A kinase-phosphatase signaling module with BSK8 and BSL2 involved in regulation of sucrose-phosphate synthase

J Proteome Res. 2014 Jul 3;13(7):3397-409. doi: 10.1021/pr5003164. Epub 2014 Jun 20.

Abstract

External supply of sucrose to carbon-starved Arabidopsis seedlings induced changes in phosphorylation of Brassinosteroid Signaling Kinase 8 (BSK8) at two different sites. Serine S(20) lies within a phosphorylation hotspot at the N-terminal region of the protein, while S(213) is located within the kinase domain of BSK8. Upon sucrose supply phosphorylation of BSK8(S20) and BSK8(S213) showed opposite behavior with increasing phosphorylation of S(213) and decreased phosphorylation of S(20) at 5 min after sucrose supply. Here we aim to systematically analyze the effects of BSK8 mutations on downstream cellular regulatory events and characterize molecular functions of BSK8 and its phosphorylation. Comparative phosphoproteomic profiling of a bsk8 knockout mutant and wild type revealed potential targets in sucrose metabolism. Activity of sucrose-phosphate synthase (SPS) was decreased by phosphorylation at S(152), and SPS phosphorylation inversely correlated with sucrose-induced BSK8 activity. Furthermore, BSK8 was found to interact with BSL2, a Kelch-type phosphatase. On the basis of a combination of kinase activity measurements, SPS activity assays, and phosphorylation site mutations in BSK8 at S(20) and S(213), we conclude that regulation of SPS by BSK8 occurs through activation of a phosphatase that in turn may dephosphorylate SPS and thus activates the enzyme.

MeSH terms

  • Amino Acid Sequence
  • Arabidopsis / cytology
  • Arabidopsis / enzymology*
  • Arabidopsis Proteins / physiology*
  • Glucosyltransferases / metabolism*
  • Mesophyll Cells / enzymology
  • Molecular Sequence Data
  • Phosphoprotein Phosphatases / physiology*
  • Phosphorylation
  • Protein Processing, Post-Translational
  • Protein Transport
  • Protein-Serine-Threonine Kinases / physiology*
  • Seedlings / cytology
  • Seedlings / enzymology
  • Signal Transduction

Substances

  • Arabidopsis Proteins
  • Glucosyltransferases
  • sucrose-phosphate synthase
  • BSK8 protein, Arabidopsis
  • Protein-Serine-Threonine Kinases
  • BSL2 protein, Arabidopsis
  • Phosphoprotein Phosphatases