Modulation of 14-3-3 Protein Interactions with Target Polypeptides by Physical and Metabolic Effectors

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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-Ser_<543>. Both plant and yeast 14-3-3 proteins were shown to bind the immobilized poptide ligand in a Mg^<2+>-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. AtpH 7.5(+Mg^<2+>), 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.
日本植物生理学会の論文