A structural basis for 14-3-3sigma functional specificity

The 14-3-3 family of proteins includes seven isotypes in mammalian cells that play numerous diverse roles in intracellular signaling. Most 14-3-3 proteins form homodimers and mixed heterodimers between different isotypes, with overlapping roles in ligand binding. In contrast, one mammalian isoform, 14-3-3sigma, expressed primarily in epithelial cells, appears to play a unique role in the cellular response to DNA damage and in human oncogenesis. The biological and structural basis for these 14-3-3sigma-specific functions is unknown. We demonstrate that endogenous 14-3-3sigma preferentially forms homodimers in cells. We have solved the x-ray crystal structure of 14-3-3sigma bound to an optimal phosphopeptide ligand at 2.4 angstroms resolution. The structure reveals the presence of stabilizing ring-ring and salt bridge interactions unique to the 14-3-3sigma homodimer structure and potentially destabilizing electrostatic interactions between subunits in 14-3-3sigma-containing heterodimers, rationalizing preferential homodimerization of 14-3-3sigma in vivo. The interaction of the phosphopeptide with 14-3-3 reveals a conserved mechanism for phospho-dependent ligand binding, implying that the phosphopeptide binding cleft is not the critical determinant of the unique biological properties of 14-3-3sigma. Instead, the structure suggests a second ligand binding site involved in 14-3-3sigma-specific ligand discrimination. We have confirmed this by site-directed mutagenesis of three sigma-specific residues that uniquely define this site. Mutation of these residues to the alternative sequence that is absolutely conserved in all other 14-3-3 isotypes confers upon 14-3-3sigma the ability to bind to Cdc25C, a ligand that is known to bind to other 14-3-3 proteins but not to sigma.     

Phosphorylated Nitrate Reductase and 14-3-3 Proteins : Site of Interaction, Effects of Ions, and Evidence for an AMP-Binding Site on 14-3-3 Proteins

The inactivation of phosphorylated nitrate reductase (NR) by the binding of 14-3-3 proteins is one of a very few unambiguous biological functions for 14-3-3 proteins. We report here that serine and threonine residues at the +6 to +8 positions, relative to the known regulatory binding site involving serine-543, are important in the interaction with GF14ω, a recombinant plant 14-3-3. Also shown is that an increase in ionic strength with KCl or inorganic phosphate, known physical effectors of NR activity, directly disrupts the binding of protein and peptide ligands to 14-3-3 proteins. Increased ionic strength attributable to KCl caused a change in conformation of GF14ω, resulting in reduced surface hydrophobicity, as visualized with a fluorescent probe. Similarly, it is shown that the 5′ isomer of AMP was specifically able to disrupt the inactive phosphorylated NR:14-3-3 complex. Using the 5′-AMP fluorescent analog trinitrophenyl-AMP, we show that there is a probable AMP-binding site on GF14ω.     

14-3-3zeta C-terminal stretch changes its conformation upon ligand binding and phosphorylation at Thr232

14-3-3 proteins are abundant binding proteins involved in many biologically important processes. 14-3-3 proteins bind to other proteins in a phosphorylation-dependent manner and function as scaffold molecules modulating the activity of their binding partners. In this work, we studied the conformational changes of 14-3-3 C-terminal stretch, a region implicated in playing a role in the regulation of 14-3-3. Time-resolved fluorescence and molecular dynamics were used to investigate structural changes of the C-terminal stretch induced by phosphopeptide binding and phosphorylation at Thr232, a casein kinase I phosphorylation site located within this region. A tryptophan residue placed at position 242 was exploited as an intrinsic fluorescence probe of the C-terminal stretch dynamics. Other tryptophan residues were mutated to phenylalanine. Time-resolved fluorescence measurements revealed that phosphopeptide binding changes the conformation and increases the flexibility of 14-3-3zeta C-terminal stretch, demonstrating that this region is directly involved in ligand binding. Phosphorylation of 14-3-3zeta at Thr232 resulted in inhibition of phosphopeptide binding and suppression of 14-3-3-mediated enhancement of serotonin N-acetyltransferase activity. Time-resolved fluorescence of Trp242 also revealed that phosphorylation at Thr232 induces significant changes of the C-terminal stretch conformation. In addition, molecular dynamic simulations suggest that phosphorylation at Thr232 induces a more extended conformation of 14-3-3zeta C-terminal stretch and changes its interaction with the rest of the 14-3-3 molecule. These results indicate that the conformation of the C-terminal stretch plays an important role in the regulation of 14-3-3 binding properties.     

