Antibodies to assess phosphorylation of spinach leaf nitrate reductase on serine 543 and its binding to 14-3-3 proteins.

To monitor site-specific phosphorylation of spinach leaf nitrate reductase (NR) and binding of the enzyme to 14-3-3 proteins, serum antibodies were raised that select for either serine 543 phospho- or dephospho-NR. The dephospho-specific antibodies blocked NR phosphorylation on serine 543. The phospho-specific antibodies prevented NR binding to 14-3-3s, NR inhibition by 14-3-3s, NR dephosphorylation on serine 543, and did not precipitate 14-3-3s together with NR. Together, this confirms that 14-3-3s bind to NR at hinge 1 after it has been phosphorylated on serine 543. The amounts of individual NR forms were determined in leaf extracts by immunoblotting and immunoprecipitation. The phosphorylation state of NR on serine 543 increased 2-3-fold in leaves upon a light/ dark transition. Before the transition, one-third of NR was already phosphorylated on serine 543 but was not bound to 14-3-3s. Phosphorylation of serine 543 seems not to be enough to bind to 14-3-3s in leaves.     

Reversible light/dark modulation of spinach leaf nitrate reductase activity involves protein phosphorylation.

Spinach (Spinacia oleracea L.) leaf nitrate reductase (NADH:NR;NADH:nitrate oxidoreductase, EC 1.6.6.1) activity was found to rapidly change during light/dark transitions. The most rapid and dramatic changes were found in a form of NR which was sensitive to inhibition by millimolar concentrations of magnesium. This form of NR predominated in leaves in the dark, but was almost completely absent from leaves incubated in the light for only 30 min. When the leaves were returned to darkness, the NR rapidly became sensitive to Mg2+ inhibition. Modulation of the overall reaction involving NADH as electron donor was also found when reduced methyl viologen was the donor (MV:NR), indicating that electron transfer had been blocked, at least in part, at or near the terminal molybdenum cofactor site. Changes in activity appear to be the result of a covalent modification that affects sensitivity of NR to inhibition by magnesium, and our results suggest that protein phosphorylation may be involved. NR was phosphorylated in vivo after feeding excised leaves [32P]Pi. The NR subunit was labeled exclusively on seryl residues in both light and dark. Tryptic peptide mapping indicated three major 32P-labeled phosphopeptide (Pp) fragments. Labeling of two of the P-peptides (designated Pp1 and 3) was generally correlated with NR activity assayed in the presence of Mg2+. In vivo, partial dephosphorylation of these sites (and activation of NR assayed with Mg2+) occurred in response to light or feeding mannose in darkness. The light effect was blocked completely by feeding okadaic acid via the transpiration stream, indicating the involvement of type 1 and/or type 2A protein phosphatases in vivo. While more detailed analysis is required to establish a causal link between the phosphorylation status of NR and sensitivity to Mg2+ inhibition, the current results are highly suggestive of one. Thus, in addition to the molecular genetic mechanisms regulating this key enzyme of nitrate assimilation, NR activity may be controlled in leaves by phosphorylation/dephosphorylation of the enzyme protein resulting from metabolic changes taking place during light/dark transitions.     

Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in high nitrite excretion and NO emission from leaf and root tissue

In wild-type Nicotiana plumbaginifolia Viv. and other higher plants, nitrate reductase (NR) is regulated at the post-translational level and is rapidly inactivated in response to, for example, a light-to-dark transition. This inactivation is caused by phosphorylation of a conserved regulatory serine residue, Ser 521 in tobacco, and interaction with divalent cations or polyamines, and 14-3-3 proteins. The physiological importance of the post-translational NR modulation is presently under investigation using a transgenic N. plumbaginifolia line. This line expresses a mutated tobacco NR where Ser 521 has been changed into aspartic acid (Asp) by site-directed mutagenesis, resulting in a permanently active NR enzyme [C. Lillo et al. (2003) Plant J 35:566–573]. When cut leaves or roots of this line (S₅₂₁) were placed in darkness in a buffer containing 50 mM KNO₃, nitrite was excreted from the tissue at rates of 0.08–0.2 mol (g FW)^–1 h^–1 for at least 5 h. For the control transgenic plant (C1), which had the regulatory serine of NR intact, nitrite excretion was low and halted completely after 1–3 h. Without nitrate in the buffer in which the tissue was immersed, nitrite excretion was also low for S₅₂₁, although 20–40 mol (g FW)^–1 nitrate was present inside the tissue. Apparently, stored nitrate was not readily available for reduction in darkness. Leaf tissue and root segments of S₅₂₁ also emitted much more nitric oxide (NO) than the control. Importantly, NO emission from leaf tissue of S₅₂₁ was higher in the dark than in the light, opposite to what was usually observed when post-translational NR modulation was operating.Abbreviations NR Nitrate reductase - NO Nitric oxide - Ser Serine - WT Wild type     

