Functional analysis of the BRI1 receptor kinase by Thr-for-Ser substitution in a regulatory autophosphorylation site

Authors: OH, MAN-HO - Chungnam National University; BENDER, KYLE - University Of Illinois; Kim, Sang Yeol; WU, XIA - University Of Washington; SUELKI, LEE - Chungnam National University; ZIELINSKI, RAYMOND - University Of Illinois; CLOUSE, STEVEN - North Carolina State University; Huber, Steven

Submitted to: Frontiers in Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/9/2015
Publication Date: 7/10/2015
Citation: Oh, M., Bender, K.W., Kim, S., Wu, X., Suelki, L., Zielinski, R.E., Clouse, S.D., Huber, S.C. 2015. Functional analysis of the BRI1 receptor kinase by Thr-for-Ser substitution in a regulatory autophosphorylation site. Frontiers in Plant Physiology. 6:562.

Interpretive Summary: Plants have a large family of receptor kinases that control a number of important processes ranging from growth and development to responses to the environment and pathogens. Hence, understanding the molecular details of how receptor kinases function is not only of fundamental interest but is also expected to have implications for crop improvement. The receptor kinases are proteins that are inserted through the plasma membrane, with the extracellular portion often specialized to bind some type of special molecule, and with an intracellular portion that contains a protein kinase domain. Activated of the receptor kinase occurs in response to the targeted ligand and often involves phosphorylation of numerous serine, threonine and (in some cases) tyrosine residues via a reaction catalyzed by the protein kinase itself referred to as autophosphorylation. The function of specific sites of autophosphorylation is usually studied by directed mutagenesis of the protein to substitute the phosphorylated amino acid with a residue that is similar but cannot be phosphorylated. Alternatively, the residue can be substituted with an acidic amino acid that may mimic the phosphorylated form. While useful these approaches often do not work because of an overriding effect that is not related to phosphorylation at the site. Consequently, in the present study a more subtle modification was tested that involved substituting a threonine residue for a known serine phosphorylation site in the BRI1 receptor kinase that is required for plant growth. Surprisingly, this subtle change, referred to as the S891T directed mutation, had strong functional impacts on BRI1 kinase activity. The results suggest that the S891T directed mutant protein has a slower rate of autophosphorylation but ultimately attains the same maximum activity as the wild type protein in vitro, and perhaps as a result the directed mutant does not function in an equivalent manner to the wild type receptor kinase in transgenic plants. These results suggest that the rate of activation/autophosphorylation may be an important parameter for receptor kinase function in vivo. Furthermore, in broad terms the results establish that serine-threonine substitutions are worth exploring at known phosphosites as the residues may not be functionally equivalent and therefore may provide a new general strategy to engineer receptor kinase function.

Technical Abstract: BRI1 becomes highly phosphorylated in vivo upon perception of the ligand, brassinolide, as a result of autophosphorylation and transphosphorylation by its co-receptor kinase, BAK1. Important autophosphorylation sites include those involved in activation of kinase activity and those that are inhibitory, such as Ser-891. The inhibitory sites are autophosphorylated after kinase activation has been achieved and are postulated to contribute to deactivation of the kinase. The function of phosphosites is usually tested by substituting a non-phosphorylatable residue or an acidic residue that can act as a phosphomimetic. What has typically not been examined is substitution of a Thr for a Ser phosphosite (or vice versa). In the present study with BRI1, we substituted Thr at the Ser-891 phosphosite to generate the S891T directed mutant. The recombinant Flag-BRI1 (S891T) cytoplasmic domain protein (the S891T protein) was catalytically active and phosphorylation occurred at the engineered Thr-891 site. However, the S891T recombinant protein autophosphorylated more slowly than the wild type protein during expression in E. coli. As a result, activation of peptide kinase activity (measured in vitro) was delayed as was transphosphorylation of bacterial proteins in situ. Stable transgenic expression of BRI1 (S891T)-Flag in Arabidopsis bri1-5 plants did not fully rescue the brassinosteroid (BR) phenotype indicating that BR signaling was constrained. Our working model is that restricted signaling in the S891T plants occurs as a result of the reduced rate of activation of the mutant BRI1 kinase by autophosphorylation. These results provide the platform for future studies to critically test this new model in vivo and establish Ser-Thr substitutions at phosphosites as an interesting approach to consider with other protein kinases.