PHOSPHORYLATED NITRATE REDUCTASE AND 14-3-3-PROTEINS - SITE OF INTERACTION, EFFECTS OF IONS, AND EVIDENCE FOR AN AMP-BINDING SITE ON 14-3-3-PROTEINS

The inactivation of phosphorylated nitrate reductase (NR) by the bindi ng of 14-3-3 proteins is one of a very few unambiguous biological func tions for 14-3-3 proteins. We report here that serine and threonine re sidues at the +6 to +8 positions, relative to the known regulatory bin ding site involving serine-543, are important in the interaction with GF14 omega, a recombinant plant 14-3-3. Also shown is that an increase in ionic strength with KCl or inorganic phosphate, known physical eff ecters of NR activity, directly disrupts the binding of protein and pe ptide ligands to 14-3-3 proteins. Increased ionic strength attributabl e to KCl caused a change in conformation of GF14 omega, resulting in r educed surface hydrophobicity, as visualized with a fluorescent probe. Similarly, it is shown that the 5' isomer of AMP was specifically abl e to disrupt the inactive phosphorylated NR:14-3-3 complex. Using the 5'-AMP fluorescent analog trinitrophenyl-AMP, we show that there is a probable AMP-binding site on GF14 omega.