An increase in phosphoinositide-specific phospholipase C activity precedes induction of C4 phosphoenolpyruvate carboxylase phosphorylation in illuminated and NH4Cl-treated protoplasts from Digitaria sanguinalis

A Ca2+-dependent phosphoinositide-specific phospholipase C (PI-PLC) activity has been characterized in the microsomal fraction of Digitaria sanguinalis mesophyll cell protoplasts. Microsomal PI-PLC was found to be inhibited in vitro by a mammalian anti-PLC-delta1 antibody and by the aminosteroide U-73122, an inhibitor of PI-PLC activity in animal cells. In Western blot experiments, the antibody recognized an 85 kDa protein in both microsomal protein extracts from mesophyll protoplasts and rat brain protein extracts containing the authentic enzyme. The involvement of the microsomal PI-PLC in the light-dependent transduction pathway leading to the phosphorylation of C4 phosphoenolpyruvate carboxylase (PEPC) was investigated in D. sanguinalis protoplasts. A transient increase in the PI-PLC reaction product inositol-1,4,5-trisphosphate (Ins(1,4, 5)P3) was observed in situ during early induction of the C4 PEPC phosphorylation cascade. U-73122, but not the inactive analogue U-73343, efficiently blocked the transient accumulation of Ins(1,4, 5)P3, and both the increase in C4 PEPC kinase activity and C4 PEPC phosphorylation in illuminated and weak base-treated protoplasts. Taken together, these data suggest that PI-PLC-based signalling is a committed step in the cascade controlling the regulation of C4 PEPC phosphorylation in C4 leaves.     

Metabolite control of Sorghum C4 phosphoenolpyruvate carboxylase catalytic activity and phosphorylation state

Kinetic analyses were performed on the nonphosphorylated and in vitro phosphorylated forms of recombinant Sorghum C4 phospho enolpyruvate carboxylase (C4 PEPC). The native enzyme was purified by immunoaffinity chromatography and its integrity demonstrated by Western blot analyses using anti N- and C-terminus antibodies. At suboptimal pH (7.1 to 7.3) and PEP concentration (2.5 mM), phosphorylation, positive metabolite effectors e.g., glucose-6-phosphate, glycine and dihydroxyacetone-phosphate, or an increase in pH strongly activated the enzyme and lowered the inhibitory effect of L-malate. C4 PEPC phosphorylation strengthened the effect of the positive effectors thereby decreasing further the enzyme's sensitivity to this inhibitor. L-malate also decreased the phosphorylation rate of C4 PEPC, a process antagonized by positive metabolite effectors. This was shown both in vitro, in a reconstituted phosphorylation assay containing the catalytic subunit of a cAMP-dependent protein kinase or the Sorghum leaf PEPC-PK and in situ, during induction of C4 PEPC phosphorylation in mesophyll cell protoplasts.     

In-situ evidence for the involvement of calcium and bundle-sheath-derived photosynthetic metabolites in the C4 phosphoenolpyruvate-carboxylase kinase signal-transduction chain

