14‐3‐3 proteins associate with the regulatory phosphorylation site of spinach leaf nitrate reductase in an isoform‐specific manner and reduce dephosphorylation of Ser‐543 by endogenous protein phosphatases

Abstract

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: ω > χ > ν ⋙ gf, ψ.