not dissociate detectably at concentrations as low as 2 μM and showed only dimer (M_{w, app}, weight average apparent molecular weight). (D) The 293 cells were transfected with the indicated combinations of luciferase-tagged IKKγ (full length or CoZi) and FLAG-tagged IKKγ (only full length) either wild type or mutant in position 270. Proteins were precipitated with an antibody against Flag and eluted with Flag peptide. The ratio between luciferase activity in eluates and lysates is shown. The expression of Flag-tagged proteins was analyzed by Western blot. (E) Lysates from F40 cells transduced with IKKγ^WT or IKKγ^K270A were fractionated over Superdex 200. Fractions were tested for IKKγ.

Fig. 6.

Model of IKK activation. Shown are IKK complexes containing two kinase and two IKKγ subunits. Indicated are the association of IKKγ subunits, the binding of CC2 to LZ, and the binding of Ub to CoZi. Ubiquitylation also is shown, but it is not meant to indicate modification of a specific lysine residue. (A) Upon stimulation, IKKγ^WT binds Ub and becomes ubiquitylated, and kinase activity is induced. In contrast, IKKγ^D304N and IKKγ^E308V do not bind Ub and are not ubiquitylated by TRAF6, and kinase activity is not induced. Although IKKγ^R331P is able to bind Ub, it still fails to be ubiquitylated. Introducing an activating mutation, K270A, into any of those alleles restores their ubiquitylation by TRAF6 and creates mutants constitutively activating NF-κB without repairing the Ub-binding defect of IKKγ^D304N and IKKγ^E308V. (B) In unstimulated cells, kinases bound to IKKγ^WT remain catalytically inactive, whereas kinases bound to IKKγ^K270A are active. The heightened affinity of CoZi^K270A domains for homophilic interactions suggests a mechanism for inducing kinase activity in wild-type cells, where binding of Ub chains to CoZi^WT may be required to stabilize weaker homophilic CoZi^WT interactions.