Journal
FRONTIERS IN PLANT SCIENCE
Volume 3, Issue -, Pages -Publisher
FRONTIERS MEDIA SA
DOI: 10.3389/fpls.2012.00262
Keywords
BRI1; BAKI; PEPR1; FLS2; CDPK; phosphorylation motif
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Funding
- National Science Foundation [IOS-1022177, MCB-0740211, MCB-1021363]
- US Department of Agriculture (USDA)-Agricultural Research Service (ARS)
- Division Of Integrative Organismal Systems [1022177] Funding Source: National Science Foundation
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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 CDPK beta) 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.
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