Dual binding of 14-3-3 protein regulates Arabidopsis nitrate reductase activity

Abstract

14-3-3 proteins represent a family of ubiquitous eukaryotic proteins involved in numerous signal transduction processes and metabolic pathways. One important 14-3-3 target in higher plants is nitrate reductase (NR), whose activity is regulated by different physiological conditions. Intra-molecular electron transfer in NR is inhibited following 14-3-3 binding to a conserved phospho-serine motif located in hinge 1, a surface exposed loop between the catalytic molybdenum and central heme domain. Here we describe a novel 14-3-3 binding site within the NR N-terminus, an acidic motif conserved in NRs of higher plants, which significantly contributes to 14-3-3-mediated inhibition of NR. Deletion or mutation of the N-terminal acidic motif resulted in a significant loss of 14-3-3 mediated inhibition of Ser534 phosphorylated NR-Mo-heme (residues 1-625), a previously established model of NR regulation. Co-sedimentation and crosslinking studies with NR peptides comprising each of the two binding motifs demonstrated direct binding of either peptide to 14-3-3. Surface plasmon resonance spectroscopy disclosed high-affinity binding of 14-3-3ω to the well-known phospho-hinge site and low-affinity binding to the N-terminal acidic motif. A binding groove-deficient 14-3-3ω variant retained interaction to the acidic motif, but lost binding to the phospho-hinge motif. To our knowledge, NR is the first enzyme that harbors two independent 14-3-3 binding sites with different affinities, which both need to be occupied by 14-3-3ω to confer full inhibition of NR activity under physiological conditions.

A 14-3-3 Family Protein from Wild Soybean (Glycine Soja) Regulates ABA Sensitivity in Arabidopsis

“…14-3-3 proteins are key regulators of multiple signal transduction cascades relating to diverse physiological and biological processes through hundreds of different protein-protein interactions [ 53 – 58 ]. The development of bioinformatics, especially the whole genome sequencing, dramatically assisted for the genome wide survey of plant 14-3-3 proteins, for example in Arabidopsis [ 59 , 60 ], rice [ 61 , 62 ], tomato [ 63 ], cotton [ 64 ], and populous [ 47 ].…”

CHAPTER 1. Molybdenum and Tungsten-Containing Enzymes: An Overview

“…interestingly, the phosphorylation of a serine residue in the linker region between the molybdenum and haem domains, 346,347 followed by the binding of a specific regulatory protein, effectively inhibits the enzyme through the inhibition of the intramolecular electron transfer. § § §, [353][354][355][356] another member of the so family is the recently described marC (the fourth mammalian molybdoenzyme, after Xo, ao and so). 357 its physiological function is not known, but it is probably involved in the detoxification of mutagenic and toxic aromatic hydroxyl-amines, such as N-hydroxylated dna base derivates, 358,359 through the catalysis of the reduction of N-and S-hydroxylated compounds at the molybdenum centre (eqn (1.21)), [360][361][362][363] among other possible roles.…”

“…352 this phosphorylation creates a recognition site that recruits a specific regulatory protein (one member of the 14-3-3 family), whose binding effectively inhibits the enzyme. [353][354][355][356] (i.e. at the haem of so or the Fad of nar); therefore, intramolecular electron transfer (Mo → haem or Fad → haem → Mo) is, once again, an integral aspect of catalysis.…”

Enzymatic characterization of recombinant nitrate reductase expressed and purified from Neurospora crassa

“…6, supplementary Tables 1-4), while there was no phosphorylation detectable for recombinant NR purified from E. coli (data not shown). Very recently Chi and co-workers demonstrated that for Arabidopsis NR the interaction with the 14-3-3 protein is mediated by the N-terminus of the enzyme (Chi et al, 2015). We suggest that the Neurospora NR N-terminus may also be involved in enzyme activity regulation by a yet unknown phosphorylation dependent mechanism.…”