title: Transphosphorylation of E. coli Proteins during Production of Recombinant Protein Kinases Provides a Robust System to Characterize Kinase Specificity.
abstract: 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.
authors.affiliation: Department of Plant Biology, University of Illinois Urbana, IL, USA.
authors.forename: Xia
authors.initials: X
authors.lastname: Wu
authors.forename: Man-Ho
authors.initials: MH
authors.lastname: Oh
authors.forename: Hyoung Seok
authors.initials: HS
authors.lastname: Kim
authors.forename: Daniel
authors.initials: D
authors.lastname: Schwartz
authors.forename: Brian S
authors.initials: BS
authors.lastname: Imai
authors.forename: Peter M
authors.initials: PM
authors.lastname: Yau
authors.forename: Steven D
authors.initials: SD
authors.lastname: Clouse
authors.forename: Steven C
authors.initials: SC
authors.lastname: Huber