Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors

Gurdeep S. Athwal, Christian R. Lombardo, Joan L. Huber, Shane C. Masters, Haian Fu, Steven C. Huber

Research output: Contribution to journalArticlepeer-review


The proteins commonly referred to as 14-3-3s have recently come to prominence in the study of protein:protein interactions, having been shown to act as allosteric or steric regulators and possibly scaffolds. The binding of 14-3-3 proteins to the regulatory phosphorylation site of nitrate reductase (NR) was studied in real-time by surface plasmon resonance, using primarily an immobilized synthetic phosphopeptide based on spinach NR-Ser543. Both plant and yeast 14-3-3 proteins were shown to bind the immobilized peptide ligand in a Mg2+-stimulated manner. Stimulation resulted from a reduction in K(D) and an increase in steady-state binding level (R(eq)). As shown previously for plant 14-3-3s, fluorescent probes also indicated that yeast BMH2 interacted directly with cations, which bind and affect surface hydrophobicity. Binding of 14-3-3s to the phosphopeptide ligand occurred in the absence of divalent cations when the pH was reduced below neutral, and the basis for enhanced binding was a reduction in K(D). At pH 7.5 (+Mg2+), AMP inhibited binding of plant 14-3-3s to the NR based peptide ligand. The binding of AMP to 14-3-3s was directly demonstrated by equilibrium dialysis (plant), and from the observation that recombinant plant 14-3-3s have a low, but detectable, AMP phosphatase activity.

Original languageEnglish (US)
Pages (from-to)523-533
Number of pages11
JournalPlant and Cell Physiology
Issue number4
StatePublished - 2000
Externally publishedYes


  • AMP binding
  • BIAcore
  • Hydrolytic activity
  • Metal binding
  • Protein:protein interaction
  • Spinacia oleracea

ASJC Scopus subject areas

  • Physiology
  • Plant Science
  • Cell Biology


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