Submitted to: Plant Physiology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 5/16/2013
Publication Date: 9/1/2013
Citation: Bajwa, V.S., Wang, X., Blackburn, R.K., Goshe, M.B., Mitra, S.K., Williams, E.L., Bishop, G.J., Krasnyanski, S., Allen, G., Huber, S.C., Clouse, S.D. 2013. Identification and functional analysis of tomato BRI1 and BAK1 receptor kinase phosphorylation sites. Plant Physiology. 163:30-42. Interpretive Summary: Brassinosteroids are plant hormones that are required for normal growth and development and stress tolerance. Consequently, there is a great deal of interest in understanding, at the molecular level, how brassinosteroids control these plant processes. Much of the molecular work to date has come from studies with the model plant, Arabidopsis. It is known that brassinosteroid signaling begins with two proteins, referred to as BRI1 and BAK1, which are receptor kinases embedded in the outer cell membrane and transmit signals from the outside of the cell to the nucleus resulting in changes in gene expression. The receptor kinases activated when the brassinosteroids they recognize are present. Once activated in this manner, the BRI1 protein kinase can phosphorylate (attach phosphate molecules to) other proteins and this way the signal is transmitted ultimately to the nucleus. As a foundation to study brassinosteroid signaling in tomato, the recombinant tomato BRI and BAK1 were analyzed in vitro for protein kinase and autophosphorylation activity. Numerous sites of autophosphorylation were identified by mass spectrometry and the functional significance of key phosphosites involved in receptor activation was confirmed in transgenic plants. In general the results with tomato are consistent with the model that the BRI1 and BAK1 receptor kinases activate by autophosphorylation and reciprocal transphosphorylation of one another. These findings provide the basis to study the role of specific phosphosites in tomato that have the potential to increase crop productivity.
Technical Abstract: Brassinosteroids (BRs) are essential plant hormones that are perceived at the cell surface by a membrane bound receptor kinase, BRASSINOSTEROID INSENSITIVE 1 (BRI1). BRI1 interacts with BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) to initiate a signal transduction pathway in which autophosphorylation and transphosphorylation of BRI1 and BAK1, as well as phosphorylation of multiple downstream substrates, plays a critical role. Detailed mechanisms of BR signaling have been examined in Arabidopsis thaliana but the role of BRI1 and BAK1 phosphorylation in crop plants is unknown. As a foundation for understanding the mechanism of BR signaling in tomato, we used liquid chromatography-tandem mass spectrometry to identify multiple in vitro phosphorylation sites of the tomato BRI1 and BAK1 cytoplasmic domains. Kinase assays showed that both tomato BRI1 and BAK1 are active in autophosphorylation, as well as transphosphorylation of each other and specific peptide substrates with a defined sequence motif. Site-directed mutagenesis revealed that the highly conserved kinase domain activation loop residue T1054 was essential for tomato BRI1 autophosphorylation and peptide substrate phosphorylation in vitro. Furthermore, analysis of transgenic lines expressing full-length tomato BRI1-Flag constructs in the weak tomato bri1 allele, curl3-abs1, demonstrated that T1054 is also essential for normal BRI1 signaling and tomato growth in planta. Finally, we cloned the tomato ortholog of Transforming Growth Factor Interacting Protein (TRIP1), which was previously shown to be a BRI1 interacting protein and kinase domain substrate in Arabidopsis, and found that tomato TRIP-1 is a substrate of both tomato BRI1 and BAK1 kinases in vitro.