The carboxyl-terminal region of IkappaB kinase gamma (IKKgamma) is required for full IKK activation

Abstract

IkappaB kinase gamma (IKKgamma) (also known as NEMO, Fip-3, and IKKAP-1) is the essential regulatory component of the IKK complex; it is required for NF-kappaB activation by various stimuli, including tumor necrosis factor alpha (TNF-alpha), interleukin 1 (IL-1), phorbol esters, lipopolysaccharides, and double-stranded RNA. IKKgamma is encoded by an X-linked gene, deficiencies in which may result in two human genetic disorders, incontinentia pigmenti (IP) and hypohidrotic ectodermal dysplasia with severe immunodeficiency. Subsequent to the linkage of IKKgamma deficiency to IP, we biochemically characterized the effects of a mutation occurring in an IP-affected family on IKK activity and NF-kappaB signaling. This particular mutation results in premature termination, such that the variant IKKgamma protein lacks its putative C-terminal Zn finger and, due to decreased mRNA stability, is underexpressed. Correspondingly, IKK and NF-kappaB activation by TNF-alpha and, to a lesser extent, IL-1 are reduced. Mutagenesis of the C-terminal region of IKKgamma was performed in an attempt to define the role of the putative Zn finger and other potential functional motifs in this region. The mutants were expressed in IKKgamma-deficient murine embryonic fibroblasts (MEFs) at levels comparable to those of endogenous IKKgamma in wild-type MEFs and were able to associate with IKKalpha and IKKbeta. Substitution of two leucines within a C-terminal leucine zipper motif markedly reduced IKK activation by TNF-alpha and IL-1. Another point mutation resulting in a cysteine-to-serine substitution within the putative Zn finger motif affected IKK activation by TNF-alpha but not by IL-1. These results may explain why cells that express these or similar mutant alleles are sensitive to TNF-alpha-induced apoptosis despite being able to activate NF-kappaB in response to other stimuli.

FIG. 1.

Molecular identification of an IP-linked mutation. (A) Pedigree of the IP-affected family being studied. Red and black circles denote carrier females; black squares denote nonaffected males; blue squares denote undiagnosed and deceased males; and red squares denote carrier and deceased males. (B) Genomic structure and organization of the human IKKγ gene (31). Coding and noncoding exons are shown in red and blue, respectively. G6PD, glucose-6-phosphate dehydrogenase. (C) Nucleotide (seq) and protein (prot) sequences of normal and mutant (IP) IKKγ alleles at the region of the mutation. The nucleotide sequence shows a 13-base duplication following a cytosine tract found in the IP-affected patient. The protein sequence shows the addition of four novel amino acids after proline 393 of human IKKγ, which results in a truncated protein that lacks all of the putative Zn finger. (D) Sequence of the putative Zn finger.

FIG. 2.

Characterization of the variant IKKγ polypeptides encoded by the IP allele. (A) Reduced expression of truncated IKKγ polypeptides detected in IP fibroblasts. Whole-cell extracts from WT and IP fibroblasts were subject to immunoblot analysis with MAb c73-429 and rabbit polyclonal antibody 3294. (B) Interaction of WT- and IP-encoded IKKγ polypeptides with IKKα and IKKβ. Extracts of WT and IP male fibroblasts were immunoprecipitated with control immunoglobulin G (lane 1) or the following antibodies: MAb IMG-324 (lane 2), MAb c73-1794 (lane 3), MAb c73-429 (lane 4), MAb c73-764 (lane 5), and polyclonal antibody 3294 (lane 6), all against IKKγ; anti-IKKα antibody M-280 (lane 7); and MAb B78-1 against IKKα (lane 8). The samples were then probed with antibodies against IKKα (IMG-136) and IKKβ (MAb 10AG2; Upstate Biotechnology Inc.). As shown by the signals in lanes 7 and 8, the two extracts contained similar amounts of IKKα and IKKβ. (C) WT (top panel) and IP (bottom panel) cell lysates were chromatographed on a Superose 6 gel filtration column. The levels of the different IKK subunits present in each fraction were determined by immunoblotting with anti-IKKα (MAb IMG-136) and anti-IKKγ (MAb c73-1794). Positions at which molecular weight markers (in thousands) eluted from this column are indicated. Inp, input.