Identification of the leaf vacuole as a major nitrate storage pool

Highly purified vacuoles were isolated from protoplasts derived from green barley (Hordeum vulgare var. Numar) leaves, in order to determine their role as a NO₃⁻ storage sink. α-Mannosidase and acid phosphatase activities were used as markers to identify vacuoles, α-mannosidase being the more suitable. Nitrate and α-mannosidase, which were released from vacuoles destroyed during lysis of protoplasts, moved at unequal rates in the density gradient used for vacuole isolation. Purified vacuoles retained less NO₃⁻ than α-mannosidase during a single washing. Empirically determined corrections were used to account for NO₃⁻ movement in estimating the percentage of total cellular nitrate found in the vacuole. Vacuoles from plants grown in the presence of NO₃⁻ contained 58% of the total cellular NO₃⁻ and therefore represent a major NO₃⁻ storage pool.     

Identification in vitro of a post-translational regulatory site in the hinge 1 region of Arabidopsis nitrate reductase.

Nitrate reductase (NR) is rapidly inactivated by phosphorylation of serine residues in response to loss of light or reduction in CO2 levels. To identify sites within NR protein that play a role in this post-translational regulation, a heterologous expression system and an in vitro inactivation assay for Arabidopsis NR were developed. Protein extracts containing NR kinases and inhibitor proteins were prepared from an NR-defective mutant that had lesions in both the NIA1 and NIA2 NR genes of Arabidopsis. Active NR protein was produced in a Pichia pastoris expression system. Incubation of these two preparations resulted in a Mg-ATP-dependent inactivation of NR that was reversed with EDTA. Mutant forms of NR were constructed, produced in P. pastoris, and tested in the in vitro inactivation assay. Six conserved serine residues in the hinge 1 region of NR, which separates the molybdenum cofactor and heme domains, were specifically targeted for mutagenesis because they are located in a potential regulatory region identified as a target for NR kinases in spinach. A change in Ser-534 to aspartate was found to block NR inactivation; changes in the other five serines had no effect. The aspartate that replaced Ser-534 did not appear to mimic a phosphorylated serine but simply prevented the NR from being inactivated. These results identify Ser-534, located in the hinge 1 of NR and conserved among higher plants NRs, as an essential site for post-translational regulation in vitro.     

Identification of Ser-55 as a major protein kinase A phosphorylation site on the 70-kDa subunit of neurofilaments. Early turnover during axonal transport.

The 70-kDa neurofilament protein subunit (NF-L) is phosphorylated in vivo on at least three sites (L1 to L3) (Sihag, R. K. and Nixon, R. A. (1989) J. Biol. Chem. 264, 457-464). The turnover of phosphate groups on NF-L during axonal transport was determined after the neurofilaments in retinal ganglion cells were phosphorylated in vivo by injecting mice intravitreally with [32P]orthophosphate. Two-dimensional phosphopeptide maps of NF-L from optic axons of mice 10 to 90 h after injection showed that radiolabel decreased faster from peptides L2 and L3 than from L1 as neurofilaments were transported. To identify phosphorylation sites on peptide L2, axonal cytoskeletons were phosphorylated by protein kinase A in the presence of heparin. After the isolated NF-L subunits were digested with alpha-chymotrypsin, 32P-peptides were separated by high performance liquid chromatography on a reverse-phase C8 column. Two-dimensional peptide mapping showed that the alpha-chymotrypsin 32P-peptide accepting most of the phosphates from protein kinase A migrated identically with the in vivo-labeled phosphopeptide L2. The sequence of this peptide (S-V-R-R-S-Y) analyzed by automated Edman degradation corresponded to amino acid residues 51-56 of the NF-L sequence. A synthetic 13-mer (S-L-S-V-R-R-S-Y-S-S-S-S-G) corresponding to amino acid residues 49-61 of NF-L was also phosphorylated by protein kinase A. alpha-Chymotryptic digestion of the 13-mer generated a peptide which contained most of the phosphates and co-migrated with the phosphopeptide L2 on two-dimensional phosphopeptide maps. Edman degradation of the phosphorylated 13-mer identified serine residue 55 which is located within a consensus phosphorylation sequence for protein kinase A as the major site of phosphorylation. Since protein kinase A-mediated phosphorylation influences intermediate filament assembly/disassembly in vitro, we propose that the phosphopeptide L2 region is a neurofilament-assembly domain and that the cycle of phosphorylation and dephosphorylation of Ser-55 on NF-L, which occurs relatively early after subunit synthesis in vivo, regulaaes a step in neurofilament assembly or initial interactions during axonal transport.     

Identification of a novel phosphorylation site, Ser-170, as a regulator of bad pro-apoptotic activity.

