Enhanced Access to the Human Phosphoproteome with Genetically Encoded Phosphothreonine

Protein phosphorylation is a ubiquitous post-translational modification that governs signaling cascades and protein-protein interactions. Orthogonal translation systems repurpose evolutionarily divergent aminoacyl-tRNA synthetase and tRNA pairs for the co-translational insertion of a modified amino acid. Subsequent advancements over the last decade have enabled the insertion of phospho-amino acids, bypassing a priori knowledge requirements of upstream kinases for the study of phosphoproteins. Here we optimized a pThrOTS and corresponding E. coli strain for pThr protein production. We then produced a peptidome library containing ~57,000 known threonine/phosphothreonine phosphosites using oligonucleotide library synthesis. We were able to identify approximately ~20% of the peptides encoded by the pThr library, and ~44% of the peptides encoded by the Thr library with mass spectrometry. Robust, genetically encoded phosphothreonine revealed a new activation and inhibition mechanism for the kinase CHK2. Proteome-wide surveys of interactions between active CHK2 and our peptidome library identified novel substrates and motif elements. Finally, we developed a novel technique, Hi-P+, for directly linking kinase substrate discovery to phospho-binding domain recognition, unveiling multi-level interaction networks with phosphosite resolution. This new methodology enables kinase-specific, proteome-wide surveys of multiple phosphorylation-dependent protein-protein interactions.