Testing an optimized peptide substrate for TPL-2/NF-κB1 p105/ABIN-2.
(A) The primary and secondary amino acid preferences for phosphorylation by the recombinant TPL-2/NF-κB1 p105/ABIN-2 complex are shown. (B) The sequences and scansite scores for the MKK1 activation loop and optimized TPL-2tide peptide substrates. (C) Time-course experiment comparing TPL-2/NF-κB1 p105/ABIN-2 complex phosphorylation of MKK1 and TPL-2tide peptides (50 µM final concentration). Assays were performed with 30 nM the recombinant TPL-2/NF-κB1 p105/ABIN-2 complex in TPL-2 kinase buffer plus 1 mM ATP and 0.02 µCi/µL [γ-32P]ATP. Peptides were transferred onto streptavidin-coated membranes, which were then extensively washed. Incorporation of 32P into peptides was quantified by phosphorimaging. Linear regression was fitted with GraFit version 7.0.3. Values are means ± SD for three replicate reactions. (D) Kinase assays as in (C) comparing TPL-2tide peptide phosphorylation by wild-type (WT) or kinase-inactive (D270A) TPL-2/NF-κB1 p105/ABIN-2 complex (15 min). (E and F) TPL-230–404 and TPL-2/NF-κB1 p105/ABIN-2 were titrated (1 : 1.66) in 384-well plates. A substrate solution containing ATP and TPL-2tide (E) or S5 peptide (F) was then added. The plates were analyzed using a Sciex API6500 (E) or API4000 (F) Triple Quad with RapidFire™ Technology. The graph shows total peak area counts for the MRM transition 953.6/904.3 Da (E; phosphorylated TPL-2tide peptide) and 612.9/120 Da (F; phosphorylated S5 peptide) for the indicated enzyme at increasing concentrations. Linear regression was fitted using GraFit software. In E and F, representative graphs from one experiment are shown, including standard deviation of triplicate assays.