Phosphorylation-dependent interactions between enzymes of plant metabolism and 14-3-3 proteins

Plant J. 1999 Apr;18(1):1-12. doi: 10.1046/j.1365-313x.1999.00417.x.

Abstract

Far-Western overlays of soluble extracts of cauliflower revealed many proteins that bound to digoxygenin (DIG)-labelled 14-3-3 proteins. Binding to DIG-14-3-3s was prevented by prior dephosphorylation of the extract proteins or by competition with 14-3-3-binding phosphopeptides, indicating that the 14-3-3 proteins bind to phosphorylated sites. The proteins that bound to the DIG-14-3-3s were also immunoprecipitated from extracts with anti-14-3-3 antibodies, demonstrating that they were bound to endogenous plant 14-3-3 proteins. 14-3-3-binding proteins were purified from cauliflower extracts, in sufficient quantity for amino acid sequence analysis, by affinity chromatography on immobilised 14-3-3 proteins and specific elution with a 14-3-3-binding phosphopeptide. Purified 14-3-3-binding proteins included sucrose-phosphate synthase, trehalose-6-phosphate synthase, glutamine synthetases, a protein (LIM17) that has been implicated in early floral development, an approximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium-dependent protein kinase that was capable of phosphorylating and rendering nitrate reductase (NR) sensitive to inhibition by 14-3-3 proteins. In contrast to the phosphorylated NR-14-3-3 complex which is activated by dissociation with 14-3-3-binding phosphopeptides, the total sugar-phosphate synthase activity in plant extracts was inhibited by up to 40% by a 14-3-3-binding phosphopeptide and the phosphopeptide-inhibited activity was reactivated by adding excess 14-3-3 proteins. Thus, 14-3-3 proteins are implicated in regulating several aspects of primary N and C metabolism. The procedures described here will be valuable for determining how the phosphorylation and 14-3-3-binding status of defined target proteins change in response to extracellular stimuli.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 14-3-3 Proteins
  • Amino Acid Sequence
  • Brassica / enzymology
  • Brassica / metabolism*
  • Carbohydrate Metabolism
  • Carrier Proteins / isolation & purification
  • Carrier Proteins / metabolism
  • Chromatography, Affinity
  • Molecular Sequence Data
  • Phosphoproteins / isolation & purification
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Plant Proteins / chemistry
  • Plant Proteins / isolation & purification
  • Plant Proteins / metabolism*
  • Precipitin Tests
  • Proteins / chemistry
  • Proteins / isolation & purification
  • Proteins / metabolism*
  • Tyrosine 3-Monooxygenase*

Substances

  • 14-3-3 Proteins
  • Carrier Proteins
  • Phosphoproteins
  • Plant Proteins
  • Proteins
  • Tyrosine 3-Monooxygenase