Divalent cation effects on interactions between multiple Arabidopsis 14-3-3 isoforms and phosphopeptide targets

Oscillations in cellular divalent cation concentrations are key events that can trigger signal transduction cascades. Common cellular divalent cations, such as calcium and magnesium, interact with 14-3-3 proteins. The metal ion interaction causes a conformational change in the 14-3-3 proteins, which is manifested as an increase in hydrophobicity. In this study, the effect of divalent cations on the interaction between 14-3-3 proteins and target peptides was investigated using surface plasmon resonance and isothermal titration calorimetry. The binding between ten recombinant Arabidopsis 14-3-3 isoforms and two synthetic target peptides was observed in the presence of various physiologically relevant concentrations of calcium or magnesium, from 1 microM to 1 mM or from 1 microM to 5 mM, respectively. The synthetic target peptides were based on sequences from Arabidopsis nitrate reductase (NR2) and the plasma membrane proton pump (AHA2) representing fundamentally different target classes. Isoforms representing every branch of the Arabidopsis 14-3-3 phylogenetic tree were tested. The general result for all cases is that an increased concentration of divalent cations in solution causes an increase in the concentration of 14-3-3 protein interacting with the respective phosphopeptide.     

Phosphorylation-dependent interactions between enzymes of plant metabolism and 14-3-3 proteins

Far-Western overlays of soluble extracts of cauliflower revealed many proteins that bound to digoxygenin (DIG)-labelled 14-3-3 proteins. Binding to DIG-14-3-3s was prevented by prior dephosphorylation of the extract proteins or by competition with 14-3-3-binding phosphopeptides, indicating that the 14-3-3 proteins bind to phosphorylated sites. The proteins that bound to the DIG-14-3-3s were also immunoprecipitated from extracts with anti-14-3-3 antibodies, demonstrating that they were bound to endogenous plant 14-3-3 proteins. 14-3-3-binding proteins were purified from cauliflower extracts, in sufficient quantity for amino acid sequence analysis, by affinity chromatography on immobilised 14-3-3 proteins and specific elution with a 14-3-3-binding phosphopeptide. Purified 14-3-3-binding proteins included sucrose–phosphate synthase, trehalose-6-phosphate synthase, glutamine synthetases, a protein (LIM17) that has been implicated in early floral development, an approximately 20 kDa protein whose mRNA is induced by NaCl, and a calcium-dependent protein kinase that was capable of phosphorylating and rendering nitrate reductase (NR) sensitive to inhibition by 14-3-3 proteins. In contrast to the phosphorylated NR-14-3-3 complex which is activated by dissociation with 14-3-3-binding phosphopeptides, the total sugar–phosphate synthase activity in plant extracts was inhibited by up to 40% by a 14-3-3-binding phosphopeptide and the phosphopeptide-inhibited activity was reactivated by adding excess 14-3-3 proteins. Thus, 14-3-3 proteins are implicated in regulating several aspects of primary N and C metabolism. The procedures described here will be valuable for determining how the phosphorylation and 14-3-3-binding status of defined target proteins change in response to extracellular stimuli.     

