Joan L. Huber

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

Three lines of evidence indicate that the 14‐3‐3 proteins that inactivate the phosphorylated form of spinach leaf NADH:nitrate reductase (NR) bind to the enzyme at the regulatory phosphorylation site (Ser‐543). First, a phosphorylated synthetic peptide based on the regulatory site can prevent and also reverse the inactivation of phospho‐NR caused by 14‐3‐3 proteins. Second, sequence‐specific and phosphorylation‐dependent binding of the aforementioned synthetic peptide to the 14‐3‐3 proteins was demonstrated in vitro. Third, 14‐3‐3 proteins were required for the ATP‐dependent phosphorylation of NR (as assessed by activity measurements) in the presence of NR‐kinase and leaf protein phosphatases. Lastly, we demonstrate specificity of recombinant Arabidopsis 14‐3‐3 isoforms in the interaction with phospho‐NR: ω > χ > ν ⋙ gf, ψ.

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

The control of nitrate reductase (NR) activity by reversible protein phosphorylation is a two-stage process: the enzyme is reversibly phosphorylated, and the phosphorylated form then binds an inhibitor protein. Recent results indicate that the phosphorylation reaction is Ca 2+ -dependent and that the inhibitor is a member of the 14-3-3 family of proteins. These results suggest the convergence of several signal transduction pathways in the control of nitrate assimilation. Recent results also implicate metabolites and 5′-AMP as possible effectors regulating the phosphorylation status of NR in vivo .

Protein phosphorylation as a mechanism for regulation of spinach leaf sucrose-phosphate synthase activity☆

Studies were conducted to determine whether protein phosphorylation may be a mechanism for regulation of spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS), shown previously to be light-dark regulated by some type of covalent modification. Radioactive phosphate was incorporated into the 120-kDa subunit of SPS during labeling of excised leaves with [32P]Pi, as shown by immunoprecipitation and denaturing gel electrophoresis of the enzyme. Conditions which activated the enzyme (illumination of leaves or mannose treatment of leaf discs in darkness) reduced the incorporation of radiolabel into SPS in the in vivo system. The partially purified SPS protein could also be phosphorylated in vitro using [gamma-32P]ATP. In the in vitro system, the incorporation of radiolabel into the 120-kDa subunit of SPS was dependent on time and magnesium concentration, and was closely paralleled by inactivation of the enzyme. These results provide the first evidence to establish protein phosphorylation as a mechanism for the covalent regulation of SPS activity.

Control of plant enzyme activity by reversible protein phosphorylation

Publisher Summary Protein phosphorylation is considered one of the most studied posttranslational-modification mechanisms affecting protein structure and function. This chapter reviews the regulation of plant enzymes by protein phosphorylation. It focuses on plant enzymes known to be regulated by reversible phosphorylation and explores how this mechanism of post-translational modification may serve to coordinate flux through major metabolic pathways. Most of the enzymes known to be phosphorylated are leaf proteins and the phosphorylation status of many of the phosphoproteins is altered in response to light. Phosphorylation of numerous chloroplast stromal proteins can be observed when intact spinach chloroplasts are provided [ 32 P]P i in the light or when stromal extracts are given [ γ - 32 P] in vitro . One chloroplast stromal enzyme that undergoes reversible phosphorylation is pyruvate P i dikinase (PPDK). This enzyme is found in highest activities in leaves of C 4 species and in certain crassulacean acid metabolism (CAM) plants, where it plays an important role in photosynthesis by catalyzing the regeneration of phosphoenolpyruvate (PEP), which is the primary CO 2 acceptor. The light or dark modulation of PPDK activity in leaves can be mimicked in vitro by an ADP-dependent inactivation and a P i -dependent activation.

Regulation of Maize Leaf Nitrate Reductase Activity Involves Both Gene Expression and Protein Phosphorylation

Nitrate reductase (NR; EC 1.6.6.1) activity increased at the beginning of the photoperiod in mature green maize (Zea mays L.) leaves as a result of increased enzyme protein level and protein dephosphorylation. In vitro experiments suggested that phosphorylation of maize leaf NR affected sensitivity to Mg2+ inhibition, as shown previously in spinach. When excised leaves were fed 32P-labeled inorganic phosphate, NR was phosphorylated on seryl residues in both the light and dark. Tryptic peptide mapping of NR labeled in vivo indicated three major 32P-phosphopeptide fragments, and labeling of all three was reduced when leaves were illuminated. Maize leaf NR mRNA levels that were low at the end of the dark period peaked within 2 h in the light and decreased thereafter, and NR activity generally remained high. It appears that light signals, rather than an endogenous rhythm, account primarily for diurnal variations in NR mRNA levels. Overall, regulation of NR activity in mature maize leaves in response to light signals appears to involve control of gene expression, enzyme protein synthesis, and reversible protein phosphorylation.