Bad is a pro-apoptotic member of the Bcl-2 family of proteins that is thought to exert a death-promoting effect by heterodimerization with Bcl-X(L), nullifying its anti-apoptotic activity. Growth factors may promote cell survival at least partially through phosphorylation of Bad at one or more of Ser-112, -136, or -155. Our previous work showed that Bad is also phosphorylated in response to cytokines at another site, which we now identify as Ser-170. The functional role of this novel phosphorylation site was assessed by site-directed mutagenesis and analysis of the pro-apoptotic function of Bad in transiently transfected HEK293 and COS-7 cells or by stable expression in the cytokine-dependent cell line, MC/9. In general, mutation of Ser-170 to Ala results in a protein with increased ability to induce apoptosis, similar to the S112A mutant. Mutation of Ser-170 to Asp, mimicking a constitutively phosphorylated site, results in a protein that is virtually unable to induce apoptosis. Similarly, the S112A/S170D double mutant does not cause apoptosis in HEK293 and MC/9 cell lines. These data strongly suggest that phosphorylation of Bad at Ser-170 is a critical event in blocking the pro-apoptotic activity of Bad.     

On the regulation of spinach nitrate reductase

A coupled assay has been worked out to study spinach (Spinacea oleracea L.) nitrate reductase under low, more physiological concentrations of NADH. In this assay the reduction of nitrate is coupled to the oxidation of malate catalyzed by spinach NAD-malate dehydrogenase. The use of this coupled system allows the assay of nitrate reductase activity at steady-state concentrations of NADH below micromolar. We have used this coupled assay to study the kinetic parameters of spinach nitrate reductase and to reinvestigate the putative regulatory role of adenine nucleotides, inorganic phosphate, amino acids, and calcium and calmodulin.     

Chloride inhibition of spinach nitrate reductase

Initial rate studies of spinach (Spinacia oleracea L.) nitrate reductase showed that NADH:nitrate reductase activity was ionic strength dependent with elevated ionic concentration resulting in inhibition. In contrast, NADH:ferricyanide reductase was markedly less ionic strength dependent. At pH 7.0, NADH:nitrate reductase activity exhibited changes in the V_max and K_m for NO₃⁻ yielding V_max values of 6.1 and 4.1 micromoles NADH per minute per nanomoles heme and K_m values of 13 and 18 micromolar at ionic strengths of 50 and 200 millimolar, respectively. Control experiments in phosphate buffer (5 millimolar) yielded a single K_m of 93 micromolar. Chloride ions decreased both NADH:nitrate reductase and reduced methyl viologen:nitrate reductase activities, suggesting involvement of the Mo center. Chloride was determined to act as a linear, mixed-type inhibitor with a K_i of 15 millimolar for binding to the native enzyme and 176 millimolar for binding to the enzyme-NO₃⁻ complex. Binding of Cl⁻ to the enzyme-NO₃⁻ complex resulted in an inactive E-S-I complex. Electron paramagnetic resonance spectra showed that chloride altered the observed Mo(V) lineshape, confirming Mo as the site of interaction of chloride with nitrate reductase.     

Mutation of the regulatory phosphorylation site of tobacco nitrate reductase results in constitutive activation of the enzyme in vivo and nitrite accumulation

In wild-type Nicotiana plumbaginifolia and other higher plants, nitrate reductase (NR) is rapidly inactivated/activated in response to dark/light transitions. Inactivation of NR is believed to be caused by phosphorylation at a special conserved regulatory Ser residue, Ser 521, and interactions with divalent cations and inhibitory 14-3-3 proteins. A transgenic N. plumbaginifolia line (S(521)) was constructed where the Ser 521 had been changed by site-directed mutagenesis into Asp. This mutation resulted in complete abolishment of inactivation in response to light/dark transitions or other treatments known to inactivate NR. During prolonged darkness, NR in wild-type plants is in the inactivated form, whereas NR in the S(521) line is always in the active form. Differences in degradation rate between NR from S(521) and lines with non-mutated NR were not found. Kinetic constants like Km values for NADH and NO3(-) were not changed, but a slightly different pH profile was observed for mutated NR as opposed to non-mutated NR. Under optimal growth conditions, the phenotype of the S(521) plants was not different from the wild type (WT). However, when plants were irrigated with high nitrate concentration, 150 mM, the transgenic plants accumulated nitrite in darkness, and young leaves showed chlorosis.     