Affinity purification of diverse plant and human 14-3-3-binding partners

Many proteins that bind to a 14-3-3 column in competition with a 14-3-3-binding phosphopeptide have been purified from plant and mammalian cells and tissues. New 14-3-3 targets include enzymes of biosynthetic metabolism, vesicle trafficking, cell signalling and chromatin function. These findings indicate central regulatory roles for 14-3-3s in partitioning carbon among the pathways of sugar, amino acid, nucleotide and protein biosynthesis in plants. Our results also suggest that the current perception that 14-3-3s bind predominantly to signalling proteins in mammalian cells is incorrect, and has probably arisen because of the intensity of research on mammalian signalling and for technical reasons.     

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.     

Interaction of 14-3-3 with a nonphosphorylated protein ligand, exoenzyme S of Pseudomonas aeruginosa

The 14-3-3 proteins are a family of conserved, dimeric proteins that interact with a diverse set of ligands, including molecules involved in cell cycle regulation and apoptosis. It is well-established that 14-3-3 binds to many ligands through phosphoserine motifs. Here we characterize the interaction of 14-3-3 with a nonphosphorylated protein ligand, the ADP-ribosyltransferase Exoenzyme S (ExoS) from Pseudomonas aeruginosa. By using affinity chromatography and surface plasmon resonance, we show that the zeta isoform of 14-3-3 (14-3-3zeta) can directly bind a catalytically active fragment of ExoS in vitro. The interaction between ExoS and 14-3-3zeta is of high affinity, with an equilibrium dissociation constant of 7 nM. ExoS lacks any known 14-3-3 binding motif, but to address the possibility that 14-3-3 binds a noncanonical phosphoserine site, we assayed ExoS for protein-bound phosphate by using mass spectrometry. No detectable phosphoproteins were found. A phosphopeptide ligand of 14-3-3, pS-Raf-259, was capable of inhibiting the binding of 14-3-3 to ExoS, suggesting that phosphorylated and nonphosphorylated ligands may share a common binding site, the conserved amphipathic groove. It is conceivable that 14-3-3 proteins may bind both phosphoserine and nonphosphoserine ligands in cells, possibly allowing kinase-dependent as well as kinase-independent regulation of 14-3-3 binding.     

Deletion of the nitrate reductase N-terminal domain still allows binding of 14-3-3 proteins but affects their inhibitory properties

Nitrate reductase (NR) is post-translationally regulated by phosphorylation and binding of 14-3-3 proteins. Deletion of 56 amino acids in the amino-terminal domain of NR was previously shown to impair this type of regulation in tobacco (Nicotiana plumbaginifolia) (L. Nussaume, M. Vincentez, C. Meyer, J.-P. Boutin, M. Caboche [1995] Plant Cell 7: 611-621), although both full-length NR and deleted NR (DeltaNR) appeared to be phosphorylated in darkness (C. Lillo, S. Kazazaic, P. Ruoff, C. Meyer [1997] Plant Physiol 114: 1377-1383). We show here that in the presence of Mg(2+) and phosphatase inhibitors, NR and endogenous 14-3-3 proteins copurify through affinity chromatography. Assay of NR activity and western blots showed that endogenous 14-3-3 proteins copurified with both NR and DeltaNR. Electron transport in the heme-binding domain of DeltaNR was inhibited by Mg(2+)/14-3-3, whereas this was not the case for NR. This may indicate a different way of binding for 14-3-3 in the DeltaNR compared with NR. The DeltaNR was more labile than NR, in vitro. Lability was ascribed to the molybdopterin binding domain, and apparently an important function of the 56 amino acids is stabilization of this domain.     

14-3-3 protein C-terminal stretch occupies ligand binding groove and is displaced by phosphopeptide binding

