Functional redundancy of the nuclear factor kappa B inhibitors I kappa B alpha and I kappa B beta - PubMed (original) (raw)

Functional redundancy of the nuclear factor kappa B inhibitors I kappa B alpha and I kappa B beta

J D Cheng et al. J Exp Med. 1998.

Abstract

The transcription factor NF-kappaB is sequestered in the cytoplasm by the inhibitor proteins of the IkappaB family. Each member of the IkappaB exhibits structural and biochemical similarities as well as differences. In an effort to address the functional redundancy of two closely related IkappaB molecules, IkappaBalpha and IkappaBbeta, we generated knock-in mice by replacing the IkappaBalpha gene with the IkappaBbeta gene. The knock-in mice do not express IkappaBalpha, but express a T7-tagged IkappaBbeta under the promoter and regulatory sequence of ikba. Unlike the IkappaBalpha-deficient mice, which display severe postnatal developmental defects and die by postnatal day 8, homozygous knock-in mice survive to adulthood, are fertile, and exhibit no apparent abnormalities. Furthermore, thymocytes and embryonic fibroblasts from the knock-in animals exhibit an inducible NF-kappaB response similar to that of wild-type animals. These results indicate that IkappaBalpha and IkappaBbeta share significant similarities in their biochemical activity, and that they acquired their different functions from divergent expression patterns during evolution.

PubMed Disclaimer

Figures

Figure 1

Figure 1

Targeted replacement of IκBα with the IκBβ gene. (A) The targeting vector pPNT-abki is shown at the top. Black boxes and thickened lines represent IκBβ exons and introns. Open boxes and thin lines represent IκBα exons and introns. Homologous recombination takes place between the 5′ and 3′ flanking regions of the ikba gene, resulting in replacement of the entire IκBα locus by IκBβ. Additionally, the ATG start codon of the IκBβ was also replaced with the T7-Tag sequence. N, NotI; Nc, NcoI; H, HindIII; E, EcoRV. (B) Southern blot analysis of DNA isolated from ES cell lines. EcoRV-digested DNA from cells that have undergone homologous recombination yielded an 11-kb signal when hybridized to an external probe. (C) Immunoprecipitation analysis of recombinant knock-in ES cells. Equal numbers of wild-type and +/ki ES cells were labeled with [35S]methionine and immunoprecipitated with antiserum against IκBβ (lanes 1 and 2) or T7-Tag (lanes 3 and 4). (D) Analysis of homozygous mouse tail DNA. (Left) Southern blot analysis of wild-type, heterozygous, and homozygous mouse tail DNA; (right) PCR analysis of tail DNA using IκBα primers and neomycin primers. Amplification of IκBα yields a 490-bp PCR product, whereas amplification of the neomycin gene yields a 239-bp PCR product.

Figure 2

Figure 2

Expression of NF-κB/IκB proteins and NF-κB binding activity in the knock-in mice. (A) Western blot analysis of splenocytes from wild-type, heterozygous, and homozygous mice with antisera against members of the NF-κB and IκB family. (B) Immunoprecipitation of cRel- and RelA-associated IκB proteins. Homozygous (ki/ki) and wild-type (+/+) thymocytes were labeled with [35S]methionine and immunoprecipitated with RelA and cRel antiserum in nondenaturing conditions. The immunoprecipitates were then denatured and sequentially reprecipitated with IκBα, T7-Tag, and IκBβ antisera. (C) EMSA for basal NF-κB activity in thymus, spleen, brain, and MEFs of the knock-in mice. Whole cell extracts were incubated with a palindromic κB-specific probe.

Figure 3

Figure 3

Signal dependent NF-κB activation in ki/ki thymocytes. (A) NF-κB binding activity from wild-type (+/+) and ki/ki thymocytes stimulated with PMA/PHA. Cells were stimulated for the indicated periods of time and EMSA was performed using the palindromic κB sequence with 5 μg of nuclear extracts. Oct-1 binding activity was used as control. (B) Western blot analysis of +/+ and ki/ki thymocytes stimulated with PMA/PHA. Similar to IκBα, T7-Tag–IκBβ is induced and reaccumulates after NF-κB stimulation. 20 μg of cytoplasmic extracts were used per lane and a sister blot was probed with antilactic dehydrogenase (LDH) antibodies as control for loading.

Figure 4

Figure 4

Signal dependent NF-κB activation in ki/ki embryo fibroblasts. (A) NF-κB binding activty of wild-type (+/+) and ki/ki fibroblasts stimulated with TNF-α for the indicated periods of time. EMSA was performed using 5 μg of nuclear extracts. Oct-1 binding was used in parallel as control. (B) Western blot analysis of +/+ and ki/ki fibroblasts stimulated with TNF-α. The T7-Tag–IκBβ reaccumulates in ki/ki fibroblasts after TNF-α stimulation. IκBα, T7-Tag–IκBβ, and IκBβ levels were determined on blots with 20 μg of cytoplasmic extracts.

Figure 4

Figure 4

Signal dependent NF-κB activation in ki/ki embryo fibroblasts. (A) NF-κB binding activty of wild-type (+/+) and ki/ki fibroblasts stimulated with TNF-α for the indicated periods of time. EMSA was performed using 5 μg of nuclear extracts. Oct-1 binding was used in parallel as control. (B) Western blot analysis of +/+ and ki/ki fibroblasts stimulated with TNF-α. The T7-Tag–IκBβ reaccumulates in ki/ki fibroblasts after TNF-α stimulation. IκBα, T7-Tag–IκBβ, and IκBβ levels were determined on blots with 20 μg of cytoplasmic extracts.

Figure 5

Figure 5

Postinduction repression of NF-κB in ki/ki embryo fibroblasts. (A) βF-κB binding activity of wild-type (+/+) and ki/ki fibroblasts treated with TNF-α for 30 min, after which cells were washed and medium without TNF-α was added to the cells for the indicated period. Nuclear extracts were prepared at the indicated periods after removal of TNF-α. A loading control using the Oct-1 probe was also performed. (B) Western blot analysis of +/+ and ki/ki fibroblasts treated with TNF-α. 20 μg each of the total cell extracts were analyzed for levels of IκBα, T7-Tag–IκBβ, and the endogenous IκBβ proteins.

Similar articles

Cited by

References

    1. Gilmore TD. Introduction: the Rel/NF-κB signal transduction pathway. Cancer Biol. 1997;8:61–62. - PubMed
    1. Whiteside ST, Israël S. IκB proteins: structure, function and regulation. Cancer Biol. 1997;8:75–82. - PubMed
    1. Israel A. A role for phosphorylation and degradation in the control of NF-κB activity. Trends Genet. 1995;11:203–205. - PubMed
    1. Beg AA, Baldwin AS., Jr The IκB proteins: multifunctional regulators of Rel/NF-κB transcription factors. Genes Dev. 1993;7:2064–2070. - PubMed
    1. Beg AA, Sha WC, Bronson RT, Baltimore D. Constitutive NF-κB activation, enhanced granulopoiesis, and neonatal lethality in IκBα-deficient mice. Genes Dev. 1995;9:2736–2746. - PubMed

MeSH terms

Substances

LinkOut - more resources