title: Protein phosphatase 2A B55 and A regulatory subunits interact with nitrate reductase and are essential for nitrate reductase activation.
abstract: Posttranslational activation of nitrate reductase (NR) in Arabidopsis (Arabidopsis thaliana) and other higher plants is mediated by dephosphorylation at a specific Ser residue in the hinge between the molybdenum cofactor and heme-binding domains. The activation of NR in green leaves takes place after dark/light shifts, and is dependent on photosynthesis. Previous studies using various inhibitors pointed to protein phosphatases sensitive to okadaic acid, including protein phosphatase 2A (PP2A), as candidates for activation of NR. PP2As are heterotrimeric enzymes consisting of a catalytic (C), structural (A), and regulatory (B) subunit. In Arabidopsis there are five, three, and 18 of these subunits, respectively. By using inducible artificial microRNA to simultaneously knock down the three structural subunits we show that PP2A is necessary for NR activation. The structural subunits revealed overlapping functions in the activation process of NR. Bimolecular fluorescence complementation was used to identify PP2A regulatory subunits interacting with NR, and the two B55 subunits were positive. Interactions of NR and B55 were further confirmed by the yeast two-hybrid assay. In Arabidopsis the B55 group consists of the close homologs B55α and B55β. Interestingly, the homozygous double mutant (b55α × b55β) appeared to be lethal, which shows that the B55 group has essential functions that cannot be replaced by other regulatory subunits. Mutants homozygous for mutation in Bβ and heterozygous for mutation in Bα revealed a slower activation rate for NR than wild-type plants, pointing to these subunits as part of a PP2A complex responsible for NR dephosphorylation.
authors: University of Stavanger, Centre for Organelle Research, Faculty of Science and Technology, N-4036 Stavanger, Norway.
authors: Behzad
authors: B
authors: Heidari
authors: Polina
authors: P
authors: Matre
authors: Dugassa
authors: D
authors: Nemie-Feyissa
authors: Christian
authors: C
authors: Meyer
authors: Odd Arne
authors: OA
authors: Rognli
authors: Simon G
authors: SG
authors: Møller
authors: Cathrine
authors: C
authors: Lillo
mesh_terms: D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D050901:Nitrate Reductase / Q000235:genetics / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010766:Phosphorylation; D054648:Protein Phosphatase 2 / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
chemical_list: D029681:Arabidopsis Proteins; D019319:Okadaic Acid; D050901:Nitrate Reductase; D054648:Protein Phosphatase 2; C489810:type 2A protein serine-threonine phosphatase 55 kDa B, Arabidopsis