FIG. 3.

Analysis of IKK and NF-κB activation in WT and IP fibroblasts. (A) WT and IP human male fibroblasts were treated or not treated with different doses (0.1, 1, and 10 ng/ml) of TNF-α or IL-1 for 5 min. Cells were lysed, and IKK activity (KA) was measured by an immunocomplex kinase assay with an anti-IKKα (M-280) and glutathione S-transferase-IκBα(1-54) as a substrate. The samples were separated by sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and autoradiographed. The membrane was reprobed (Blot: IKKα) with MAb IMG-136 against IKKα as a loading control. (B) Same as in panel A, except that cells were treated with saturating doses of TNF-α (10 ng/ml) or IL-1 (10 ng/ml) for different times as indicated prior to the determination of kinase activity. (C) Induction of NF-κB DNA binding activity in WT and IP fibroblasts treated as described above. Whole-cell extracts (10 μg per sample) were incubated with an NF-κB probe. The same extracts were also incubated with an NF-1 probe used as a control. DNA binding activity was determined by EMSAs. (D) Western blot analysis of IκBα, p50, and p65 in whole-cell extracts prepared from WT and IP fibroblasts treated as described for panel B.

FIG. 4.

Mutational analysis of the IKKγ C-terminal region. (A) Scheme showing the structural features of full-length IKKγ and the amino acid substitutions introduced into different functional motifs (C-C, coiled coil; αH, α-helical region; ZF, Zn finger; SH3, SH3 binding site). The mutantsgenerated were as follows: M1 (L315P), M2 (L322P), M3 (L326P), M4 (L329P), M5 (L336P), M6 (C389S and C393S), and M7 (P360A and P363A). (B) IKKγ-deficient MEFs were reconstituted with WT IKKγ or the different amino acid substitution mutants described in panel A. Levels of expression of IKKγ in the different reconstituted cells were similar to those found in WT MEFs (control) that were not infected by any retrovirus. The membrane was reprobed with MAb IMG-136 against IKKα as a loading control (bottom panel). (C) Amino acid substitutions within the C-terminal region of IKKγ do not affect binding to IKKα or IKKβ. IKKγ was immunoprecipitated from reconstituted cells or WT fibroblasts with a polyclonal antibody directed against its N terminus followed by immunoblotting with antibodies against IKKα and IKKβ. The membrane was reprobed with a rabbit polyclonal antibody against IKKγ as a loading control (bottom panel).

FIG. 5.

Effects of amino acid substitutions within the C-terminal region of IKKγ on IKK activation by TNF-α or IL-1. IKKγ-deficient cells that were either mock infected or reconstituted with either WT or amino acid substitution mutants of IKKγ were left untreated or treated with either TNF-α (10 ng/ml) or IL-1 (10 ng/ml). Immunocomplex kinase assays were performed on cells lysed at the indicated times as described in the legend to Fig. 3B. The membrane was reprobed with MAb IMG-136 against IKKα as a loading control (data not shown).

FIG. 6.

Effects of amino acid substitutions within the C-terminal region of IKKγ on susceptibility to TNF-α-induced apoptosis. WT mouse fibroblasts or Ikkγ⁻ fibroblasts that were either mock reconstituted or reconstituted with WT or C-terminal substitution mutants of IKKγ as well as WT and IP human fibroblasts were treated with TNF-α (50 ng/ml). After a 24-h incubation period, cells were fixed, stained with 4′,6′-diamidino-2-phenylindole (DAPI), and mounted with a coverslip. Values shown are percentages of apoptotic nuclei per field scored by using a fluorescence microscope; error bars indicate standard deviations.