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 journalArticle

Abstract

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
Volume41
Issue number4
DOIs
StatePublished - Jan 1 2000

Fingerprint

14-3-3 Proteins
polypeptides
Nitrate Reductase
Peptides
nitrate reductase
Phosphopeptides
Adenosine Monophosphate
Ligands
proteins
cations
peptides
yeasts
surface plasmon resonance
5'-Nucleotidase
Proteins
Fungal Proteins
Spinacia oleracea
Surface Plasmon Resonance
Divalent Cations
protein-protein interactions

Keywords

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

ASJC Scopus subject areas

  • Physiology
  • Plant Science
  • Cell Biology

Cite this

Athwal, G. S., Lombardo, C. R., Huber, J. L., Masters, S. C., Fu, H., & Huber, S. C. (2000). Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors. Plant and Cell Physiology, 41(4), 523-533. https://doi.org/10.1093/pcp/41.4.523

Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors. / Athwal, Gurdeep S.; Lombardo, Christian R.; Huber, Joan L.; Masters, Shane C.; Fu, Haian; Huber, Steven C.

In: Plant and Cell Physiology, Vol. 41, No. 4, 01.01.2000, p. 523-533.

Research output: Contribution to journalArticle

Athwal, GS, Lombardo, CR, Huber, JL, Masters, SC, Fu, H & Huber, SC 2000, 'Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors', Plant and Cell Physiology, vol. 41, no. 4, pp. 523-533. https://doi.org/10.1093/pcp/41.4.523
Athwal, Gurdeep S. ; Lombardo, Christian R. ; Huber, Joan L. ; Masters, Shane C. ; Fu, Haian ; Huber, Steven C. / Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors. In: Plant and Cell Physiology. 2000 ; Vol. 41, No. 4. pp. 523-533.
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