14-3-3-PROTEIN 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

Three lines of evidence indicate that the 14-3-3 proteins that inactiv ate 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 sit e can prevent and also reverse the inactivation of phospho-NR caused b y 14-3-3 proteins, Second, sequence-specific and phosphorylation-depen dent binding of the aforementioned synthetic peptide to the 14-3-3 pro teins was demonstrated in vitro, Third, 14-3-3 proteins were required for the ATP-dependent phosphorylation of IVR (as assessed by activity measurements) in the presence of NR-kinase and leaf protein phosphatas es, Lastly, we demonstrate specificity of recombinant Arabidopsis 14-3 -3 isoforms in the interaction with phospho-NR: omega > chi > upsilon >>> phi, psi.