Proteomics shed light on the brassinosteroid signaling mechanisms

Abstract

Large numbers of receptor-like kinases (RLKs) play key roles in plant development and defense by perceiving extracellular signals. The mechanisms of ligand-induced kinase activation and downstream signal transduction have been studied for only a few RLK pathways, among which the brassinosteroid (BR) pathway is the best characterized. Recently, proteomics studies identified new components that bridge the last gap in the genetically defined BR-signaling pathway, establishing the first complete pathway from an RLK to transcription factors in plants. Furthermore, analyses of phosphorylation events, mostly by mass spectrometry, provided insights into the mechanistic details of receptor kinase activation and regulation of downstream components by phosphorylation. This review focuses on recent progress in understanding BR signal transduction made by proteomics studies.

Figure 1. In vivo and in vitro phosphorylated residues on BRI1 and BAK1 intracellular domains

JM, juxtamembrane domain; CT, C-terminal domain. Activation loop within the kinase domain is highlighted. Red letters indicate the phosphorylated residues that are critical for BRI1 or BAK1 kinase activity. S/T to A or Y to F substitution of these residues reduces or abolishes BRI1 or BAK1 kinase activity. Residues at which phosphorylation seems to inhibit the kinase activity are in blue color. BRI1 and BAK1 transphosphorylated residues are underlined.

Figure 2

BR-induced post-translational modifications of BAK1 (A) and BSKs (B) were detected as shifted spots in 2-D DIGE of plasma membrane fractions. Plasma membrane proteins of untreated control sample labeled with Cy3 (green) and BR-treated samples labeled with Cy5 (red) were separated in a 2-D DIGE gel. White arrows show spots that disappear (green spots) or and black arrows show spots that appear (red spots) after BR treatment. (A). An area of the 2-D DIGE gel showing BAK1 spots. (B) An gel area showing the two rows of spots that contain BSK1 and BSK2. From left to right are acidic to basic pH.

Figure 3

A diagram of the BR signal transduction pathway in Arabidopsis. The left side of the dashed line (A) shows the inactive pathway when BR level is low, and the right side (B) shows the pathway when activated by BR. Although there is evidence for the formation of heterotetramer between BRI1 and BAK1, for simplicity a simple heterodimer model is shown. Components are in blue color when inactive and red color when active. (A) When BR level is low, BRI1 is associated with BKI1, which inhibits BRI1, as well as BSK1, which is to be phosphorylated upon activation of BRI1. BAK1 and BSU1 remain inactive, whereas BIN2 phosphorylates BZR1 and BZR2/BES1, which inhibits DNA binding, promotes cytoplasmic localization through the 14-3-3 proteins, and accelerates degradation by the proteasome. (B) BR binding to the extracellular domain of BRI1 activates BRI1 through disassociation of BKI1 and oligomerization/transphosphorylation between BRI1 and BAK1. Activated BRI1 phosphorylates Ser230 of BSK1, which then interacts with and presumably activates BSU1. BSU1 dephosphorylates BIN2 at Tyr200 to inactivate BIN2 and stop phosphorylation of BZR1/2, leading to accumulation of unphosphorylated BZR1/2, likely with help of an unknown phosphatase (PPase) that dephosphorylates BZR1/2. Unphosphorylated BZR1/BZR2 accumulate in the nucleus, where they directly regulate BR-responsive gene expression and plant development.