Proteomics studies of brassinosteroid signal transduction using prefractionation and two-dimensional DIGE.
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.
Tang
Wenqiang
W
Department of Plant Biology, Carnegie Institution of Washington, Stanford, California 94305, USA.
Deng
Zhiping
Z
Oses-Prieto
Juan A
JA
Suzuki
Nagi
N
Zhu
Shengwei
S
Zhang
Xin
X
Burlingame
Alma L
AL
Wang
Zhi-Yong
ZY
D017360:Arabidopsis / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D002776:Cholestanes / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005456:Fluorescent Dyes / Q000032:analysis; D008565:Membrane Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism; D010937:Plant Growth Regulators / Q000378:metabolism*; D010940:Plant Proteins / Q000032:analysis* / Q000302:isolation & purification / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction
D002776:Cholestanes; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D010750:Phosphoproteins; D010937:Plant Growth Regulators; D010940:Plant Proteins