Fig. 4.

IKKα phosphorylates NIK on Ser⁸⁰⁹, Ser⁸¹², and Ser⁸¹⁵. (A) Truncation analysis implicates amino acid residues 742 to 817 of NIK as encompassing the region that contains IKKα target residues. HEK 293T cells were cotransfected with plasmids encoding FLAG-tagged truncations of NIK spanning the C-terminal 200 residues (c200, residues 742 to 942) or 125 residues (c125, residues 817 to 942) with or without plasmid encoding IKKα. In all cases, NIK was included to stimulate the kinase activity of IKKα. (B) MS/MS spectrum of 981.1 (3+). Matching band y-ions, and one immonium ion have been marked. “P” indicates peaks resulting from neutral loss of phosphoric acid. The backbone fragmentation necessary to generate the b- (“\”) and y-ions (“/”) have been marked in the peptide sequence spanning residues 810 to 827 of NIK. (C) Ser→Ala mutations at residues Ser⁸⁰⁹, Ser⁸¹², or Ser⁸¹⁵ lead to enhanced stability of NIK. NIK^−/− fibroblasts were retrovirally reconstituted with pBABE-empty (−), Ser→Ala point mutations of NIK at given residues (8××), or combined mutation of all six serines (all). (D) Diagram of NIK indicating its TRAF3-binding motif (TRAF3-BM), kinase domain (KD), and expansion of the area harboring IKKα target serine residues (highlighted). (E) Combined Ser→Ala mutation of NIK at residues Ser⁸⁰⁹, Ser⁸¹², and Ser⁸¹⁵ generates a mutant protein, henceforth referred to as NIK-3SA, that interacts with IKKα. HEK 293T cells were transfected with plasmid encoding IKKα in combination with either empty vector (−), FLAG-tagged wild-type NIK (NIK-WT), NIK-aly, or NIK-3SA. Lysates subjected to immunoprecipitation with antibody against FLAG were analyzed by Western blotting for IKKα. (F) The NIK-3SA mutant retains kinase activity as assessed by transfecting HEK 293T cells with a 2xκB-luciferase reporter, CMV-βgal, and either pCDNA3-emtpy (−), NIK-WT (WT), the NIK KK430/431AA kinase-defective mutant (KD), or NIK-3SA (3SA). Mean raw light units (RLU) normalized to lacZ activity are shown for triplicate samples. Abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; G, Gly; H, His; K, Lys; L, Leu; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; and W, Trp.