Biochemical and Biophysical Research Communications
NF-κB p65 transactivation domain is involved in the NF-κB-inducing kinase pathway☆,☆☆
Section snippets
Materials and methods
Plasmid constructs. Construction of mammalian expression vectors, pM-p65, pM-p65 (286–551), pM-p65 (521–551), and pcDNA3.1-p65, was previously described [26]. pM-p65 (1–286) was generated by amplifying the corresponding p65 fragment by PCR using the oligo-nucleotide primers 5′-CGGGATCCCGATGGACGAACTGTTCCCCCTCAT-3′ and 5′-GCTCTAGAGCGAATTCCATGGGCTCACTGAGCT-3′ containing BamHI and XbaI sites. pM-p65 (521–551:S529A) was generated by PCR using the oligo-nucleotides 5′
Synergism between NIK and IKKα or IKKβ in the NF-κB activation cascade
We first examined the effects of overexpression of NIK, IKKα, and IKKβ in the NF-κB activation pathway, either alone or in combination, using the transient luciferase assay with an NF-κB-dependent reporter plasmid (4κBw-luc). As shown in Fig. 1A, TNF alone stimulated NF-κB-dependent gene expression by 21-fold and overexpression of NIK activated the gene expression by 23-fold. Overexpression of IKKα alone did not significantly activate NF-κB-dependent gene expression whereas IKKβ stimulated the
Discussion
It is widely accepted that the nuclear translocation is a hallmark of the transcriptional activation of NF-κB and its intracellular localization is governed by IκBs [1], [2]. However, recent studies revealed that both NF-κB and IκB shuttle in and out of the nucleus [33], [34]. In addition, Birbach et al. [33] reported that the treatment of cells with a nuclear export blocker leptomycin B resulted in the nuclear accumulation of NIK and IKKα but not IKKβ, indicating that NIK and IKKα also shuttle
Acknowledgements
We thank Dr. H. Nakano, Dr. D. Wallach, Dr. S. Hatakeyama, and Dr. T. Kimura for their generosity in providing the expression vectors encoding IKKα and IKKβ, NIK, Myc-IκBα, Ran and Ran (Q69L), respectively.
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This work was supported in part by grants-in-aid from the Ministry of Health, Labour and Welfare, the Ministry of Education, Culture, Sports, Science and Technology of Japan and the Japanese Health Sciences Foundation.
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Abbreviations: NF-κB, nuclear factor kappa B; TA1, transactivation domains 1; NIK, NF-κB-inducing kinase; RHD, Rel homology domain; NLS, nuclear localization signal; Ran, Ras-related protein; TNF, tumor necrosis factor; IL-1β, interleukin-1β; IKK, IκB kinase; GFP, green fluorescent protein; luc, luciferase; NTF2, nuclear transport factor 2.