Functional Characterization of Specific BRI1 Phosphorylation Sites In vivo

The BR-insensitive bri1-5 mutant is a weak allele that shows intermediate dwarfism and a smaller rosette size than wild-type due to a C69Y amino acid substitution in the extracellular domain of BRI1 [5]. Transgenic constructs containing the BRI1 promoter, the entire coding region and an in-frame C-terminal FLAG epitope tag fully rescue the bri1-5 phenotype to wild-type. Substitutions of Ala for specific Thr or Ser residues within the wild-type construct that were shown to be phosphorylated by LC-MS/MS analysis were generated and transformed into bri1-5. Multiple independent transgenic lines for each construct were evaluated visually for phenotype compared with untrans-formed wild-type and the bri1-5 mutant and transgene expression and native BRI1 RNA levels were monitored. Substitutions in the activation loop dramatically affected the ability of the construct to rescue bri1-5. T1039A and S1042A both showed a phenotype that was intermediate between bri1-5 and wild-type, while T1049A and S1044A were incapable of bri1-5 rescue in any of the transgenic lines examined, which is consistent with their nearly complete loss of kinase activity using in vitro biochemical assays [21].

Substitution of Ala for Thr or Ser in the juxtamembrane and C-terminal region of BRI1 did not appear to affect the general ability of the transgene to rescue the bri1-5 mutant to an apparently normal wild-type size, consistent with our in vitro observation that none of these mutations affected general autophosphorylation of the BRI1 cytoplasmic domain. However, based on our in vitro peptide substrate assays, we expected that some of these mutations might show an altered phenotype due to inability to phosphorylate a downstream substrate to the extent required for normal BR signaling. It is possible that the juxtamembrane and/or C-terminal mutations may have subtle phenotypes due to altered phosphorylation of substrates in a specific branch pathway of BR signal transduction, and we are testing this possibility using a detailed morphometric analysis of several growth parameters in homozygous T3 lines for each mutation. Based on in vitro and in vivo data and on conservation of function in other LRR RLKs, we propose that BL-dependent phosphorylation of T1049 and S1044 are required for activation and function of BRI1 in planta, while phosphorylation of individual juxtamembrane and carboxy-terminal Ser and Thr residues might affect further phosphorylation of BRI1 cytoplasmic substrates, but apparently is not required for general kinase activation [21].

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