14-3-3 proteins are important regulators of numerous cellular signaling circuits. They bind to phosphorylated protein ligands and regulate their functions by a number of different mechanisms. The C-terminal part of the 14-3-3 protein is known to be involved in the regulation of 14-3-3 binding properties. The structure of this region is unknown; however, a possible location of the C-terminal stretch within the ligand binding groove of the 14-3-3 protein has been suggested. To fully understand the role of the C-terminal stretch in the regulation of the 14-3-3 protein binding properties, we investigated the physical location of the C-terminal stretch and its changes upon the ligand binding. For this purpose, we have used Forster resonance energy transfer (FRET) measurements and molecular dynamics simulation. FRET measurements between Trp242 located at the end of the C-terminal stretch and a dansyl group attached at two different cysteine residues (Cys25 or Cys189) indicated that in the absence of the ligand, the C-terminal stretch occupies the ligand binding groove of 14-3-3 protein. Our data also showed that phosphopeptide binding displaces the C-terminal stretch from the ligand binding groove. Intramolecular distances calculated from FRET measurements fit well with distances obtained from molecular dynamics simulation of full-length 14-3-3zeta protein.     

The C-terminal tail of Arabidopsis 14-3-3omega functions as an autoinhibitor and may contain a tenth alpha-helix

The eukaryotic regulatory protein 14-3-3 is involved in many important plant cellular processes including regulation of nitrate assimilation through inhibition of phosphorylated nitrate reductase (pNR) in darkened leaves. Divalent metal cations (Me2+) and some polyamines interact with the loop 8 region of the 14-3-3 proteins and allow them to bind and inhibit pNR in vitro. The role of the highly variant C-terminal regions of the 14-3-3 isoforms in regulation by polycations is not clear. In this study, we carried out structural analyses on the C-terminal tail of the Arabidopsis 14-3-3omega isoform and evaluated its contributions to the inhibition of pNR. Nested C-terminal truncations of the recombinant 14-3-3omega protein revealed that the removal of the C-terminal tail renders the protein partially Mg2+-independent in both pNR binding and inhibition of activity, suggesting that the C-terminus functions as an autoinhibitor. The C-terminus of 14-3-3omega appears to undergo a conformational change in the presence of polycations as demonstrated by its increased trypsin cleavage at Lys-247. C-terminal truncation of 14-3-3omega at Thr-255 increased its interaction with antibodies to the C-terminus of 14-3-3omega in non-denaturing conditions, but not in denaturing conditions, suggesting that the C-terminal tail contains ordered structures that might be disrupted by the truncation. Circular dichroism (CD) analysis of a C-terminal peptide, from Trp-234 to Lys-249, revealed that the C-terminal tail might contain a tenth alpha-helix, in agreement with the in silico predictions. The function of the putative tenth alpha-helix is not clear because substituting two prolyl residues within the predicted helix (E245P/I246P mutant), which prevented the corresponding peptide from adopting a helical conformation, did not affect the inhibition of pNR activity in the presence or absence of Mg2+. We propose that in the absence of polycations, access of target proteins to their binding groove in the 14-3-3 protein is restricted by the C-terminus, which acts as part of a gate that opens with the binding of polycations to loop 8.     

Structural basis for SARS-CoV-2 nucleocapsid (N) protein recognition by 14-3-3 proteins

14-3-3 proteins are important dimeric scaffolds that regulate the function of hundreds of proteins in a phosphorylation-dependent manner. The SARS-CoV-2 nucleocapsid (N) protein forms a complex with human 14-3-3 proteins upon phosphorylation, which has also been described for other coronaviruses. Here, we report a high-resolution crystal structure of 14-3-3 bound to an N phosphopeptide bearing the phosphoserine 197 in the middle. The structure revealed two copies of the N phosphopeptide bound, each in the central binding groove of each 14-3-3 monomer. A complex network of hydrogen bonds and water bridges between the peptide and 14-3-3 was observed explaining the high affinity of the N protein for 14-3-3 proteins.     

