Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase

Markus Bachmann, Naomasa Shiraishi, Wilbur H. Campbell, Byung Chun Yoo, Alice C. Harmon, Steven C. Huber

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)505-517
Number of pages13
JournalPlant Cell
Volume8
Issue number3
StatePublished - Mar 1 1996

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Nitrate Reductase
Spinacia oleracea
nitrate reductase
spinach
phosphorylation
Phosphorylation
phosphotransferases (kinases)
Phosphotransferases
leaves
synthetic peptides
Nitrate Reductase (NADH)
inactivation
nitrate reductase (NADH)
Peptides
Cytochromes b
protein phosphorylation
molybdenum
cytochrome b
protein kinases
Protein Kinases

ASJC Scopus subject areas

  • Plant Science
  • Cell Biology

Cite this

Bachmann, M., Shiraishi, N., Campbell, W. H., Yoo, B. C., Harmon, A. C., & Huber, S. C. (1996). Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase. Plant Cell, 8(3), 505-517.

Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase. / Bachmann, Markus; Shiraishi, Naomasa; Campbell, Wilbur H.; Yoo, Byung Chun; Harmon, Alice C.; Huber, Steven C.

In: Plant Cell, Vol. 8, No. 3, 01.03.1996, p. 505-517.

Research output: Contribution to journalArticle

Bachmann, M, Shiraishi, N, Campbell, WH, Yoo, BC, Harmon, AC & Huber, SC 1996, 'Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase', Plant Cell, vol. 8, no. 3, pp. 505-517.
Bachmann M, Shiraishi N, Campbell WH, Yoo BC, Harmon AC, Huber SC. Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase. Plant Cell. 1996 Mar 1;8(3):505-517.
Bachmann, Markus ; Shiraishi, Naomasa ; Campbell, Wilbur H. ; Yoo, Byung Chun ; Harmon, Alice C. ; Huber, Steven C. / Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase. In: Plant Cell. 1996 ; Vol. 8, No. 3. pp. 505-517.
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