Assay for I kappa B kinases using an in vivo biotinylated I kappa B protein substrate

I kappa B kinases (IKK)-1 and -2 are related kinases that are induced by st imuli such as TNF or IL-1 to phosphorylate serines 32 and 36 of I kappa B a lpha, the regulatory subunit of the transcription factor NF-kappa B. A proc edure for an IKK protein kinase assay is described that uses an in vivo bio tinylated I kappa B protein substrate, [gamma-P-33]ATP, and capture onto a streptavidin membrane. Residues 1-54 of the I kappa B alpha substrate were expressed as a fusion with glutathione S-transferase (GST) and a short (22 amino acid) biotinylation sequence that allowed modification during bacteri al expression. Using the streptavidin capture assay the phosphorylation act ivities of recombinant IKK-1 and -2 were characterized. The assay provided a convenient way to compare IKK protein and peptide substrate preferences; biotinylated GST-I kappa B alpha(1-54) was more readily phosphorylated by b oth IKK-1 and IKK-2 compared to biotinylated myelin basic protein or a 20-m er biotinylated peptide containing serines 32 and 36 of I kappa B alpha. IK K-1 had 83-fold less activity than IKK-8, and the IKK-1+2 complex had appro ximately a-fold more activity than IKK-2. IKK-1+2 and IKK-2 had similar K-m values for ATP and GST-biotin-I kappa B(1-54) and were similarly inhibited by staurosporine and two of its analogues K252a and K252b, suggesting that most of the I kappa B alpha kinase activity in the IKK-1+2 complex may be attributed to IKK-2. Several features of the assay including the broad line ar binding range of the streptavidin membranes for the protein substrate GS T-biotin-I kappa B(1-54) (1-4000 pmol of protein/cm(2)), the low background , and its capacity for both biotinylated peptides and proteins make it a us eful tool for quantitating IKK activity. These factors and the ease of expr essing in vivo biotinylated GST fusions will make this assay approach suita ble for a wide variety Of protein kinases. (C) 1999 Academic Press.