FTICR-MS analysis of 14-3-3 isoform substrate selection

The 14-3-3s are a ubiquitous class of eukaryotic proteins that participate in a second regulatory step in many phosphorylation-based signal transduction systems. The Arabidopsis family of 14-3-3 proteins represents a rather large 14-3-3 gene family. The biological motive for such diversity within a single protein family is not yet completely understood. The work presented here utilizes 14-3-3 micro-affinity chromatography in conjunction with Fourier transform ion cyclotron resonance mass spectrometry to survey the substrate sequence selectivity of two Arabidopsis 14-3-3 isoforms that represent the two major subclasses of this protein family. A method was developed to compare the relative binding of eight synthetic phosphopeptide sequences. The degree to which each phosphopeptide bound to either isoform was assigned a relative value, defined here as the binding ratio. The method provided a simple means for visualizing differences in substrate sequence selection among different 14-3-3 isoforms. A reproducible preference for specific phosphopeptide sequences was measured for both isoforms. This binding preference was consistent among the two classes of isoforms, suggesting that any pressure for isoform selectivity must reside outside the central core that interacts with the phosphopeptide sequence of the client.     

Polycations globally enhance binding of 14-3-3omega to target proteins in spinach leaves

The binding of 14-3-3omega to phosphorylated nitrate reductase (pNR) is stimulated by cations such as Mg(2+) or spermine, and decreased by 5'-AMP. In order to determine whether binding to other cellular proteins is affected similarly, far-Western overlays of extracts prepared from light- or dark-treated spinach (Spinacia oleracea) leaves were performed using digoxigenin (DIG)-labeled Arabidopsis 14-3-3omega. When separated by SDS-PAGE, approximately 25 proteins of >35 kDa could be resolved that interacted with DIG-labeled 14-3-3omega in the absence of added cations. The presence of 5 mM Mg(2+) or 0.5 mM spermine enhanced binding to most of the target proteins to a maximum of about a doubling of the observed binding. In most cases, the binding was dependent on phosphorylation of the target protein, whereas that was not necessarily the case for binding to target proteins that were unaffected by polycations. The extent of stimulation varied among the target proteins, but there was no indication that the nature of the cation activator (e.g. Mg(2+) vs. spermine(4+)) altered the specificity for target proteins. In addition, binding of DIG-labeled 14-3-3omega to some, but not all, target proteins was reduced by 5 mM 5'-AMP. Interestingly, light/dark treatment of spinach leaves affected the subsequent binding of DIG-labeled 14-3-3omega in the overlay assay to only a few of the target proteins, one of which was identified as NADH:nitrate reductase. Overall, the results suggest that the binding of 14-3-3s to targets in addition to pNR may also be regulated by polycations and 5'-AMP.     

14-3-3s regulate fructose-2,6-bisphosphate levels by binding to PKB-phosphorylated cardiac fructose-2,6-bisphosphate kinase/phosphatase

The cardiac isoform of 6-phosphofructo-2-kinase/ fructose-2,6-bisphosphatase (PFK-2), regulator of the glycolysis-stimulating fructose-2,6-bisphosphate, was among human HeLa cell proteins that were eluted from a 14-3-3 affinity column using the phosphopeptide ARAApSAPA. Tryptic mass fingerprinting and phospho-specific antibodies showed that Ser466 and Ser483 of 14-3-3-affinity-purified PFK-2 were phosphorylated. 14-3-3 binding was abolished by selectively dephosphorylating Ser483, and 14-3-3 binding was restored when both Ser466 and Ser483 were phosphorylated with PKB, but not when Ser466 alone was phosphorylated by AMPK. Furthermore, the phosphopeptide RNYpS(483)VGS blocked binding of PFK-2 to 14-3-3s. These data indicate that 14-3-3s bind to phosphorylated Ser483. When HeLa cells expressing HA-tagged PFK-2 were co-transfected with active PKB or stimulated with IGF-1, HA-PFK-2 was phosphorylated and bound to 14-3-3s. The response to IGF-1 was abolished by PI 3-kinase inhibitors. In addition, IGF-1 promoted the binding of endogenous PFK-2 to 14-3-3s. When cells were transduced with penetratin-linked AARAApSAPA, we found that this reagent bound specifically to 14-3-3s, blocked the IGF-1-induced binding of HA-PFK-2 to 14-3-3s, and completely inhibited the IGF-1-induced increase in cellular fructose-2,6-bisphosphate. These findings suggest that PKB-dependent binding of 14-3-3s to phospho-Ser483 of cardiac PFK-2 mediates the stimulation of glycolysis by growth factor.     

