To map the phosphorylation site systematically, we decided for a peptide array that displays 73 peptides containing all serine and threonine residues of DDX3X (Supplementary Figure 5).
Incubation of the peptide array with purified TBK1 produced a number of phosphorylation signals (Figure 7A). The TBK1 control peptide was phosphorylated by TBK1, suggesting that the activation loop is indeed an autophosphorylation site.
Analysing the remaining hits in the peptide array, we identified 11 potential phosphorylation sites in 9 DDX3X-derived peptides (Figure 7A). Four of these target sites (S181, S183, S240 and S269) were found in the DEAD domain that contains the ATPase activity of DDX3X. The seven remaining sites were scattered throughout the helicase domain (S429, T438, S442, S456, S520, T542 and S543). We used the information gathered in the peptide array to derive a TBK1 consensus phosphorylation site (Figure 7B). Strikingly, TBK1 showed a strong preference for serine over threonine. In fact, none of the 12 peptides displayed on the array that contain exclusively threonines was phosphorylated.
Mapping of the TBK1 phosphorylation site in DDX3X. (A) Peptides derived from DDX3X or control peptides derived from TBK1 or IRF3 along with alanine mutants were incubated with TBK1 in the presence of [γ-32P]ATP. Each array contained a total of 82 peptides spotted in triplicate. (B) Phosphorylation sites obtained from the peptide array (see also text) were used to build a TBK1 phosphorylation consensus sequence.