Identification of Ser-543 as the Major Regulatory Phosphorylation Site in Spinach Leaf Nitrate Reductase

Abstract

Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.

The inhibitor protein of phosphorylated nitrate reductase from spinach (Spinacia oleracea) leaves is a 14‐3‐3 protein

“…The action of IP in the inactivation process of NR might be of a very similar nature. IP has no effect on NR activity as long as the enzyme is not phosphorylated [9], indicating that phosphorylation may be required for the protein interaction between IP and NR. Whether phosphorylation of Ser-543 itself or a change in NR conformation caused by phosphorylation is required for the protein interaction with IP remains to be determined.…”

“…Spinach leaf NR is 32P-labeled exclusively on seryl residues in vivo when leaves are fed [3zP]Pi [4]. Recently, the n~ajor regulatory phosphorylation site in spinach leaf NR was i~entified as Ser-543, which is located in the hinge 1 region b,~tween the cytochrome b domain and the molybdenum-cof,~ctor binding domain [8,9].…”

“…FI .maih shuber@cropservl .cropsci.ncsu.edu Abbreviations: GF14o), G-box binding factor of the 14-3-3 protein family; IP, inhibitor protein; NR, NADH :nitrate reductase 13]. The interaction of IP with phospho-NR specifically inhibited electron flow between the heme and the molybdenum cofactor domains [9], most probably by altering the conformation of NR. Results from experiments with partially degraded NR [8] and a transgenic tobacco expressing a truncated form of NR [14] suggest that the N-terminal domain of the protein (i.e.…”

THE AMP-ACTIVATED/SNF1 PROTEIN KINASE SUBFAMILY: Metabolic Sensors of the Eukaryotic Cell?

“…the tuber) by the Figure 5 Alignment of sequences around the phosphorylation sites on 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases, sucrose phosphate synthases, and nitrate reductases from a number of higher plant species. For each enzyme, the phosphorylation site has been established for the species at the top of the list (138,(140)(141)(142). Other sequences are from the databases.…”

14‐3‐3 proteins associate with the regulatory phosphorylation site of spinach leaf nitrate reductase in an isoform‐specific manner and reduce dephosphorylation of Ser‐543 by endogenous protein phosphatases

“…E-mail: shuber@cropserv 1 .cropsci.ncsu.edu Abbreviations: IP, inhibitor protein; NR, NADH:nitrate reductase; NRk, NR kinase; NR-PP, NR-protein phosphatase. [13,14]. Thus, the most likely possibility is that the 14-3-3 protein(s) binds directly to the regulatory phosphorylation site of NR as is thought to occur with other proteins that interact with 14-3-3s [12].…”