Identification of in vitro phosphorylation sites in the Arabidopsis thaliana somatic embryogenesis receptor-like kinases

Proteomics. 2009 Jan;9(2):368-79. doi: 10.1002/pmic.200701059.

Abstract

The Arabidopsis thaliana somatic embryogenesis receptor-like kinase (SERK) family consists of five leucine-rich repeat receptor-like kinases (LRR-RLKs) with diverse functions such as brassinosteroid insensitive 1 (BRI1)-mediated brassinosteroid perception, development and innate immunity. The autophosphorylation activity of the kinase domains of the five SERK proteins was compared and the phosphorylated residues were identified by LC-MS/MS. Differences in autophosphorylation that ranged from high activity of SERK1, intermediate activities for SERK2 and SERK3 to low activity for SERK5 were noted. In the SERK1 kinase the C-terminally located residue Ser-562 controls full autophosphorylation activity. Activation loop phosphorylation, including that of residue Thr-462 previously shown to be required for SERK1 kinase activity, was not affected. In vivo SERK1 phosphorylation was induced by brassinosteroids. Immunoprecipitation of CFP-tagged SERK1 from plant extracts followed by MS/MS identified Ser-303, Thr-337, Thr-459, Thr-462, Thr-463, Thr-468, and Ser-612 or Thr-613 or Tyr-614 as in vivo phosphorylation sites of SERK1. Transphosphorylation of SERK1 by the kinase domain of the main brassinosteroid receptor BRI1 occurred only on Ser-299 and Thr-462. This suggests both intra- and intermolecular control of SERK1 kinase activity. Conversely, BRI1 was transphosphorylated by the kinase domain of SERK1 on Ser-887. BRI1 kinase activity was not required for interaction with the SERK1 receptor in a pull down assay.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / genetics
  • Arabidopsis / metabolism*
  • Arabidopsis Proteins / chemistry
  • Arabidopsis Proteins / genetics*
  • Arabidopsis Proteins / metabolism*
  • Catalytic Domain / physiology
  • Escherichia coli / metabolism
  • Mutagenesis, Site-Directed
  • Phosphorylation / physiology
  • Protein Kinases / chemistry
  • Protein Kinases / genetics*
  • Protein Kinases / metabolism*
  • Protein-Serine-Threonine Kinases / chemistry
  • Protein-Serine-Threonine Kinases / genetics
  • Protein-Serine-Threonine Kinases / metabolism
  • Repetitive Sequences, Amino Acid
  • Serine / metabolism
  • Signal Transduction
  • Tandem Mass Spectrometry
  • Threonine / metabolism

Substances

  • Arabidopsis Proteins
  • Threonine
  • Serine
  • Protein Kinases
  • BAK1 protein, Arabidopsis
  • SERK1 protein, Arabidopsis
  • BRI1 protein, Arabidopsis
  • Protein-Serine-Threonine Kinases