IN VITRO PHOSPHORYLATION AND INACTIVATION OF SPINACH LEAF SUCROSE-PHOSPHATE SYNTHASE BY AN ENDOGENOUS PROTEIN KINASE

(1) Partially purified preparations of spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS) contain an endogenous protein kinase that phosphorylates and inactivates the enzyme with [gamma-32P]ATP. (2) The kinetic effect of phosphorylation is to alter affinities for substrates and the effector inorganic phosphate without affecting maximum velocity. (3) Two-dimensional peptide mapping of tryptic digests of in vitro labeled SPS yielded two phosphopeptides (designated sites 5 and 7). Labeling of the two sites occurred equally with time, and both correlated with inactivation. Maximum inactivation was associated with incorporation of 1.5 to 2.0 mol P/mol SPS tetramer, and about 70% of the phosphoryl groups were incorporated into one of the sites (phosphopeptide 7). (4) Phosphorylation and inactivation were strongly inhibited by NaCl, and the presence of salt alters some characteristics of the kinase reaction. In the absence of salt, the apparent Km for Mg.ATP was estimated to be 5 microM. (5) The dependence of the rate of phosphorylation on SPS concentration suggested that SPS and the protein kinase are distinct enzymes, but have some tendency to associate especially in the presence of ethylene glycol. (6) Ca2+/EGTA and polyamines have no effect on the rate of phosphorylation, whereas polycations (polylysine, polybrene and protamine) are inhibitory. (7) Of the metabolic intermediates tested, Glc 6-P inhibited phosphorylation and inactivation of the enzyme. The inhibition was not antagonized by inorganic phosphate, which suggests that Glc 6-P may be an effector of the kinase, rather than the target protein. Regulation by Glc 6-P may be of physiological significance.

Metabolic activators of spinach leaf nitrate reductase: Effects on enzymatic activity and dephosphorylation by endogenous phosphatases

Nitrate reductase (NR; EC 1.6.6.1) in spinach (Spinacia oleracea L.) leaves was inactivated in the dark and reactivated by light in vivo. When extracted from dark leaves, NR activity was lower and more strongly inhibited by Mg2+ relative to the enzyme extracted from leaves harvested in the light. When dark extracts were desalted at pH 6.5 and preincubated at 25° C prior to assay, enzyme activity (assayed either in the presence or absence of Mg2+) remained essentially constant, i.e. there was no spontaneous reactivation in vitro. However, addition of certain metabolites resulted in a time- and concentration-dependent activation of NR in vitro. Effective activators included inorganic phosphate (Pi), 5′-AMP, and certain of its derivatives such as FAD and pyridine nucleotides (both oxidized and reduced forms). All of the activators increased NR activity as assayed in the absence of Mg2+, whereas some activators (e.g. Pi, 5′-AMP and FAD) also reduced Mg2+ inhibition. The reduction of Mg2+ inhibition was also time-dependent and was almost completely prevented by a combination of okadaic acid plus KF, suggesting the involvement of dephosphorylation catalyzed by endogenous phosphatase(s). In contrast, the activation of NR (assayed minus Mg2+) was relatively insensitive to phosphatase inhibitors, indicating a different mechanism was involved. Compounds that were not effective activators of NR included sulfate, ribose-5-phosphate, adenosine 5′-monosulfate, coenzyme A, ADP and ATP. We postulate that NR can exist in at least two states that differ in enzymatic activity. The activators appear to interact with the NR molecule at a site distinct from the NADH active site, and induce a slow conformational change (hysteresis) that increases NR activity (assayed in the absence of Mg2+). Possibly as a result of the conformational change caused by certain activators, the regulatory phospho-seryl groups are more readily dephosphorylated by endogenous phosphatases, thereby reducing sensitivity to Mg2+ inhibition. Preliminary results suggest that light/dark transitions in vivo may alter the distribution of NR molecules between the low- and high-activity forms.

Regulation of Plant Metabolism by Protein Phosphorylation. Possible Regulation of Sucrose-Phosphate Synthase by 14-3-3 Proteins

Reversible protein phosphorylation is known to be an important regulatory mechanism for several plant enzymes involved in primary carbon and nitrogen metabolism (1,2). Our laboratory has been studying two enzymes of sucrose metabolism, sucrose-phosphate synthase (SPS) and sucrose synthase (SuSy), and an enzyme of N-metabolism, NADH:nitrate reductase (NR), which are known to be phosphorylated in vivo. Interestingly, the effect of phosphorylation is somewhat different for the three enzymes. With SuSy, phosphorylation of the single, major phosphorylation site (Ser-15; ref. 3) has little effect on activity but appears to be one of the factors that controls its association with the plasma membrane (4) and actin cytoskeleton (5). Thus, phosphorylation affects intracellular localization of the enzyme. With NR, phosphorylation of Ser-543 has no direct effect on activity but completes the motif required for binding of a 14-3-3 inhibitor protein (6,7). Thus, phosphorylation affects enzyme activity indirectly by affecting protein:protein interactions. With SPS, phosphorylation appears to directly affect enzyme activity, at least in some cases. Current work suggests that SPS may have three regulatory phosphorylation sites: i) Ser-158, which is largely (or solely) responsible for light/dark modulation (8); ii) Ser-424, which is thought to be responsible for osmotic stress activation of SPS (9), and iii) Ser-229, which may constitute a binding site for a 14-3-3 inhibitor protein (10).