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-33P]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 Km 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/cm2), 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.