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 B55a and B55b. Interestingly, the homozygous double mutant (b55a 3 b55b) 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 Bb and heterozygous for mutation in Ba 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.Nitrate reductase (NR), a key enzyme in nitrogen assimilation, reduces nitrate to nitrite in the cytosol. The nitrite formed is further reduced to ammonium in the plastids, whereafter ammonium is incorporated into amino acids. Regulation of NR takes place at the transcriptional as well as posttranslational level. At both levels signals from the chloroplasts are involved in initiating an increase in NR activity levels, but these signals and their processing are still unknown (Jonassen et al.,

Atypical Protein Phosphatase 2A Gene Families Do Not Expand via Paleopolyploidization

“…Two or more B56 isoforms regulate dephosphorylation of the BAK1 coreceptor (Segonzac et al, 2014), the brassinosteroid-signaling transcription factor BZR1 (Tang et al, 2011), and the brassinosteroid receptor BRI1 (Wang et al, 2016). Both Arabidopsis B55 isoforms interact with nitrate reductase (Heidari et al, 2011), and at least two B72 (clade II) isoforms interact with 3-hydroxy-3-methylglutaryl-CoA reductase (Leivar et al, 2011). These data suggest that, in plants at least, many PP2A subunit isoforms share some functional overlap.…”

Regulatory Subunit B′γ of Protein Phosphatase 2A Prevents Unnecessary Defense Reactions under Low Light in Arabidopsis

“…Of the regulatory B subunits, the B$-type PP2A subunit TONNEAU2 was found to regulate the dynamic organization of the cortical cytoskeleton (Camilleri et al, 2002), whereas a metabolic role for B55-type subunits a and b in the activation of nitrate reductase was recently reported (Heidari et al, 2011). Lately, vital roles for PP2A in the regulation of cell elongation and growth through brassinosteroid signaling (Tang et al, 2011) and the TARGET OF RAPAMYCIN (TOR) pathway (Ahn et al, 2011) were elucidated, and a role for the catalytic PP2A-C2 subunit as a negative regulator of abscisic acid signaling has also been demonstrated (Pernas et al, 2007).…”

Okadaic acid and microcystin insensitive PPP-family phosphatases may represent novel biotechnology targets

“…20 As is the case with PP1 and PP2A, regulatory proteins may be central to determining SLP protein phosphatase substrate specificity. [21][22][23][24] …”