Identification of BZR1-interacting proteins as potential components of the brassinosteroid signaling pathway in Arabidopsis through tandem affinity purification

Mol Cell Proteomics. 2013 Dec;12(12):3653-65. doi: 10.1074/mcp.M113.029256. Epub 2013 Sep 9.

Abstract

Brassinosteroids (BRs) are essential phytohormones for plant growth and development. BRs are perceived by the cell surface receptor kinase BRI1, and downstream signal transduction through multiple components leads to activation of the transcription factors BZR1 and BZR2/BES1. BZR1 activity is highly controlled by BR through reversible phosphorylation, protein degradation, and nucleocytoplasmic shuttling. To further understand the molecular function of BZR1, we performed tandem affinity purification of the BZR1 complex and identified BZR1-associated proteins using mass spectrometry. These BZR1-associated proteins included several known BR signaling components, such as BIN2, BSK1, 14-3-3λ, and PP2A, as well as a large number of proteins with previously unknown functions in BR signal transduction, including the kinases MKK5 and MAPK4, histone deacetylase 19, cysteine proteinase inhibitor 6, a DEAD-box RNA helicase, cysteine endopeptidases RD21A and RD21B, calmodulin-binding transcription activator 5, ubiquitin protease 12, cyclophilin 59, and phospholipid-binding protein synaptotagmin A. Their interactions with BZR1 were confirmed by in vivo and in vitro assays. Furthermore, MKK5 was found to phosphorylate BZR1 in vitro. This study demonstrates an effective method for purifying proteins associated with low-abundance transcription factors, and identifies new BZR1-interacting proteins with potentially important roles in BR response.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / drug effects*
  • Arabidopsis / genetics
  • Arabidopsis / metabolism
  • Arabidopsis Proteins / genetics
  • Arabidopsis Proteins / metabolism*
  • Brassinosteroids / pharmacology*
  • Chromatography, Affinity
  • DNA-Binding Proteins
  • Gene Expression Regulation, Plant*
  • Mass Spectrometry
  • Mitogen-Activated Protein Kinase Kinases / genetics
  • Mitogen-Activated Protein Kinase Kinases / metabolism
  • Molecular Sequence Annotation
  • Nuclear Proteins / genetics
  • Nuclear Proteins / metabolism*
  • Phosphorylation
  • Plant Growth Regulators / pharmacology*
  • Protein Binding
  • Protein Interaction Mapping
  • Protein Isoforms / genetics
  • Protein Isoforms / metabolism
  • Protein Kinases / genetics
  • Protein Kinases / metabolism
  • Protein Transport
  • Proteolysis
  • Signal Transduction*

Substances

  • Arabidopsis Proteins
  • BES1 protein, Arabidopsis
  • BZR1 protein, Arabidopsis
  • Brassinosteroids
  • DNA-Binding Proteins
  • Nuclear Proteins
  • Plant Growth Regulators
  • Protein Isoforms
  • Protein Kinases
  • BRI1 protein, Arabidopsis
  • MKK5 protein, Arabidopsis
  • Mitogen-Activated Protein Kinase Kinases