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ARS Home » Midwest Area » Urbana, Illinois » Soybean/maize Germplasm, Pathology, and Genetics Research » Research » Publications at this Location » Publication #182294
Title: IDENTIFICATION AND FUNCTIONAL ANALYSIS OF IN VIVO PHOSPHORYLATION SITES OF THE ARABIDOPSIS BRASSINOSTEROID INSENSITIVE 1 RECEPTOR KINASE

Author
item WANG, X - NC STATE UNIVERSITY
item GOSHE, M - NC STATE UNIVERSITY
item SODERBLOM, E - NC STATE UNIVERSITY
item PHINNEY, B - MI STATE UNIVERSITY
item KUCHAR, J - MI STATE UNIVERSITY
item LI, J - UNIVERSITY OF OK
item ASAMI, T - RIKEN, JAPAN
item Huber, Steven
item CLOUSE, S - NC STATE UNIVERSITY

Submitted to: The Plant Cell
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/20/2005
Publication Date: 5/1/2005
Citation: Wang, X., Goshe, M.B., Soderblom, E.J., Phinney, B.S., Kuchar, J., Li, J., Asami, T., Huber, S.C., Clouse, S.D. 2005. Identification and functional analysis of in vivo phosphorylation sites of the arabidopsis brassinosteroid insensitive 1 receptor kinase. The Plant Cell. 17:1685-1703.

Interpretive Summary: Brassinosteroids (BRs) are important plant growth hormones that are required for many aspects of normal plant growth and development, including cell elongation, vascular differentiation, seed germination, and senescence. Current thinking is that BR perception occurs when it binds to a ‘receptor-like kinase’ known as BRI1 (Brassinosteroid-Insensitive 1), a protein in the plasma membrane that has an extracellular domain that interacts with the hormone and an intracellular protein kinase domain that transmits the hormone signal. The present study shows that in vivo, BR promotes the association of BRI1 with another receptor-like kinase known as BAK1 (BRI1-Associated Receptor Kinase-1). Moreover, when the two receptor-like kinases interact in the presence of BR, both kinase domains are phosphorylated on multiple threonine and serine residues. Eleven in vivo phosphorylation sites on BRI1 were identified and several of these sites were shown to be important for BR signal transduction. These results provide new information about the mechanism of receptor kinase action in plants. The Arabidopsis genome contains more than 600 receptor-like kinases that are thought to play major roles in signal transduction controlling a range of physiological processes in plants. The results with BRI1 provide a model that may help to understand plant receptor-like kinases in general.

Technical Abstract: Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID INSENSITIVE 1 (BRI1) and BRI1 ASSOCIATED RECEPTOR KINASE 1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent homo or heterodimerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in BR signaling share this mechanism, we used co-immunoprecipitation of epitope-tagged proteins to show that in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels and that phosphorylation of both BRI1 and BAK1 on Thr residues was BR-dependent. Immunoprecipitation of epitope-tagged BRI1 from Arabidopsis plants followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS), identified S838, S858, T872 and T880 in the juxtamembrane region, T982 in the kinase domain, and S1168 in carboxy terminal region as in vivo phosphorylation sites of BRI1. Ion-trap MS data also strongly suggested that an additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivo. We also identified four specific BAK1 autophosphorylation sites in vitro using LC/MS/MS. Site-directed mutagenesis of identified and predicted BRI1 phosphorylation sites revealed that the highly conserved activation loop residue T-1049 and either S-1044 or T-1045, were essential for kinase function in vitro and normal BRI1 signaling in planta. Mutations in the juxtamembrane or carboxy terminal regions had little effect on autophosphorylation but dramatically affect phosphorylation of a peptide substrate in vitro. Our findings are consistent with many aspects of the animal receptor kinase model in which ligand-dependent autophosphorylation of the activation loop generates a functional kinase, while phosphorylation of non-catalytic intracellular domains is required for recognition and/or phosphorylation of downstream substrates.