Divalent cations and polyamines bind to loop 8 of 14‐3‐3 proteins, modulating their interaction with phosphorylated nitrate reductase

Summary Binding of 14‐3‐3 proteins to nitrate reductase phosphorylated on Ser 543 (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 Mg 2+ 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+  ≈ putrescine 2+  ≈ agmatine 2+  ≈ N 1 ‐acetylspermidine 2+ , 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 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.