Three lines of evidence indicate that the 14-3-3 proteins that inactivate the phosphorylated form of spinach leaf NADH:nitrate reductase (NR) bind to the enzyme at the regulatory phosphorylation site (Ser-543). First, a phosphorylated synthetic peptide based on the regulatory site can prevent and also reverse the inactivation of phospho-NR caused by 14-3-3 proteins. Second, sequence-specific and phosphorylation-dependent binding of the aforementioned synthetic peptide to the 14-3-3 proteins was demonstrated in vitro. Third, 14-3-3 proteins were required for the ATP-dependent phosphorylation of NR (as assessed by activity measurements) in the presence of NR-kinase and leaf protein phosphatases. Lastly, we demonstrate specificity of recombinant Arabidopsis 14-3-3 isoforms in the interaction with phospho-NR: to > Z > a) >>> ~, ¥.

We report an Mg 2+ -dependent interaction between spinach leaf sucrose-phosphate synthase (SPS) and endogenous 14-3-3 proteins, as evidenced by co-elution during gel filtration and co-immunoprecipitation. The content of 14-3-3s associated with an SPS immunoprecipitate was inversely related to activity, and was specifically reduced when tissue was pretreated with 5-aminoimidazole-4-carboxamide riboside, suggesting metabolite control in vivo. A synthetic phosphopeptide based on Ser-229 was shown by surface plasmon resonance to bind a recombinant plant 14-3-3, and addition of the phosphorylated SPS-229 peptide was found to stimulate the SPS activity of an SPS:14-3-3 complex. Taken together, the results suggest a regulatory interaction of 14-3-3 proteins with Ser-229 of SPS.z 1998 Federation of European Biochemical Societies.

SummaryBinding 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 GF14w, which encodes the Arabidopsis 14-3-3 isoform w, indicated that loop 8 (residues 208±219) is the likely cationbinding site. Directed mutagenesis of loop 8, which contains the EF hand-like region identi®ed in earlier studies, was performed to test the role of speci®c 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 af®nity for the cation. Mutations and Cterminal 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.