title: Identification and functional analysis of tomato BRI1 and BAK1 receptor kinase phosphorylation sites.
abstract: Brassinosteroids (BRs) are plant hormones that are perceived at the cell surface by a membrane-bound receptor kinase, BRASSINOSTEROID INSENSITIVE1 (BRI1). BRI1 interacts with BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) to initiate a signal transduction pathway in which autophosphorylation and transphosphorylation of BRI1 and BAK1, as well as phosphorylation of multiple downstream substrates, play critical roles. Detailed mechanisms of BR signaling have been examined in Arabidopsis (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 (Solanum lycopersicum), 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 threonine-1054 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 threonine-1054 is also essential for normal BRI1 signaling and tomato growth in planta. Finally, we cloned the tomato ortholog of TGF-β Receptor Interacting Protein (TRIP1), which was previously shown to be a BRI1-interacting protein and kinase domain substrate in Arabidopsis, and found that tomato TRIP1 is a substrate of both tomato BRI1 and BAK1 kinases in vitro.
authors.affiliation: Department of Horticultural Science , North Carolina State University, Raleigh, North Carolina 27695, USA.
authors.forename: Vikramjit S
authors.initials: VS
authors.lastname: Bajwa
authors.forename: Xiaofeng
authors.initials: X
authors.lastname: Wang
authors.forename: R Kevin
authors.initials: RK
authors.lastname: Blackburn
authors.forename: Michael B
authors.initials: MB
authors.lastname: Goshe
authors.forename: Srijeet K
authors.initials: SK
authors.lastname: Mitra
authors.forename: Elisabeth L
authors.initials: EL
authors.lastname: Williams
authors.forename: Gerard J
authors.initials: GJ
authors.lastname: Bishop
authors.forename: Sergei
authors.initials: S
authors.lastname: Krasnyanski
authors.forename: George
authors.initials: G
authors.lastname: Allen
authors.forename: Steven C
authors.initials: SC
authors.lastname: Huber
authors.forename: Steven D
authors.initials: SD
authors.lastname: Clouse
mesh_terms: D001665:Binding Sites; D007700:Kinetics; D018551:Solanum lycopersicum / Q000201:enzymology*; D010766:Phosphorylation; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D010940:Plant Proteins; D017346:Protein Serine-Threonine Kinases