Phosphorylation of Arabidopsis Ubiquitin Ligase ATL31 Is Critical for Plant Carbon/Nitrogen Nutrient Balance Response and Controls the Stability of 14-3-3 Proteins

Ubiquitin ligase plays a fundamental role in regulating multiple cellular events in eukaryotes by fine-tuning the stability and activity of specific target proteins. We have previously shown that ubiquitin ligase ATL31 regulates plant growth in response to nutrient balance between carbon and nitrogen (C/N) in Arabidopsis. Subsequent study demonstrated that ATL31 targets 14-3-3 proteins for ubiquitination and modulates the protein abundance in response to C/N-nutrient status. However, the underlying mechanism for the targeting of ATL31 to 14-3-3 proteins remains unclear. Here, we show that ATL31 interacts with 14-3-3 proteins in a phosphorylation-dependent manner. We identified Thr²⁰⁹, Ser²⁴⁷, Ser²⁷⁰, and Ser³⁰³ as putative 14-3-3 binding sites on ATL31 by motif analysis. Mutation of these Ser/Thr residues to Ala in ATL31 inhibited the interaction with 14-3-3 proteins, as demonstrated by yeast two-hybrid and co-immunoprecipitation analyses. Additionally, we identified in vivo phosphorylation of Thr²⁰⁹ and Ser²⁴⁷ on ATL31 by MS analysis. A peptide competition assay showed that the application of synthetic phospho-Thr²⁰⁹ peptide, but not the corresponding unphosphorylated peptide, suppresses the interaction between ATL31 and 14-3-3 proteins. Moreover, Arabidopsis plants overexpressing mutated ATL31, which could not bind to 14-3-3 proteins, showed accumulation of 14-3-3 proteins and growth arrest in disrupted C/N-nutrient conditions similar to wild-type plants, although overexpression of intact ATL31 resulted in repression of 14-3-3 accumulation and tolerance to the conditions. Together, these results demonstrate that the physiological role of phosphorylation at 14-3-3 binding sites on ATL31 is to modulate the binding ability and stability of 14-3-3 proteins to control plant C/N-nutrient response.     

Deciphering the role of 14-3-3 proteins

14-3-3 Proteins are found to bind to a growing number of eukaryotic proteins and evidence is accumulating that 14-3-3 proteins serve as modulators of enzyme activity. Several 14-3-3 protein recognition motifs have been identified and an increasing number of target proteins have been found to contain more than one binding site for a 14-3-3 protein. It is thus possible that 14-3-3 dimers function as clamps that simultaneously bind to two motifs within a single binding partner. Phosphorylation of a number of binding motifs has been shown to increase the affinity for 14-3-3 proteins but other mechanisms also regulate the association. It has recently been demonstrated that fusicoccin induces a tight association between 14-3-3 proteins and the plant plasma membrane H⁺-ATPase. Phorbol esters and other hydrophobic molecules may have a similar effect on the association between 14-3-3 proteins and specific binding partners.     

Biological Significance of Divalent Metal Ion Binding to 14-3-3 Proteins In Relationship to Nitrate Reductase Inactivation

In this report we address two questions regarding the regulationof phosphorylated nitrate reductase (pNR; EC 1.6.6.1 [EC] ) by 14-3-3proteins. The first concerns the requirement for millimolarconcentrations of a divalent cation in order to form the inactivepNR: 14-3-3 complex at pH 7.5. The second concerns the reducedrequirement for divalent cations at pH 6.5. In answering thesequestions we highlight a possible general mechanism involvedin the regulation of 14-3-3 binding to target proteins. We showthat divalent cations (e.g. Ca²⁺, Mg²⁺ and Mn²⁺) bind directlyto 14-3-3s, and as a result cause a conformational change, manifestedas an increase in surface hydrophobicity. A similar change isalso obtained by decreasing the pH from pH 7.5 to pH 6.5, inthe absence of divalent cations, and we propose that protonationof amino acid residues brings about a similar effect to metalion binding. A possible regulatory mechanism, where the 14-3-3protein has to be "primed" prior to binding a target protein,is discussed. ¹Co-operative investigations of the U.S. Department of Agriculture,Agricultural Research Service, and the North Carolina AgriculturalResearch Service, Raleigh, NC. This work was supported by agrant from the U.S. Department of Agriculture-National ResearchInitiative (Grant 93-37305-9231 to JLH and SCH). Mention ofa trademark or proprietary product does not constitute a guaranteeor warranty of the product by the USDA or the North CarolinaAgricultural Service. Nor does it imply its approval to theexclusion of other products that might also be suitable.     

