Post-translational regulation of nitrate reductase activity: a role for Ca2+ and 14-3-3 proteins

Huber, Steven C.; Bachmann, M.; Huber, Joan L.

doi:10.1016/s1360-1385(96)10046-7

Cited by 139 publications

(108 citation statements)

References 40 publications

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“…also in Drosophila, mammals, and cyanobacteria (Dunlap, 1998;Nishiwaki et al, 2000). NR is known to be phosphorylated at a specific site, and this leads to binding of so-called 14-3-3 proteins and inhibition of NR activity (MacKintosh et al, 1995;Huber et al, 1996). In addition, phosphorylation is thought to influence degradation rate of the NR protein itself (Kaiser and Huber, 1997;Cotelle et al, 2000).…”

Section: Characterization Of the Nr Oscillatory Systemmentioning

confidence: 99%

The Nitrate Reductase Circadian System. The Central Clock Dogma Contra Multiple Oscillatory Feedback Loops

2001

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Section: Characterization Of the Nr Oscillatory Systemmentioning

confidence: 99%

The Nitrate Reductase Circadian System. The Central Clock Dogma Contra Multiple Oscillatory Feedback Loops

2001

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“…For PKC, the phosphorylation reaction was performed in a total volume of 150 mL, containing peptide substrate at various concentrations, 60 mm Tris/HCl (pH 7.5), 0.0013% Triton X-100, 0.75 mm CaCl 2 All phosphorylation reactions were carried out at 30 8C. For each peptide, the reaction time and enzyme amount were adjusted so that the initial rates of the phosphorylation reaction could be measured.…”

Section: Phosphorylation Of Synthetic Peptidesmentioning

confidence: 99%

Peptide phosphorylation by calcium‐dependent protein kinase from maize seedlings

Loog

Toomik

Sak

et al. 2000

European Journal of Biochemistry

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Ca2+ -dependent protein kinase (CDPK-1) was purified from maize seedlings, and its substrate specificity studied using a set of synthetic peptides derived from the phosphorylatable sequence RVLSRLHS 15 VRER of maize sucrose synthase 2. The decapeptide LARLHSVRER was found to be efficiently phosphorylated as a minimal substrate. The same set of peptides were found to be phosphorylated by mammalian protein kinase Cb (PKC), but showed low reactivity with protein kinase A (PKA). Proceeding from the sequence LARLHSVRER, a series of cellulose-membrane-attached peptides of systematically modified structure was synthesised. These peptides had hydrophobic (Ala, Leu) and ionic (Arg, Glu) amino acids substituted in each position. The phosphorylation of these substrates by CDPK-1 was measured and the substrate specificity of the maize protein kinase characterised by the consensus sequence motif A/L -5 X -4 R -3 X -2 X -1 SX +1 R +2 Z +3 R +4 , where X denotes a position with no strict amino acid requirements and Z a position strictly not tolerating arginine compared with the other three varied amino acids. This motif had a characteristic sequence element RZR at positions +2 to +4 and closely resembled the primary structure of the sucrose synthase phosphorylation site. The sequence surrounding the phosphorylatable serine in this consensus motif was similar to the analogous sequence K/RXXS/TXK/R proposed for mammalian PKC, but different from the consensus motif RRXS/TX for PKA. Recently it has been shown that one of the key enzymes of sucrose metabolism in maize, sucrose synthase type 2, can be phosphorylated at Ser15 in vivo [6,7]. This reaction seems to have regulatory significance, as the catalytic properties of the phosphorylated enzyme were affected [6].The phosphorylatable serine residue of sucrose synthase 2 is surrounded by the peptide sequence RVLSRLHS 15 VRER, with several positively charged amino acids before and after the Ser15 residue. This means that the substrate specificity of the relevant plant protein kinase(s) should follow similar principles to that of a large group of mammalian protein kinases, showing selectivity for positively charged amino acids [8]. The most extensively investigated members of this group are PKC and protein kinase A (PKA).In the present work, the substrate specificity of a protein kinase purified from maize seedlings was investigated using synthetic peptides derived from the maize sucrose synthase 2 phosphorylation site sequence, which were phosphorylated by this kinase in a Ca 2+ -dependent manner. The influence of peptide length on phosphorylation effectiveness was studied, and the sequence LARLHSVRER was found to be the minimal peptide substrate for this enzyme. Using this sequence, a series of structurally varied analogues were synthesized as cellulosemembrane-attached (SPOTs TM membranes) peptides. In these peptides, all the positions were substituted with hydrophobic (Leu, Ala) and charged (Arg, Glu) amino acids. By systematically varying the peptide structure, we were able to o...

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“…A phosphopeptide containing the 14-3-3-binding ' consensus ' phosphopeptide completely blocked the inhibition of phosphorylated NR by 14-3-3s, while a dephosphorylated peptide or unrelated phosphopeptides had no effect (Moorhead et al, 1996). The 14-3-3s also hinder the dephosphorylation of serine-543 on NR by the NR phosphatase (Bachmann et al, 1996). Together, these results show that 14-3-3s bind directly to the phosphorylation site of NR, and that the interaction is probably in dynamic equilibrium.…”

Section:      1 4-3-3 mentioning

confidence: 95%

Regulation of plant nitrate assimilation: from ecophysiology to brain proteins

MacKintosh

1998

New Phytologist

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Nitrogenous inorganic compounds impact plants as nutrients, signals and toxins. We are dissecting a regulatory network that controls nitrate assimilation at the level of nitrate reductase (NR) activity. The identification of protein kinase cascades, protein phosphatases and 14-3-3 proteins as regulators of NR are giving clues about how plants sense their nutrient availability, and use the information to signal changes in their metabolism and developmental strategies to cope with supplies. We hope that understanding these controls might lead to the design of transgenic plants with deregulated signalling networks, which would make them more efficient in using nitrogen fertilizers, and improving quality and yield of crops. There are circumstantial indications that gaseous anthropogenic nitrogenous emissions might also have complex regulatory influences on plant growth and development.

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Post-translational regulation of nitrate reductase activity: a role for Ca2+ and 14-3-3 proteins

Cited by 139 publications

References 40 publications

The Nitrate Reductase Circadian System. The Central Clock Dogma Contra Multiple Oscillatory Feedback Loops

The Nitrate Reductase Circadian System. The Central Clock Dogma Contra Multiple Oscillatory Feedback Loops

Peptide phosphorylation by calcium‐dependent protein kinase from maize seedlings

Regulation of plant nitrate assimilation: from ecophysiology to brain proteins

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