Abstract
Brassinosteroids (BRs) regulate multiple aspects of plant growth and development and require an active BRASSINOSTEROID-INSENSITIVE1 (BRI1) and BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1) for hormone perception and signal transduction. Many animal receptor kinases exhibit ligand-dependent oligomerization followed by autophosphorylation and activation of the intracellular kinase domain. To determine if early events in BR signaling share this mechanism, we used coimmunoprecipitation 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 thaliana followed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) identified S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sites of BRI1. MS analysis 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 C-terminal regions had only small observable effects on autophosphorylation and in planta signaling but dramatically affected phosphorylation of a peptide substrate in vitro. These 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, whereas phosphorylation of noncatalytic intracellular domains is required for recognition and/or phosphorylation of downstream substrates.
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Claims 8
Claim · model-written summary
BRI1 and BAK1 association was affected by endogenous and exogenous brassinosteroid levels.
- Taxon
- Arabidopsis thaliana
Arabidopsis thalianae1 - Gene
- BRI1 and BAK1e0
- Compound
- BRe0
- Outcome
- in vivo association of BRI1 and BAK1e0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“in vivo association of BRI1 and BAK1 was affected by endogenous and exogenous BR levels”
e1“Arabidopsis thaliana”
Claim · model-written summary
Threonine phosphorylation of BRI1 and BAK1 depended on brassinosteroids.
- Taxon
- Arabidopsis thalianaArabidopsis thalianae1
- Gene
- BRI1 and BAK1e0
- Compound
- BRe0
- Outcome
- phosphorylation of both BRI1 and BAK1 on Thr residuese0
Not reportedDirection
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“phosphorylation of both BRI1 and BAK1 on Thr residues was BR dependent”
e1“Arabidopsis thaliana”
Claim · model-written summary
Six specific in vivo BRI1 phosphorylation sites were identified.
- Taxon
- Arabidopsis thalianaArabidopsis thalianae1
- Gene
- BRI1e0
- Outcome
- S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sitese0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“identified S-838, S-858, T-872, and T-880 in the juxtamembrane region, T-982 in the kinase domain, and S-1168 in C-terminal region as in vivo phosphorylation sites of BRI1”
e1“Arabidopsis thaliana”
Claim · model-written summary
MS suggested another two juxtamembrane and three activation loop residues are phosphorylated in vivo.
- Taxon
- Arabidopsis thalianaArabidopsis thalianae1
- Gene
- BRI1e2
- Outcome
- additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivoe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“an additional two residues in the juxtamembrane region and three sites in the activation loop of kinase subdomain VII/VIII were phosphorylated in vivo”
e1“Arabidopsis thaliana”
e2“epitope-tagged BRI1 from Arabidopsis thaliana”
Claim · model-written summary
Four specific BAK1 autophosphorylation sites were identified in vitro.
- Taxon
- Arabidopsis thalianaArabidopsis thalianae1
- Gene
- BAK1e0
- Outcome
- BAK1 autophosphorylation sitese0
- Value
- four specific BAK1 autophosphorylation sitese0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“four specific BAK1 autophosphorylation sites in vitro”
e1“Arabidopsis thaliana”
Claim · model-written summary
BRI1 T-1049 and either S-1044 or T-1045 were essential for in vitro kinase function and normal in planta signalling.
- Taxon
- Arabidopsis thalianaArabidopsis thalianae1
- Gene
- T-1049 and either S-1044 or T-1045e0
- Outcome
- kinase function in vitro and normal BRI1 signaling in plantae0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“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”
e1“Arabidopsis thaliana”
Claim · model-written summary
Juxtamembrane or C-terminal mutations had small effects on autophosphorylation and in planta signalling.
- Taxon
- Arabidopsis thalianaArabidopsis thalianae1
- Treatment
- Mutations in the juxtamembrane or C-terminal regionse0
- Outcome
- autophosphorylation and in planta signalinge0
- Direction
- only small observable effectse0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Mutations in the juxtamembrane or C-terminal regions had only small observable effects on autophosphorylation and in planta signaling”
e1“Arabidopsis thaliana”
Claim · model-written summary
The same mutations dramatically affected peptide substrate phosphorylation in vitro.
- Taxon
- Arabidopsis thalianaArabidopsis thalianae1
- Treatment
- Mutations in the juxtamembrane or C-terminal regionse0
- Outcome
- phosphorylation of a peptide substrate in vitroe0
- Direction
- dramatically affectede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“Mutations in the juxtamembrane or C-terminal regions had only small observable effects on autophosphorylation and in planta signaling but dramatically affected phosphorylation of a peptide substrate in vitro”
e1“Arabidopsis thaliana”
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