Poster: Signaling, cell-to-cell
Abs #
421: Functional Analysis of BRI1 Receptor Kinase Phosphorylation Sites Involved in Brassinosteroid Signaling
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Presenter: |
Clouse, Steven D., steve_clouse@ncsu.edu |
Authors | Wang, Xiaofeng (A) Goshe, Michael B. (A) Phinney, Brett S. (B) Kuchar, Jason (B) Li, Jia (C) Asami, Tadao (D) Yoshida, Shigeo (D) Huber, Steven C. (E) Clouse, Steven D. (A) | | Affiliations: |
(A): North Carolina State University
(B): Michigan State University
(C): University of Oklahoma
(D): RIKEN, Japan
(E): University of Illinois
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Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active Brassinosteroid Insensitive 1 (BRI1) receptor serine/threonine kinase for hormone perception and signal transduction. A common mechanistic property associated with many receptor kinases is ligand-dependent homo or heterodimerization followed by autophosphorylation and activation of the intracellular kinase domain. To thoroughly characterize plant receptor kinase function, it is essential to identify specific autophosphorylation sites and their functional significance. We previously identified twelve sites of in vitro autophosphorylation in the BRI1 kinase domain (Oh et al., Plant Physiology 124:751-766, 2000). We now show using immunoprecipitation of epitope-tagged BRI1 followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS), that BRI1 is phosphorylated on multiple Ser and Thr residues in vivo. The function of each identified site in BRI1 is being assessed by in vitro mutagenesis followed by monitoring the ability of the mutated gene to rescue a weak bri1 allele in planta. Quantitative changes in phosphorylation status of specific in vivo sites under different endogenous and exogenous BR levels are being examined by isotope dilution techniques and isotope-coded affinity tagging followed by LC-MS/MS analysis. The activation of many protein kinases occurs by autophosphorylation of one to three residues within the activation loop of subdomain VIII, which is thought to allow substrate access to the catalytic site in subdomain VIb. We have now confirmed that BRI1 autophosphorylates at three residues within the activation loop in vivo and thus may have an activation mechanism similar to that observed in many animal kinases.