Phosphorylation and 14-3-3 binding of Arabidopsis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

Fructose 2,6-bisphosphate (fru-2,6-P2) is a signalling metabolite that regulates photosynthetic carbon partitioning in plants. The content of fru-2,6-P2 in Arabidopsis leaves varied in response to photosynthetic activity with an abrupt decrease at the start of the photoperiod, gradual increase through the day, and modest decrease at the start of the dark period. In Arabidopsis suspension cells, fru-2,6-P2 content increased in response to an unknown signal upon transfer to fresh culture medium. This increase was blocked by either 2-deoxyglucose or the protein phosphatase inhibitor, calyculin A, and the effects of calyculin A were counteracted by the general protein kinase inhibitor K252a. The changes in fru-2,6-P2 at the start of dark period in leaves and in the cell experiments generally paralleled changes in nitrate reductase (NR) activity. NR is inhibited by protein phosphorylation and binding to 14-3-3 proteins, raising the question of whether fructose-6-phosphate,2-kinase/fructose-2,6-bisphosphatase protein from Arabidopsis thaliana (AtF2KP), which both generates and hydrolyses fru-2,6-P2, is also regulated by phosphorylation and 14-3-3s. Consistent with this hypothesis, AtF2KP and NR from Arabidopsis cell extracts bound to a 14-3-3 column, and were eluted specifically by a synthetic 14-3-3-binding phosphopeptide (ARAApSAPA). 14-3-3s co-precipitated with recombinant glutathione S-transferase (GST)-AtF2KP that had been incubated with Arabidopsis cell extracts in the presence of Mg-ATP. 14-3-3s bound directly to GST-AtF2KP that had been phosphorylated on Ser220 (SLSASGpSFR) and Ser303 (RLVKSLpSASSF) by recombinant Arabidopsis calcium-dependent protein kinase isoform 3 (CPK3), or on Ser303 by rat liver mammalian AMP-activated protein kinase (AMPK; homologue of plant SNF-1 related protein kinases (SnRKs)) or an Arabidopsis cell extract. We have failed to find any direct effect of 14-3-3s on the F2KP activity in vitro to date. Nevertheless, our findings indicate the possibility that 14-3-3 binding to SnRK1-phosphorylated sites on NR and F2KP may regulate both nitrate assimilation and sucrose/starch partitioning in leaves.     

Modulation of nitrate reductase: some new insights, an unusual case and a potentially important side reaction

The mechanism of the post-translational modulation of nitrate reductase activity (NR, EC 1.6.6.1) is briefly summarized, and it is shown that by this mechanism nitric oxide production through NR is also rapidly modulated. New and partly unexpected details on the modulation mechanism have been obtained by using immunological techniques. The phosphorylation state of NR has been assessed with peptide antibodies raised against the serine phosphorylation motive of spinach NR. By co-immunoprecipitation experiments, 14-3-3 binding to phospho-NR and the function of Mg(2+) in that process has been elucidated. Conflicting data on the role of NR phosphorylation and 14-3-3 binding in controlling NR proteolysis are discussed. A possible role of other NR inactivating proteins is also briefly considered and the regulation of NR of Ricinus communis is described as an interesting special case that differs from the 'normal' mechanism in several important aspects.