Ser-59 is the major phosphorylation site in alphaB-crystallin accumulated in the brains of patients with Alexander's disease

The phosphorylation state of alphaB-crystallin accumulated in the brains of two patients with Alexander's disease (one infantile and one juvenile type) was determined by means of SDS-PAGE or isoelectric focusing of soluble and insoluble fractions of brain extracts and subsequent western blot analysis with specific antibodies against alphaB-crystallin and each of three phosphorylated serine residues. The level of mammalian small heat shock protein of 25-28 kDa (Hsp27) in the same fraction was also estimated by western blot analysis. The majority of alphaB-crystallin was detected in the insoluble fraction of brain homogenates and phosphorylation was preferentially observed at Ser-59 in both cases. A significant level of phosphorylation at Ser-45 but not Ser-19 was also detected. Hsp27 was found at considerable levels in the insoluble fractions. alphaB-crystallin and phosphorylated forms were detected in the cerebrospinal fluid of patient with the juvenile type. AlphaB-crystallin and phosphorylated forms were also detectable at considerable levels in the insoluble fraction of brain homogenates from patients with Alzheimer's disease and aged controls. The phosphorylation site was mostly at Ser-59 in all cases. Immunohistochemically, alphaB-crystallin was stained in Rosenthal fibers in brains of patients with Alexander's disease and their peripheral portions were immunostained with antibodies recognizing phosphorylated Ser-59. These results indicate that the major phosphorylation site in alphaB-crystallin in brains of patients with Alexander's disease or Alzheimer's disease as well as in aged controls is Ser-59.     

Limited proteolysis of the nitrate reductase from spinach leaves

The functional structure of assimilatory NADH-nitrate reductase from spinach leaves was studied by limited proteolysis experiments. After incubation of purified nitrate reductase with trypsin, two stable products of 59 and 45 kDa were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The fragment of 45 kDa was purified by Blue Sepharose chromatography. NADH-ferricyanide reductase and NADH-cytochrome c reductase activities were associated with this 45-kDa fragment which contains FAD, heme, and NADH binding fragment. After incubation of purified nitrate reductase with Staphylococcus aureus V8 protease, two major peaks were observed by high performance liquid chromatography size exclusion gel filtration. FMNH2-nitrate reductase and reduced methyl viologen-nitrate reductase activities were associated with the first peak of 170 kDa which consists of two noncovalently associated (75-90-kDa) fragments. NADH-ferricyanide reductase activity, however, was associated with the second peak which consisted of FAD and NADH binding sites. Incubation of the 45-kDa fragment with S. aureus V8 protease produced two major fragments of 28 and 14 kDa which contained FAD and heme, respectively. These results indicate that the molybdenum, heme, and FAD components of spinach nitrate reductase are contained in distinct domains which are covalently linked by exposed hinge regions. The molybdenum domain appears to be important in the maintenance of subunit interactions in the enzyme complex.     

Inhibition of nitrate reductase from spinach (Spinacea oleracea L.) leaf by adenosine nucleotides

NADH-dependent nitrate reductase of spinach showed ping-pongKinetics. Inhibition by ADP in the absence of thiols increasedduring a transient phase during which Kinetics changed fromweak (K₁ 20 mM), non-competitive and linear to comparativelystrong (K₁ 0.09 mM at 10 mM ADP), mixed with respect to NADHand non-linear (n_H = 1.7). Thiol compounds transformed the inhibitionto competitive (K₁ 0.18 mM at 10 mM ADP) and non-linear (n_H= 2.3). Effective concentrations increased in order: GSH>mercaptoethanol= thioglycollate>cysteine, and were increased by dissolvedoxygen or disulphide. Non-competitive inhibition by ribose-5-phosphate(K₁ 3 nua) was abolished by glutathione. Cyclic 3',5'-AMP inhibitednon-linearly (nH=2.4) and comparably to or more than ADP (K₁0.22 mM at 2.5 mM) but predominantly competitively except atlow NADH concentrations. Cyclic 2',3'-AMP showed similar butweaker inhibition (K₁ 1.4 mM) and more nearly linear (n_H =1.3).5'-AMP inhibited competitively (K₁ 1.2 mM at 10 mM) (n_H = 1.3).5'-ATP showed weak mixed inhibition (K₁ 5 nun at 10 mM) (n_H= l.3). Activity with BV° was scarcely inhibited by ADP,but phosphate activation with BV° or NADH were both inhibitedby ADP. Physiological significance of inhibition by ADP is discussedand might lie between 3% in light and 25% in dark in specialcircumstances of thiol supply. ¹ Present address: Department of Soil Microbiology, RothamstedExperimental Station, Harpenden, Herts. (Received May 22, 1975; )     

Effects of denaturing agents on spinach nitrate reductase

The effects of various denaturing agents (temperature, pH, urea, guanidine hydrochloride and sodium dodecyl sulphate) on the 3 enzymic activities of th     

Regulation of spinach-leaf nitrate reductase by reversible phosphorylation.

Okadaic acid and microcystin (but not the inactive methyl esters of these toxins) prevented the rapid light-induced activation of nitrate reductase (NR) in intact spinach leaves. In vitro, nitrate reductase was inactivated by a protein kinase and activated by PP2A. The role of reversible protein phosphorylation in regulation of light-coupled cytoplasmic metabolism is discussed.