Proteomics Studies of Brassinosteroid Signal Transduction Using Prefractionation and Two-dimensional DIGE

Abstract

Signal transduction involves posttranslational modifications and protein-protein interactions, which can be studied by proteomics. In Arabidopsis, the steroid hormone (brassinosteroid (BR)) binds to the extracellular domain of a receptor kinase (BRI1) to initiate a phosphorylation/ dephosphorylation cascade that controls gene expression and plant growth. Here we detected early BR signaling events and identified early response proteins using prefractionation and two-dimensional (2-D) DIGE. Proteomic changes induced rapidly by BR treatments were detected in phosphoprotein and plasma membrane (PM) fractions by 2-D DIGE but not in total protein extracts. LC-MS/MS analysis of gel spots identified 19 BR-regulated PM proteins and six proteins from phosphoprotein fractions. These include the BAK1 receptor kinase and BZR1 transcription factor of the BR signaling pathway. Both proteins showed spot shifts consistent with BR-regulated phosphorylation. In addition, in vivo phosphorylation sites were identified for BZR1, two tetratricopeptide repeat proteins, and a phosphoenolpyruvate carboxykinase (PCK1). Overexpression of a novel BR-induced PM protein (DREPP) partially suppressed the phenotypes of a BR-deficient mutant, demonstrating its important function in BR responses. Our study demonstrates that prefractionation coupled with 2-D DIGE is a powerful approach for studying signal transduction. Molecular & Cellular Proteomics 7:728 -738, 2008.Perception and response to extracellular signals are crucial for growth and survival of all organisms. Signals are transduced intracellularly through mechanisms that include posttranslational protein modification and protein-protein interaction. Detection of such signaling events using proteomics methods can identify proteins involved in signal transduction but is technically challenging because of the low abundance of signaling proteins. Brassinosteroids (BRs) 1 are plant hormones that play important roles in multiple plant developmental processes. Mutant plants with a defect in BR synthesis or signal transduction show a wide range of phenotypes including dwarfism, reduced fertility, light-grown morphology in the dark, and delayed senescence (1). BRs structurally resemble animal steroid hormones but act through a distinct signaling mechanism (2). Although animal steroid hormones are perceived by nuclear receptors, BRs are perceived by a cell surface receptor-like kinase named BRI1 (2, 3). BRs bind to the extracellular domain of BRI1 to activate its kinase and downstream BR signal transduction (4), which involves another receptor-like kinase named BAK1 (5, 6); a GSK3-like kinase, BIN2 (7); a phosphatase, BSU1 (8); and two transcription factors, BZR1 and BZR2 (also known as BES1) (9, 10). In the absence of BR, the inhibitory BIN2 kinase phosphorylates BZR1 and BZR2, and phosphorylation inhibits the function of BZR1 and BZR2 through multiple mechanisms, which include inhibition of DNA binding activity, degradation by the proteasome, and cytoplasmic retention by the 14-3-3 proteins that b...

Visualization of BRI1 and BAK1(SERK3) Membrane Receptor Heterooligomers during Brassinosteroid Signaling

“…This variation could be due to the presence of a mixture of phosphorylated and nonphosphorylated receptors after ligand application, each with different affinity and stability. The presence of multiple forms of SERK3 after ligand application was seen by Tang et al (2008a). These pitfalls of coimmunoprecipitation were also noted during an investigation of animal receptor signaling systems, and as a consequence, receptor recruitment models as originally proposed often resulted in revised models including preformed receptor oligomers (Springael et al, 2005;Wang and Norcross, 2008).…”

Identification of BZR1-interacting Proteins as Potential Components of the Brassinosteroid Signaling Pathway in Arabidopsis Through Tandem Affinity Purification

“…The extracted digests were vacuumdried and resuspended in 20 l of 1% formic acid in water. The digests were analyzed by nano-flow High-Performance Liquid Chromatography (HPLC)-electrospray tandem mass spectrometry (LC-MS/MS) as described (30,31). The separation was performed using a 75 mϫ150 mm C18 PepMap column (LC Packings, Sunnyvale, CA) in an Ultimate HPLC system linked with a FAMOS autosampler (LC Packings, San Francisco, CA).…”

A Na+/Ca2+ Exchanger-like Protein (AtNCL) Involved in Salt Stress in Arabidopsis

“…The images were processed using LSM 4.2 software (Carl Zeiss Micro Imaging, Inc.). Plant Membrane Fractionation and Western Blotting-Protein extracts were prepared from the wild-type or transgenic seedlings and separated into soluble and membrane fractions by ultracentrifugation as described previously (52). The fractions were then analyzed by Western blotting using anti-GFP serum as the primary antibody.…”