title: Single amino acid variation in barley 14-3-3 proteins leads to functional isoform specificity in the regulation of nitrate reductase.
abstract: The highly conserved family of 14-3-3 proteins function in the regulation of a wide variety of cellular processes. The presence of multiple 14-3-3 isoforms and the diversity of cellular processes regulated by 14-3-3 suggest functional isoform specificity of 14-3-3 isoforms in the regulation of target proteins. Indeed, several studies observed differences in affinity and functionality of 14-3-3 isoforms. However, the structural variation by which isoform specificity is accomplished remains unclear. Because other reports suggest that specificity is found in differential expression and availability of 14-3-3 isoforms, we used the nitrate reductase (NR) model system to analyse the availability and functionality of the three barley 14-3-3 isoforms. We found that 14-3-3C is unavailable in dark harvested barley leaf extract and 14-3-3A is functionally not capable to efficiently inhibit NR activity, leaving 14-3-3B as the only characterized isoform able to regulate NR in barley. Further, using site directed mutagenesis, we identified a single amino acid variation (Gly versus Ser) in loop 8 of the 14-3-3 proteins that plays an important role in the observed isoform specificity. Mutating the Gly residue of 14-3-3A to the alternative residue, as found in 14-3-3B and 14-3-3C, turned it into a potent inhibitor of NR activity. Using surface plasmon resonance, we show that the ability of 14-3-3A and the mutated version to inhibit NR activity correlates well with their binding affinity for the 14-3-3 binding motif in the NR protein, indicating involvement of this residue in ligand discrimination. These results suggest that both the availability of 14-3-3 isoforms as well as binding affinity determine isoform-specific regulation of NR activity.
authors.affiliation: Department of Developmental Genetics, De Boelelaan 1085, 1081 HV Amsterdam, the Netherlands.
authors.forename: Mark P
authors.initials: MP
authors.lastname: Sinnige
authors.forename: Ilja
authors.initials: I
authors.lastname: Roobeek
authors.forename: Tom D
authors.initials: TD
authors.lastname: Bunney
authors.forename: Antonie J W G
authors.initials: AJ
authors.lastname: Visser
authors.forename: Jos N M
authors.initials: JN
authors.lastname: Mol
authors.forename: Albertus H
authors.initials: AH
authors.lastname: de Boer
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D000595:Amino Acid Sequence; D001665:Binding Sites; D001467:Hordeum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D050901:Nitrate Reductase / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism* / Q000502:physiology; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D048948:14-3-3 Proteins; D010940:Plant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins; D050901:Nitrate Reductase