Tetrameric oligomerization of IkappaB kinase gamma (IKKgamma) is obligatory for IKK complex activity and NF-kappaB activation

Abstract

The IkappaB kinase (IKK) complex mediates activation of transcription factor NF-kappaB by phosphorylation of IkappaB proteins. Its catalytic subunits, IKKalpha and IKKbeta, require association with the regulatory IKKgamma (NEMO) component to gain full basal and inducible kinase activity. However, the oligomeric composition of the IKK complex and its regulation by IKKgamma are poorly understood. We show here that IKKgamma predominantly forms tetramers and interacts with IKKalpha or IKKbeta in this state. We propose that tetramerization is accomplished by a prerequisite dimerization through a C-terminal coiled-coil minimal oligomerization domain (MOD). This is followed by dimerization of the dimers with their N-terminal sequences. Tetrameric IKKgamma sequesters four kinase molecules, yielding a gamma(4)(alpha/beta)(4) stoichiometry. Deletion of the MOD leads to loss of tetramerization and of phosphorylation of IKKbeta and IKKgamma, although the kinase can still interact with the resultant IKKgamma monomers and dimers. Likewise, MOD-mediated IKKgamma tetramerization is required to enhance IKKbeta kinase activity when overexpressed in 293 cells and to reconstitute a lipopolysaccharide-responsive IKK complex in pre-B cells. These data thus suggest that IKKgamma tetramerization enforces a spatial positioning of two kinase dimers to facilitate transautophosphorylation and activation.

FIG. 1.

Tetrameric oligomerization of IKKγ. (A) IKKγ-deficient 1.3E2 cells and 1.3E2 cells stably expressing IKKγ were metabolically labeled with [³⁵S]methionine. Lysates were treated with EGS, IKKγ was immunoprecipitated (IP: IKKγ), and pellets were washed and analyzed by SDS-PAGE and autoradiography. To confirm that the 200-kDa band resulting from the EGS cross-link consists exclusively of IKKγ, the band was excised from a polyacrylamide gel, and as indicated, proteins were electroeluted and treated with hydroxylamine-HCl to cleave EGS. IKKγ complexes were precipitated and separated by SDS-PAGE, and components were detected with a phosphorimager (top gels). The lack of immunoprecipitated labeled proteins in 1.3E2 cells confirms the specificity of the antibody. IKKγ expression before cross-linking was determined by Western blotting (WB). (B) Metabolically labeled HeLa cells were lysed, and proteins were cross-linked with sulfo-EGS. Immunoprecipitation with an IKKγ antibody (IP: IKKγ) was performed with (+) or without (−) preblocking with a specific peptide, as indicated above the gel. Products were analyzed by SDS-PAGE and autoradiography. The arrows in panels A and B indicate the position of IKKγ tetramers. (C) [³⁵S]methionine-labeled, N-terminally FLAG-tagged IKKγ (FLAG-IKKγ) and C-terminally HA-tagged IKKγ (IKKγ-HA) were prepared by coupled in vitro transcription and translation and cross-linked with EGS. The reaction was stopped by the addition of Tris, and immunoprecipitations (IP) with the indicated antibodies were performed. The positions of molecular mass markers (in kilodaltons) are shown to the left of the gel. (D) (Left) Recombinant IKKγ was purified from E. coli and analyzed on a Coomassie blue-stained SDS-polyacrylamide gel along with protein molecular mass markers, as indicated. (Right) Analytical ultracentrifugation analysis of purified recombinant IKKγ (Superose 6 peak fractions). Radial concentration distribution curves of IKKγ dissolved in 20 mM Tris-HCl (pH 7.5) containing 100 mM NaCl are shown. The profiles were recorded at 10,000 rpm and wavelengths of 220 (○), 225 (•), or 230 (□) nm. The three curves were fitted globally using the polymole program, resulting in a molecular mass of 192.6 ± 3.6 kDa.

FIG. 2.

Oligomerization of IKKγ is mediated by a C-terminal domain between amino acids 246 to 365. (A) Conserved motifs and structural predictions of IKKγ by PredictProtein and MultiCoil are shown at the top. The positions of alpha-helical regions (open boxes), coiled-coil domains (black bars), and zinc fingers are shown. Below the IKKγ map, a schematic summary of IKKγ deletion mutants is shown. The numbers are amino acids. IKKγ oligomerization is shown to the right as follows: +, IKKγ oligomerizes; −, IKKγ does not oligomerize; ++, IKKγ oligomerizes strongly; (+), IKKγ oligomerizes weakly; n.d., not determined. (B) Interaction of FLAG-tagged IKKγ and IKKγ deletion mutants with HA-tagged full-length IKKγ coexpressed in 293 cells. Lysates were analyzed for expression with anti-FLAG antibody (top gel). Immunoprecipitation (IP) was performed with an anti-HA antibody. Coimmunoprecipitated proteins were detected in a Western blot (WB) with anti-FLAG antibody (middle gel), and precipitated HA-tagged IKKγ (HA-IKKγ) was detected with anti-HA antibody (bottom gel). The asterisk marks a signal caused by the HA antibody heavy chain. (C) GST or GST-tagged IKKγ^246-365 and [³⁵S]methionine-labeled full-length IKKγ (input [middle and bottom gels]) were used for an interaction analysis (top gel), as indicated.

FIG. 3.