Regulation of the light activation of C₄ phosphoenolpyruvate-carboxylase (PEPC) protein kinase (PEPC-PK) and the ensuing phosphorylation of its cytosolic target protein were studied in intact mesophyll cells (MC) and protoplasts (MP) isolated from dark-adapted leaves of Digitaria sanguinalis [L.] Scop, (hairy crabgrass). The apparent in-situ phosphorylation state of PEPC (EC 4.1.1.31) was assessed by the sensitivity of its activity in desalted MC- and MP-extracts to l-malate under suboptimal assay conditions, while the activity-state of PEPC-PK was determined by in-vitro ³²P-labeling of purified maize or recombinant sorghum PEPC by these extracts. In-situ pretreatment of intact MC at pH 8.0 by illumination and calcium addition led to significant decreases in PEPC malate sensitivity and increases in PEPC-kinase activity that were negated by the addition of EGTA to the external cell medium. Similarly, in-situ pretreatment of MP with light plus NH₄Cl at pH 7.6 led to significant decreases in malate sensitivity which did not occur when a Ca²⁺ ionophore and EGTA were included in the suspension medium. In contrast, neither EGTA nor exogenous Ca²⁺ had a major direct effect on the in-vitro activity of PEPC-PK extracted from Digitaria MC and MP. Preincubation of intact MC with 5 mM 3-phosphoglycerate or pyruvate at pH 8.0 in the dark led to significant decreases in PEPC malate sensitivity and increases in PEPC-PK activity which were not observed with various other exogenous metabolites. These collective in-situ experiments with isolated C₄ MC and MP (i) support our earlier hypothesis that alkalization of cytosolic pH is involved in the PEPC-PK signal-transduction cascade (see J.-N. Pierre et al., Eur J Biochem, 1992,210: 531–537), (ii) suggest that intracellular calcium is involved in the PEPC-kinase signal-transduction chain, but at a step upstream of PEPC-PK per se, and (iii) provide direct evidence that the bundle-sheath-derived, C₄-pathway intermediates 3-PGA and/or pyruvate also play a role in this signal-transduction cascade which ultimately effects the up-regulation of PEPC in the C₄ mesophyll cytosol.Abbreviations BS bundle-sheath - CAM Crassulacean acid metabolism - DHAP dihydroxyacetone phosphate - FPLC fast-protein liquid chromatography - Glc6P glucose 6-phosphate - I_0.5 50% inhibition constant - MC mesophyll cell(s) - MP me-sophyll protoplast(s) - PEP phosphoenolpyruvate - PEPC PEP carboxylase - PEPC-PK PEPC protein-Ser/Thr kinase - 2-PGA 2-phosphoglycerate - 3-PGA 3-phosphoglycerate - PPFD photosynthetic photon flux density - Pyr pyruvate - Ser serine The authors thank Ms. Jill Myatt for her help with some of the MC preparations. This work was supported in part by grants INT-9115566 and MCB-9315928 from the U.S. National Science Foundation (to R.C.). S.M.G.D. was a recipient of an NSERC of Canada Post-Doctoral Fellowship. This paper is Journal Series No. 11 395 of the University of Nebraska Agricultural Research Division.     

Phosphoenolpyruvate Carboxylase Kinase in Tobacco Leaves Is Activated by Light in a Similar but Not Identical Way as in Maize

We have previously reported the partial purification of a Ca2+- independent phosphoenolpyruvate carboxylase (PEPC) protein-serine/threonine kinase (PEPC-PK) from illuminated leaves of N-sufficient tobacco (Nicotiana tabacum L.) plants (Y.-H. Wang, R. Chollet [1993] FEBS Lett 328: 215-218). We now report that this C3 PEPC-kinase is reversibly light activated in vivo in a time-dependent manner. As the kinase becomes light activated, the activity and L-malate sensitivity of its target protein increases and decreases, respectively. The light activation of tobacco PEPC-PK is prevented by pretreatment of detached leaves with various photosynthesis and cytosolic protein-synthesis inhibitors. Similarly, specific inhibitors of glutamine synthetase block the light activation of tobacco leaf PEPC-kinase under both photorespiratory and nonphotorespiratory conditions. This striking effect is partially and specifically reversed by exogenous glutamine, whereas it has no apparent effect on the light activation of the maize (Zea mays L.) leaf kinase. Using an in situ "activity-gel" phosphorylation assay, we have identified two major Ca2+-independent PEPC-kinase catalytic polypeptides in illuminated tobacco leaves that have the same molecular masses (approximately 30 and 37 kD) as found in illuminated maize leaves. Collectively, these results indicate that the phosphorylation of PEPC in N-sufficient leaves of tobacco (C3) and maize (C4) is regulated through similar but not identical light-signal transduction pathways.     

Illumination increases the affinity of phosphoenolpyruvate carboxylase to bicarbonate in leaves of a C4 plant, Amaranthus hypochondriacus

Illumination increased markedly the affinity to bicarbonate of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) in leaves of Amaranthus hypochondriacus L., a C4 plant. When leaves were illuminated, the apparent Km for (HCO3-) of PEPC decreased by about 50% concurrent with a 2- to 5-fold increase in Vmax and 3- to 4-fold increase in Ki for malate. The inclusion of ethoxyzolamide, an inhibitor of carbonic anhydrase, during the assay had no effect on kinetic and regulatory properties of PEPC indicating that carbonic anhydrase was not involved during light-induced sensitization of PEPC to HCO3-. Pretreatment of leaf discs with cycloheximide (CHX), a cytosolic protein synthesis inhibitor, suppressed significantly the light-enhanced decrease in apparent Km (HCO3-). Further, in vitro phosphorylation of purified dark-form PEPC by protein kinase A (PKA) decreased the apparent Km (HCO3-) of the enzyme, in addition increasing Ki (malate) as expected. Such changes, due to in vitro phosphorylation of purified PEPC by PKA, occurred only with wild-type PEPC, but not in the mutant form of maize (S15D) which is already a mimic of the phosphorylated enzyme. These results suggest that phosphorylation of the enzyme is important during the sensitization of PEPC to HCO3- by illumination in C4 leaves. Since illumination is expected to increase the cytosolic pH and the availability of dissolved HCO3- in mesophyll cells, the sensitization by light of PEPC to HCO3- could be physiologically quite significant.     

