A Direct Docking Mechanism for a Plant GSK3-like Kinase to Phosphorylate Its Substrates

Peng Peng,Jun Zhao,Yongyou Zhu,Tadao Asami,Jianming Li

doi:10.1074/jbc.m110.142547

Abstract

Glycogen synthase kinase 3 (GSK3) is a highly conserved serine/threonine protein kinase that plays important roles in a variety of physiological and developmental processes in animals. It is well known that the GSK3 kinase-catalyzed protein phosphorylation often requires a stable kinase-substrate docking interaction, which is achieved mainly by two mechanisms as follows: priming phosphorylation of a substrate by a distinct kinase to create a docking phosphate group and scaffold protein-mediated protein complex formation. Brassinosteroid-INsensitive 2 (BIN2) is an Arabidopsis GSK3-like kinase that negatively regulates brassinosteroid (BR) signaling by phosphorylating BES1 (bri1 EMS suppressor 1) and BZR1 (brassinazole-resistant 1), two highly similar transcription factors critical for bringing about characteristic BR responses. However, little is known about the biochemical mechanism by which BIN2 phosphorylates its substrates. Here, we show that BIN2 interacts directly with BZR1 through a 12-amino acid BIN2-docking motif adjacent to the C terminus of BZR1. Interestingly, this 12-amino acid motif is sufficient to allow a Drosophila GSK3 substrate Armadillo to be phosphorylated by BIN2 in vitro. Deletion of this motif inhibits the phosphorylation and subsequent degradation of BZR1 in vivo, resulting in phenotypic suppression of a hypermorphic bin2 mutation and enhanced resistance to a BR biosynthesis inhibitor. We thus concluded that BIN2 utilizes a direct kinase-substrate docking mechanism to phosphorylate BZR1 and regulate its protein stability.

Highlights

Glycogen synthase kinase 3 (GSK3) kinases are a group of highly conserved serine/threonine kinases that play key regulatory roles in many signaling pathways [1, 2]
Previous experiments demonstrated that the corresponding Arg-Ala mutation in the mammalian GSK3␤ destroys the phosphate-binding pocket and prevents the phosphorylation of GSK3 substrates that require priming phosphorylation [6], while our earlier transgenic studies showed that expression of a bin2-1-mutated Brassinosteroid-INsensitive 2 (BIN2) genomic transgene was able to block BR signaling in Arabidopsis, generating strong dwarf transgenic plants resembling the homozygous bin2-1 mutant [10]
We demonstrated that an Arabidopsis GSK3like kinase, BIN2, requires a direct kinase-substrate docking interaction to phosphorylate its substrate in vitro and in vivo

Summary

Introduction

GSK3 kinases are a group of highly conserved serine/threonine kinases that play key regulatory roles in many signaling pathways [1, 2]. BIN2 contains a conserved primed phosphatebinding site [10], its phosphorylation activity of BZR1 and BES1 in vitro does not require a priming phosphorylation or a scaffold protein but involves a direct kinase-substrate interaction [20].

Results

Conclusion