Molecular mechanisms and phosphorylation of receptor kinase in early Brassinosteroids signal transduction
Xiaofeng Wang^1,2, Man-Ho Oh³, Kevin Blackburn⁴, Jia Li⁵, Michael B Goshe⁴, Steven C Huber³, Steven D Clouse ²

¹College of Horticulture, Northwest A&F University.
²Department of Horticultural Science, North Carolina State University, Raleigh, NC 27695, USA.
³USDA/ARS, University of Illinois, Urbana, IL 61801, USA.
⁴Department of Molecular and Structural Biochemistry, North Carolina State University, Raleigh, NC 27695, USA.
⁵Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019, USA.
Corresponding  Author：Tel: (+86) 15332398998; E-mail: wangxff99@yahoo.com ; Tel: (+01) 919-5155360; E-mail: steve_clouse@ncsu.edu

Abstract
Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) for hormone perception and signal transduction. Using immunoprecipitation of epitope-tagged BRI1 and BAK1 from Arabidopsis plants followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS), we have identified multiple in vivo phosphorylation sites (Ser/Thr/Tyr) on BRI1 and BAK1. Examination of the in planta function of these sites by site-directed mutational analysis has revealed many of these sites are not only critical for the kinases function and optimal BR signaling, but also having specificity for signaling. We examined the association and phosphorylation of BRI1 and BAK1 in multiple genetic backgrounds to elucidate the detailed mechanisms of phosphorylation and dimerization in response to BR. a sequential transphosphorylation model  established in this study should enrich our knowledge of early events in BR signal transduction as well as found general modes of action for the large family of plant receptor-like kinases.

Key words: Brassinosteroid; Receptor kinase; Phosphorylation; Signal transduction; BRI1; BAK1