C-terminal dimerization is a prerequisite for IKKγ tetramerization. Labeled FLAG-tagged IKKγ mutants were prepared by in vitro translation. After EGS cross-linking, products were precipitated with anti-FLAG antibodies and analyzed by SDS-PAGE and autoradiography. The positions of molecular mass markers are indicated. (A) Analysis of full-length IKKγ and internal deletion mutants IKKγ^Δ246-302, IKKγ^Δ302-365, and IKKγ^Δ246-365. Migration of monomers and tetramers is indicated. (B) (Top) Analysis of N- and C-terminal IKKγ deletion mutants. Major and minor multimeric forms are indicated below the autoradiogram (minor forms shown in parentheses). (Bottom) Schematic presentation of major oligomerization states of IKKγ subregions. The positions of alpha-helical regions (open boxes), coiled-coil domains (black bars), and zinc finger are shown.

FIG. 4.

IKKγ binds as a tetramer to IKKβ. [³⁵S]methionine-labeled FLAG-tagged IKKγ (FLAG-IKKγ) and HA-tagged IKKβ (HA-IKKβ) were prepared by in vitro translation (input [left gel]). Equal aliquots of FLAG-tagged IKKγ were not treated or were cross-linked with EGS, followed by inactivation of EGS. Both samples were immunoprecipitated with anti-FLAG antibodies (IP: FLAG). Precipitates were washed and incubated with HA-tagged IKKβ on ice for 30 min. After an additional washing step, proteins were separated by SDS-PAGE and analyzed by autoradiography.

FIG. 5.

Physical interaction of IKKα and IKKβ with IKKγ involves overlapping but distinct N-terminal domains of IKKγ and does not require IKKγ tetramerization. (A) [³⁵S]methionine-labeled IKKγ mutants were prepared by coupled in vitro transcription and translation. Labeling efficiency was controlled by SDS-PAGE or autoradiography (top gel). 293 cells were transiently transfected with HA-tagged IKKα (HA-IKKα) or HA-tagged IKKβ (HA-IKKβ). Lysates were checked for equal expression of transfected proteins by Western blotting (WB) with anti-HA antibodies. Labeled IKKγ mutants were added to lysates with either overexpressed HA-tagged IKKα or HA-tagged IKKβ and incubated for 30 min on ice. After immunoprecipitation (IP) with anti-HA antibodies, precipitated proteins were analyzed by SDS-PAGE and autoradiography. (B) (Left) In vitro-translated, [³⁵S]methionine-labeled HA-tagged IKKβ, left untreated (−) or incubated with EGS (+), was analyzed for cross-linked IKKβ dimers after anti-HA immunoprecipitation (IP:HA), PAGE, and autoradiography. (Right) In vitro-translated, [³⁵S]methionine-labeled, FLAG-tagged IKKγ^1-196 was incubated without or with baculovirus-expressed, purified recombinant IKKβ and treated with EGS, as indicated. Cross-linked species were visualized after anti-FLAG immunoprecipitation (IP: FLAG) by PAGE and autoradiography. The positions of the monomeric and dimeric proteins are indicated by the arrows.

FIG. 6.

Activity and inducibility of the IKK complex require IKKγ tetramerization. (A) 293 cells were cotransfected with HA-tagged IKKβ (HA-IKKβ) and FLAG-tagged IKKγ wild-type or mutant proteins, as indicated. Complexes were immunoprecipitated with HA antibody (IP: HA) and tested in in vitro kinase assays (KA) with GST-IκBα^1-53 as the substrate (top gel). Aliquots of immunoprecipitated IKKβ were analyzed by Western blotting (WB) using HA antibody (middle gel), and expression of IKKγ proteins was controlled in whole-cell extracts with FLAG antibody (bottom gel). (B) 70Z/3, 1.3E2, and 1.3E2 cells stably expressing the indicated constructs were tested for LPS-induced NF-κB activation by an electrophoretic mobility shift assay (EMSA). The presence (+) and absence (−) of LPS is shown above the gel. Expression control of endogenous and ectopic IKKγ proteins and of endogenous IKKβ analyzed by Western blotting (WB) of whole-cell extracts is shown at the bottom. Immunoprecipitation (IP) of endogenous and ectopic IKKγ proteins was performed to confirm equal interaction of IKKβ with mutant and wild-type IKKγ. (C) 1.3E2 cells were cotransfected with the NF-κB reporter plasmid 6xNF-κBluc, internal control plasmid, and IKKγ or IKKγ^Δ246-365 or empty expression vector (mock), as indicated. Cells were stimulated with LPS (+) for 6 h or not treated with LPS (−), and luciferase activity was determined. (D) IKKβ autophosphorylation and phosphorylation of IKKγ. 293 cells were transfected with HA-tagged IKKβ (HA-IKKβ) and FLAG-tagged IKKγ (FLAG-IKKγ) constructs, as indicated. HA-immunoprecipitated complexes were incubated with [γ-³²P]ATP in kinase assays (KA), and phosphorylated proteins were detected by SDS-PAGE and autoradiography (top gel). Expression of IKKβ and IKKγ proteins and IKKβ precipitation were controlled by anti-HA or anti-FLAG immunoblotting of extracts (middle gel) and pellets (bottom gel), respectively. WB, Western blotting; IP, immunoprecipitation.

FIG. 7.

Dose-dependent inhibition of NF-κB activation by the MOD. 293 cells were cotransfected with the indicated amounts of expression vectors for wild-type IKKγ, IKKγ^1-196, or IKKγ^246-365, NF-κB reporter plasmid 6xNF-κBluc, and an internal control plasmid. After stimulation of the cells with TNF-α for 6 h, luciferase activity was measured.

FIG. 8.

Hypothetical model for tetrameric assembly of IKKγ by head-to-head dimerization of two dimers and interaction with two kinase dimers. The positions of zinc fingers, MODs, and kinase binding domains (KBD) are shown.