Functional investigation of the role of a plant GSK3 kinase (BIN2) in brassinosteroid signaling pathway.

Abstract

GSK3 is a highly conserved kinase that negatively regulates many cellular processes. BIN2 is an <italic>Arabidopsis</italic> GSK3-like kinase that negatively regulates brassinosteroid (BR) signaling. It was proposed that BR signals, perceived by a membrane BR receptor complex containing BRI1 and BAK1, inactivate BIN2 to relieve its inhibitory effect on downstream signaling components. Using a yeast two-hybrid approach, we discovered a potential BIN2 substrate BES1. BES1 and its closest homolog, BZR1, specifically interact with BIN2 in yeast and can be phosphorylated by BIN2 <italic>in vitro</italic>. A <italic> bes1</italic> mutant containing the P233L mutation was identified as semidominant suppressor of <italic>bri1</italic> mutations. Over-expression of the wild type <italic>BZR1</italic> gene partially complemented <italic>bin2</italic>/+ mutant, whereas overexpression of a mutated <italic>BZR1</italic> gene containing the corresponding P234L mutation rescued <italic>bin2</italic>/+ as well as a weak <italic>bri1</italic> mutation. Both BES1 and BZR1 proteins were mainly localized in the nucleus. We propose that BES1/BZR1 are two nuclear components of BR signaling that are negatively regulated by BIN2 through a phosphorylation-initiated process. Animal GSK3 kinases phosphorylate their substrates through two docking mechanisms involving priming phosphorylation and/or scaffold proteins. In this study, I have shown that BIN2 interacts directly with BZR1 through a 12-amino acid BIN2-docking motif near the C-terminus of BZR1. Deletion of this motif inhibits the phosphorylation and subsequent degradation of BZR1 <italic> in vivo</italic>, resulting in phenotypic suppression of a hypermorphic <italic> bin2</italic> mutation and enhanced hypocotyl growth. We conclude that BIN2 uses a direct kinase/substrate docking mechanism to phosphorylate and regulate the protein stability of BZR1. Phosphorylation, protein complex formation, and subcellular localization play a role in controlling GKS3 actions. My study revealed that BR induces a rapid turnover of BIN2 protein. The steady state of wild type BIN2 is very low in plants, however, treatment of <italic>BIN2:GFP</italic> transgenic plants with brassinazole (Brz), a BR biosynthetic inhibitor, stabilizes the BIN2 kinase, which is rapidly degradated upon BL treatment. Such a BR-induced degradation is BR specific and is mediated through BRI1. We propose that the BR-mediated protein degradation of BIN2 constitutes a critical step in the plant steroid signaling.