Chemical phosphoproteomic approaches reveal substrate specificity of protein phosphatases-1 and -2

The phosphatases PP1 and PP2A are responsible for the majority of dephosphorylation reactions on phosphoserine (pS) and –threonine (pT), and are involved in basically all cellular processes and many related diseases. They are thought to have no appreciable intrinsic substrate specificity, but to gain specificity only in their holoenzyme forms. Through the development of a peptide library approach and application of a complementary phosphoproteomics assay, we uncover that PP1 and PP2A show intrinsic specificity towards pT over pS, as well as toward the sequence context surrounding the phospho-site. Our substrate specificity data reveal that PP1 is a key regulator of the 14-3-3 protein binding motif, which enabled us to establish an unknown role for PP1 as a regulator of the GRB-associated-binding-protein 2 downstream (Gab2)/14-3-3 complex, exemplifying predictive potential of the data. Thus, this data should serve as a rich resource for (de)phosphorylation studies covering multiple cellular processes and phosphoproteins.