Divalent cations and polyamines bind to loop 8 of 14-3-3 proteins, modulating their interaction with phosphorylated nitrate reductase.
Binding of 14-3-3 proteins to nitrate reductase phosphorylated on Ser543 (phospho-NR) inhibits activity and is responsible for the inactivation of nitrate reduction that occurs in darkened leaves. The 14-3-3-dependent inactivation of phospho-NR is known to require millimolar concentrations of a divalent cation such as Mg2+ at pH 7.5. We now report that micromolar concentrations of the polyamines, spermidine(4+) and spermine(3+), can substitute for divalent cations in modulating 14-3-3 action. Effectiveness of the polyamines decreased with a decrease of polycation charge: spermine(4+) > spermidine(3+) >>> cadavarine(2+) approximately putrescine(2+) approximately agmatine(2+) approximately N1-acetylspermidine(2+), indicating that two primary and at least one secondary amine group were required. C-terminal truncations of GF14 omega, which encodes the Arabidopsis 14-3-3 isoform omega, indicated that loop 8 (residues 208-219) is the likely cation-binding site. Directed mutagenesis of loop 8, which contains the EF hand-like region identified in earlier studies, was performed to test the role of specific amino acid residues in cation binding. The E208A mutant resulted in a largely divalent cation-independent inhibition of phospho-NR activity, whereas the D219A mutant was fully Mg(2+)-dependent but had decreased affinity for the cation. Mutations and C-terminal truncations that affected the Mg(2+) dependence of phospho-NR inactivation had similar effects on polyamine dependence. The results implicate loop 8 as the site of divalent cation and polyamine binding, and suggest that activation of 14-3-3s occurs, at least in part, by neutralization of negative charges associated with acidic residues in the loop. We propose that binding of polyamines to 14-3-3s could be involved in their regulation of plant growth and development.
Athwal
Gurdeep S
GS
US Department of Agriculture, Agricultural Research Service, and Department of Horticultural Science, North Carolina State University, Raleigh, NC 27695-7631, USA.
Huber
Steven C
SC
D048948:14-3-3 Proteins; D029681:Arabidopsis Proteins; D001665:Binding Sites / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002413:Cations, Divalent / Q000378:metabolism / Q000494:pharmacology*; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010940:Plant Proteins / Q000378:metabolism*; D011073:Polyamines / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation; D013095:Spermidine / Q000494:pharmacology; D013096:Spermine / Q000494:pharmacology; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D048948:14-3-3 Proteins; D029681:Arabidopsis Proteins; D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; C087620:GF14 protein, Arabidopsis; D010940:Plant Proteins; D011073:Polyamines; D013096:Spermine; D014446:Tyrosine 3-Monooxygenase; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D008274:Magnesium; D013095:Spermidine