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.
Highlights
Protein phosphorylation on serine, threonine, and tyrosine residues is catalyzed by protein kinases that transfer the phosphate moiety from ATP to the modified residues (Hanks and Hunter, 1995)
In this study, we demonstrate that monitoring the transphosphorylation of E. coli proteins during production of recombinant protein kinases provides a simple and robust system to characterize kinase activity and more importantly, specificity
We reported for the first time that expression of the cytoplasmic domain of BRASSINOSTEROID-INSENSITIVE 1 (BRI1) in E. coli resulted in the transphosphorylation of numerous bacterial proteins, and 77 phosphosites were identified by LC-MS/MS analysis (Oh et al, 2012)
Summary
Threonine, and tyrosine residues is catalyzed by protein kinases that transfer the phosphate moiety from ATP to the modified residues (Hanks and Hunter, 1995). There are more than 500 kinases (Manning et al, 2002) that phosphorylate thousands of identified human phosphosites (Hornbeck et al, 2012). Such a large-scale phosphorylation network is present in plants. In Arabidopsis, a model plant with a relatively small genome, almost 1000 protein kinases have been identified (Chevalier and Walker, 2005). To elucidate the complex phosphorylation regulatory networks in eukaryotes, we need to better understand the specificity of the kinases. Advances in mass spectrometry have allowed identification of numerous phosphosites, but in most cases it is not clear which kinase(s) is responsible for the phosphorylation. Analysis of kinase specificity remains an important area of study
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