title: Modulation of 14-3-3 protein interactions with target polypeptides by physical and metabolic effectors.
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 KD and an increase in steady-state binding level (Req). 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.
authors.affiliation: US Department of Agriculture, Department of Horticultural Science, North Carolina State University, Raleigh 27695, USA.
authors.forename: G S
authors.initials: GS
authors.lastname: Athwal
authors.forename: C R
authors.initials: CR
authors.lastname: Lombardo
authors.forename: J L
authors.initials: JL
authors.lastname: Huber
authors.forename: S C
authors.initials: SC
authors.lastname: Masters
authors.forename: H
authors.initials: H
authors.lastname: Fu
authors.forename: S C
authors.initials: SC
authors.lastname: Huber
mesh_terms: D048948:14-3-3 Proteins; D000249:Adenosine Monophosphate / Q000494:pharmacology; D017360:Arabidopsis; D015374:Biosensing Techniques; D002413:Cations, Divalent / Q000494:pharmacology; D005656:Fungal Proteins; D015971:Gene Expression Regulation, Enzymologic; D008024:Ligands; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010748:Phosphopeptides / Q000378:metabolism*; D010940:Plant Proteins; D011485:Protein Binding / Q000187:drug effects; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011522:Protons; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D018724:Spinacia oleracea; D014446:Tyrosine 3-Monooxygenase
chemical_list: D048948:14-3-3 Proteins; D002413:Cations, Divalent; D005656:Fungal Proteins; D008024:Ligands; D010446:Peptide Fragments; D010748:Phosphopeptides; D010940:Plant Proteins; D011506:Proteins; D011522:Protons; D011994:Recombinant Proteins; D000249:Adenosine Monophosphate; D014446:Tyrosine 3-Monooxygenase; D009565:Nitrate Reductases; D050901:Nitrate Reductase