TBK1 has an absolute requirement for a hydrophobic residue at the 1 146368-11-8 position relative to the phosphorylation site. TBK1 also displays a strong preference for phenylalanine or tyrosine at the -2 position, and a minor preference for bulky hydrophobic residues at the 3 position. To confirm this phosphorylation motif, an optimal peptide was generated and was efficiently phosphorylated by TBK1 in vitro. In contrast, peptides in which the 1 leucine or -2 tyrosine are changed to alanine were no longer efficiently phosphorylated by TBK1. TBK1 is highly homologous to the related kinase IKKe, and also shares significant homology with the canonical IKK family member IKKb. The substrate specificities of IKKe, IKKa, and IKKb have also recently been determined using the PSPL technology. Not surprisingly, the phosphorylation motif for TBK1 is identical to that of IKKe. Interestingly, while both the noncanonical and canonical IKKs display preferences for hydrophobic residues at the 1 position and aromatic residues at the -2 position, the optimal phosphorylation motifs for these kinases differ at other positions. For example, while TBK1 prefers large aliphatic residues at the 3 position, IKKa and IKKb prefer acidic residues at 3. In addition, the canonical IKKs display a strong preference for phosphorylated residues at the -4 and -5 positions, suggesting that these kinases can be primed by upstream phosphorylation events. However, no evidence of priming phosphorylation is observed for TBK1. MK-0457 manufacturer Consistent with these data, a peptide substrate corresponding to the well-established IKKa/b phosphorylation sites on IkBa was phosphorylated by TBK1 much less efficiently than TBK1-Tide. As the PSPL assays employ degenerate peptide mixtures, it was important to confirm differences in the substrate specificities among the IKKs using individual peptide substrates. To this end, the predicted optimal IKKb substrate peptide was generated. This peptide contains the 1 leucine and -2 tyrosine which are preferred by all IKK family members, but differs from TBK1-Tide at secondary positions. Importantly, this peptide contains a phosphothreonine residue at -4. We also generated a similar peptide which is identical to IKKb-Tide-pT except that it contains an alanine at the -4 position. All
