@entity16047 becomes highly phosphorylated in vivo upon perception of the ligand, @entity16044 , 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) but given that Thr and Ser are not equivalent @entity3534 this type of substitution may represent a new approach to engineer regulatory phosphorylation. In the present study with @entity16047 , we substituted Thr at the Ser-891 phosphosite to generate the @entity69808 directed mutant. The recombinant Flag- @entity16047 ( @entity69808 ) cytoplasmic domain protein (the @entity69808 protein) was catalytically active and phosphorylation occurred at the engineered Thr-891 site. However, the @entity69808 recombinant protein autophosphorylated more slowly than the wild-type protein during expression in @entity526 . 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 @entity16047 ( @entity69808 )-Flag in @entity958 @entity16047 -5 plants did not fully rescue the @entity16656 (BR) phenotype indicating that BR signaling was constrained. Our working model is that restricted signaling in the @entity69808 plants occurs as a result of the reduced rate of activation of the mutant @entity16047 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.

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

@entity3534 :: ('MESH:D000596', 'Chemical') :: ['amino acids'] @entity958 :: ('3702', 'Species') :: ['Arabidopsis'] @entity69808 :: ('p|SUB|S|891|T', 'ProteinMutation') :: ['S891T'] @entity16044 :: ('MESH:C023623', 'Chemical') :: ['brassinolide'] @entity16656 :: ('CHEBI:22921', 'Chemical') :: ['brassinosteroid'] @entity16047 :: ('830095(Tax:3702)', 'Gene') :: ['BRI1', 'bri1'] @entity526 :: ('562', 'Species') :: ['E. coli']
@entity16047 :: (830095(Tax:3702),Gene) :: ['BRI1']
