Identification of in vitro phosphorylation sites in the Arabidopsis thaliana somatic embryogenesis receptor-like kinases.
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.
Karlova
Rumyana
R
Laboratory of Biochemistry, Wageningen University, Wageningen, The Netherlands.
Boeren
Sjef
S
van Dongen
Walter
W
Kwaaitaal
Mark
M
Aker
Jose
J
Vervoort
Jacques
J
de Vries
Sacco
S
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020134:Catalytic Domain / Q000502:physiology; D004926:Escherichia coli / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation / Q000502:physiology; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020449:Repetitive Sequences, Amino Acid; D012694:Serine / Q000378:metabolism; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry; D013912:Threonine / Q000378:metabolism
D029681:Arabidopsis Proteins; D013912:Threonine; D012694:Serine; D011494:Protein Kinases; C463660:BAK1 protein, Arabidopsis; C440035:SERK1 protein, Arabidopsis; C108270:BRI1 protein, Arabidopsis; D017346:Protein Serine-Threonine Kinases