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-b 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.Brassinosteroids (BRs) are regulators of plant growth and development that signal through the membranebound receptor kinase BRASSINOSTEROID INSEN-STIVE1 (BRI1) and its coreceptor BRI1-ASSOCIATED

An autophosphorylation site database for leucine‐rich repeat receptor‐like kinases in Arabidopsis thaliana

“…The NTE residues T872 and T880 are both autophosphorylated in Arabidopsis BRI1, and sequence alignment shows that more than 75% of RD‐type Arabidopsis LRR RLKs have a Ser or Thr residue at positions equivalent to both of these (Wang et al ., ). Functional studies in both Arabidopsis and tomato suggest that phosphorylation of T872 (or the equivalent T877 in tomato BRI1) exerts a negative regulatory effect on kinase function since a T872A mutant had increased autophosphorylation activity and dramatically enhanced phosphorylation of a peptide substrate (Wang et al ., ; Bajwa et al ., ). Moreover, structural studies suggest an important role for unphosphorylated T872 in maintaining proper three‐dimensional conformation of the BRI1 cytoplasmic domain (Bojar et al ., ).…”

“…The P + 1 loop residue corresponding to T1049 in Arabidopsis BRI1 and T455 in BAK1, almost invariantly contains a Ser/Thr residue in 100 of the most closely related Arabidopsis RLKs (Wang et al ., ). This conserved residue is also present in tomato BRI1 (T1054) and BAK1 (T459) (Bajwa et al ., ). Our previous functional analyses show that Arabidopsis BRI1 T1049 and tomato BRI1 T1054 are essential for both autophosphorylation and peptide substrate phosphorylation in vitro and for normal BRI1 signaling and plant development in vivo (Wang et al ., ; Bajwa et al ., ).…”

“…This conserved residue is also present in tomato BRI1 (T1054) and BAK1 (T459) (Bajwa et al ., ). Our previous functional analyses show that Arabidopsis BRI1 T1049 and tomato BRI1 T1054 are essential for both autophosphorylation and peptide substrate phosphorylation in vitro and for normal BRI1 signaling and plant development in vivo (Wang et al ., ; Bajwa et al ., ). Furthermore, phosphorylation at Arabidopsis BAK1 T455 is also essential for BAK1 kinase function and BR signaling in vivo (Wang et al ., ).…”

“…Besides Arabidopsis BRI1 and BAK1, targeted studies of other LRR RLKs in several species have resulted in the identification of specific phosphorylation sites within LRR RLK cytoplasmic domains, including Xa21 in rice (Xu et al ., ), FLS2 and NIK1 in Arabidopsis (Santos et al ., ; Cao et al ., ), NARK in soybean (Miyahara et al ., ), symbiosis receptor kinase in Lotus japonicus (Yoshida and Parniske, ), and BRI1 and BAK1 in tomato (Bajwa et al ., ). Moreover, global phosphoproteomic studies have revealed numerous in vivo phosphorylation sites for a number of LRR RLKs, providing a valuable resource for comparative analysis (Peck, ; Durek et al ., ; Yao et al ., ; Cheng et al ., ).…”

The growth–defense pivot: crisis management in plants mediated by LRR-RK surface receptors

“…Upon treatment of seedlings with brassinolide (BL), the most potent BR, BRI1 phosphorylates the negative regulator BRASSINOSTEROID KINASE INHIBITOR 1 (BKI1) on tyrosine 211 causing its displacement from the membrane to the cytosol where it is inactive [40]. The LRR-RK co-receptor BRI1-ASSOCIATED KINASE 1 (BAK1) can then freely associate with BRI1 to form a complex that elevates the signaling output of the pathway through a series of reciprocal transphosphorylation events [41]. In parallel, activated BRI1 phosphorylates the serine/threonine kinase BR SIGNALING KINASE 1 (BSK1) [42, 43].…”