|Kim, Hyoung Seok|
Submitted to: Frontiers in Plant Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/10/2012
Publication Date: 11/30/2012
Citation: Wu, X., Oh, M., Kim, H., Schwartz, D., Imai, B., Yau, P., Clouse, S.D., Huber, S.C. 2012. Transphosphorylation of E. coli proteins during production of recombinant protein kinases provides a robust system to characterize kinase specificity. Frontiers in Plant Science. 3:262. Interpretive Summary: Protein kinases catalyze the covalent addition of a phosphate molecule to specific residues in cellular proteins, thereby potentially altering their activity, stability, interaction with other proteins, or localization within the cell. This process is referred to as protein phosphorylation and is known to regulate most aspects of growth and development and response to the environment in both plants and animals. Therefore, protein kinases must be highly specific in terms of not only which proteins are phosphorylated but also which specific amino acids are phosphorylated. For many important plant protein kinases we lack fundamental information about these aspects of specificity. In the present study we report that when plant protein kinases are produced in bacterial cells, many bacterial proteins become phosphorylated and identifying specific phosphosites can uncover new insights about the intrinsic specificity of the protein kinase. Using this system, we determined that the receptor kinase involved in plant steroid hormone signaling, referred to as BRI1, targets serine and threonine residues surrounded by basic amino acids (lysine and arginine) at specific positions. Importantly, this experimental approach to characterize the intrinsic specificity of a protein kinase should have broad application to a wide array of plant and animal protein kinases.
Technical 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 during production. Using enrichment approaches followed by LC-MS/MS, phosphosites were identified allowing motifs associated with auto- and trans-phosphorylation 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 CDPKß) 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.