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Protein kinase specificity is of fundamental importance to pathway regulation and signal transduction. Here, we report a convenient system to monitor the activity and specificity of recombinant protein kinases expressed in  E. coli . We apply this to the study of the cytoplasmic domain of the plant receptor kinase BRASSINOSTEROID-INSENSITIVE 1 (BRI1), which functions in brassinosteroid (BR) signaling. Recombinant BRI1 is catalytically active and both autophosphorylates and transphosphorylates  E. coli  proteins  in situ . Using enrichment approaches followed by LC-MS/MS, phosphosites were identified allowing motifs associated with auto- and transphosphorylation to be characterized. Four lines of evidence suggest that transphosphorylation of  E. coli  proteins by BRI1 is specific and therefore provides meaningful results: (1) phosphorylation is not correlated with bacterial protein abundance; (2) phosphosite stoichiometry, estimated by spectral counting, is also not related to protein abundance; (3) a transphosphorylation motif emerged with strong preference for basic residues both N- and C-terminal to the phosphosites; and (4) other protein kinases (BAK1, PEPR1, FLS2, and CDPKβ) phosphorylated a distinct set of  E. coli  proteins and phosphosites. The  E. coli  transphosphorylation assay can be applied broadly to protein kinases and provides a convenient and powerful system to elucidate kinase specificity.

Transphosphorylation of E. coli Proteins during Production of Recombinant Protein Kinases Provides a Robust System to Characterize Kinase Specificity