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, spermidine⁴⁺ and spermine³⁺, can substitute for divalent cations in modulating 14-3-3 action. Effectiveness of the polyamines decreased with a decrease of polycation charge: spermine⁴⁺ > spermidine³⁺ >>> cadavarine²⁺ ≅ putrescine²⁺ ≅ agmatine²⁺ ≅ N¹-acetylspermidine²⁺, indicating that two primary and at least one secondary amine group were required. C-terminal truncations of GF14ω, which encodes the Arabidopsis 14-3-3 isoform ω, 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 Mg²⁺-dependent but had decreased affinity for the cation. Mutations and C-terminal truncations that affected the Mg²⁺ 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.