The primary amino acid sequence specificity of TPL-2.
The peptide library comprised 198 individual biotinylated peptide mixtures. Each peptide contained a central phosphoracceptor Ser or Thr, flanked by degenerate positions, comprising an equimolar mixture of the 17 amino acids, excluding Cys, Ser and Thr. In each peptide, one position was fixed (fixed residue) with one of the 20 naturally occurring unmodified amino acids, phosphor-Thr (pT) or phosphor-Tyr (pY). Peptides were incubated with 30 nM recombinant TPL-2/NF-κB1 p105/ABIN-2 complex at a final substrate concentration of 50 µM at 30°C for 1 h. Assays were performed in TPL-2 kinase buffer plus 10 µM ATP and 3 nCi/µL [γ-32P]ATP in the absence (A) or presence (B) of C34 TPL-2 inhibitor (10 µM). Following incubation, reactions were transferred onto SAM2 membranes, which were then washed extensively. Incorporation of 32P into peptides was quantified by phosphorimaging.