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Assay for IκB Kinases Using an in Vivo Biotinylated IκB Protein Substrate

Abstract IκB kinases (IKK)-1 and -2 are related kinases that are induced by stimuli such as TNF or IL-1 to phosphorylate serines 32 and 36 of IκBα, the regulatory subunit of the transcription factor NF-κB. A procedure for an IKK protein kinase assay is described that uses an in vivo biotinylated IκB protein substrate, [γ- 33 P]ATP, and capture onto a streptavidin membrane. Residues 1–54 of the IκBα substrate were expressed as a fusion with glutathione S -transferase (GST) and a short (22 amino acid) biotinylation sequence that allowed modification during bacterial expression. Using the streptavidin capture assay the phosphorylation activities of recombinant IKK-1 and -2 were characterized. The assay provided a convenient way to compare IKK protein and peptide substrate preferences; biotinylated GST–IκBα(1–54) was more readily phosphorylated by both IKK-1 and IKK-2 compared to biotinylated myelin basic protein or a 20-mer biotinylated peptide containing serines 32 and 36 of IκBα. IKK-1 had 83-fold less activity than IKK-2, and the IKK-1+2 complex had approximately 2-fold more activity than IKK-2. IKK-1+2 and IKK-2 had similar K m values for ATP and GST–biotin–IκB(1–54) and were similarly inhibited by staurosporine and two of its analogues K252a and K252b, suggesting that most of the IκBα kinase activity in the IKK-1+2 complex may be attributed to IKK-2. Several features of the assay including the broad linear binding range of the streptavidin membranes for the protein substrate GST–biotin–IκB(1–54) (1–4000 pmol of protein/cm 2 ), the low background, and its capacity for both biotinylated peptides and proteins make it a useful tool for quantitating IKK activity. These factors and the ease of expressing in vivo biotinylated GST fusions will make this assay approach suitable for a wide variety of protein kinases.