Brassinosteroids (BRs) bind to the extracellular domain of the receptor kinase BRI1 to activate a signal transduction cascade that regulates nuclear gene expression and plant development. Many components of the BR signaling pathway have been identified and studied in detail. However, the substrate of BRI1 kinase that transduces the signal to downstream components remains unknown. Proteomic studies of plasma membrane proteins lead to the identification of three homologous BR-signaling kinases (BSK1, BSK2, and BSK3). The BSKs are phosphorylated by BRI1 in vitro and interact with BRI1 in vivo. Genetic and transgenic studies demonstrate that the BSKs represent a small family of kinases that activate BR signaling downstream of BRI1. These results demonstrate that BSKs are the substrates of BRI1 kinase that activate downstream BR signal transduction.

Brassinosteroids trigger a receptor kinase-mediated signalling pathway to modulate plant development through the dephosphorylation of the BZR transcription factors, which are normally kept inactive by the kinase BIN2. The phosphatase PP2A is now found to be responsible for the dephosphorylation of BZR to trigger the signalling cascade. When brassinosteroid levels are low, the GSK3-like kinase BIN2 phosphorylates and inactivates the BZR1 transcription factor to inhibit growth in plants. Brassinosteroid promotes growth by inducing dephosphorylation of BZR1, but the phosphatase that dephosphorylates BZR1 has remained unknown. Here, using tandem affinity purification, we identified protein phosphatase 2A (PP2A) as a BZR1-interacting protein. Genetic analyses demonstrated a positive role for PP2A in brassinosteroid signalling and BZR1 dephosphorylation. Members of the B’ regulatory subunits of PP2A directly interact with BZR1’s putative PEST domain containing the site of the bzr1-1D mutation. Interaction with and dephosphorylation by PP2A are enhanced by the bzr1-1D mutation, reduced by two intragenic bzr1-1D suppressor mutations, and abolished by deletion of the PEST domain. This study reveals a crucial function for PP2A in dephosphorylating and activating BZR1 and completes the set of core components of the brassinosteroid-signalling cascade from cell surface receptor kinase to gene regulation in the nucleus.