FIGURE 4.

Lys-717 and Lys-719 in ANK7 of IκBζ are crucial for activation of the Lcn2 gene. A, amino acid sequence alignment of ANK7 in IκBζ from various species. TAD, trans-activation domain; Mm, Mus musculus; Hs, Homo sapiens; Gg, Gallus gallus; Xt, Xenopus tropicalis, Dr, Danio rerio. B, the IκBζ C-terminal boundary required for Lcn2 activation. p50-/IκBζ-deficient MEFs were transfected with the following plasmids: the luciferase reporter plasmid pGL3-Basic containing the upstream region of Lcn2 (−1031/+54); the internal control plasmid pRL-TK, and pcDNA3 for expression of FLAG-IκBζ with the indicated truncation and HA-p50. Luciferase activities were determined as described under “Experimental Procedures.” Each graph represents the mean ± S.D. obtained from three independent transfections. Cell lysates were analyzed by immunoblot with anti-FLAG, anti-HA, or anti-β-tubulin antibody. MW, molecular weight. C, the IκBζ C-terminal boundary required for interaction with NF-κB p50. GST-fused IκBζ with the indicated truncation was incubated with His-p50 and pulled down with glutathione-Sepharose-4B beads, followed by SDS-PAGE analysis with CBB staining. D, the role of Lys-717 and Lys-719 of IκBζ ANK7 in Lcn2 activation. The ability of FLAG-IκBζ with the indicated amino acid substitution was analyzed as in B. E, the role of Lys-717 and Lys-719 of IκBζ ANK7 in interaction with NF-κB p50 in vivo. FLAG-tagged IκBζ with the K717E/K719E or D451T substitution was co-expressed with HA-p50 in HEK293T cells, and proteins in the cell lysate were immunoprecipitated (IP) with anti-FLAG antibody, followed by immunoblot analysis with the indicated antibody (Blot). F, the effect of arginine substitution for Lys-717 and Lys-719 on IκBζ-mediated Lcn2 activation. The ability of FLAG-IκBζ with the indicated amino acid substitution was analyzed as in B. Positions for marker proteins are indicated in kilodaltons.