In Vivo Regulation of Wheat-Leaf Phosphoenolpyruvate Carboxylase by Reversible Phosphorylation

Regulation of C3 phosphoenolpyruvate carboxylase (PEPC) and its protein-serine/threonine kinase (PEPC-PK) was studied in wheat (Triticum aestivum) leaves that were excised from low-N-grown seedlings and subsequently illuminated and/or supplied with 40 mM KNO3. The apparent phosphorylation status of PEPC was assessed by its sensitivity to L-malate inhibition at suboptimal assay conditions, and the activity state of PEPC-PK was determined by the in vitro 32P labeling of purified maize dephospho-PEPC by [[gamma]-32P]ATP/Mg. Illumination ([plus or minus]NO3-) for 1 h led to about a 4.5-fold increase in the 50% inhibition constant for L-malate, which was reversed by placing the illuminated detached leaves in darkness (minus NO3-). A 1 -h exposure of excised leaves to light, KNO3, or both resulted in relative PEPC-PK activities of 205, 119, and 659%, respectively, of the dark/0 mM KNO3 control tissue. In contrast, almost no activity was observed when a recombinant sorghum phosphorylation-site mutant (S8D) form of PEPC was used as protein substrate in PEPC-PK assays of the light plus KNO3 leaf extracts. In vivo labeling of wheat-leaf PEPC by feeding 32P-labeled orthophosphate showed that PEPC from light plus KNO3 tissue was substantially more phosphorylated than the enzyme in the dark minus-nitrate immunoprecipitates. Immunoblot analysis indicated that no changes in relative PEPC-protein amount occurred within 1 h for any of the treatments. Thus, C3 PEPC activity in these detached wheat leaves appears to be regulated by phosphorylation of a serine residue near the protein's N terminus by a Ca2+ -independent protein kinase in response to a complex interaction in vivo between light and N.     

Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase (A Cardinal Event Influencing the Photosynthesis Rate in Sorghum and Maize)

C4 leaf phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is subject to a day/night regulatory phosphorylation cycle. By using the cytoplasmic protein synthesis inhibitor cycloheximide (CHX), we previously reported that the reversible in vivo light activation of the C4 PEPC protein-serine kinase requires protein synthesis. In the present leaf gas-exchange study, we have examined how and to what extent the CHX-induced inhibition of PEPC protein kinase activity/PEPC phosphorylation in the light influences C4 photosynthesis. Detached Sorghum vulgare and maize (Zea mays) leaves fed 10 [mu]M CHX showed a gradual but marked decrease in photosynthetic CO2 assimilation capacity. A series of control experiments designed to assess deleterious secondary effects of the inhibitor established that this reduction in C4 leaf CO2 assimilation was not due to (a) an increased stomatal resistance to CO2 diffusion, (b) a decrease in the activation state of other photoactivated C4 cycle enzymes, and (c) a perturbation of the Benson-Calvin C3 cycle, as evidenced by the absence of an inhibitory effect of CHX on leaf photosynthesis by a C3 grass (Triticum aestivum). It is notable that the CHX-induced decrease in CO2 assimilation by illuminated Sorghum leaves was highly correlated with a decrease in the apparent phosphorylation status of PEPC and a concomitant change in carbon isotope discrimination consistent with a shift from a C4 to a C3 mode of leaf CO2 fixation. These collective findings indicate that the light-dependent activation of the PEPC protein-serine kinase and the resulting phosphorylation of serine-8 or serine-15 in Sorghum or maize PEPC, respectively, are fundamental regulatory events that influence leaf C4 photosynthesis in vivo.     

Phosphorylation of phosphoenolpyruvate carboxylase is not essential for high photosynthetic rates in the C4 species Flaveria bidentis

Phosphoenolpyruvate carboxylase (PEPC; EC4.1.1.31) plays a key role during C(4) photosynthesis. The enzyme is activated by metabolites such as glucose-6-phosphate and inhibited by malate. This metabolite sensitivity is modulated by the reversible phosphorylation of a conserved serine residue near the N terminus in response to light. The phosphorylation of PEPC is modulated by a protein kinase specific to PEPC (PEPC-PK). To explore the role PEPC-PK plays in the regulation of C(4) photosynthetic CO(2) fixation, we have transformed Flaveria bidentis (a C(4) dicot) with antisense or RNA interference constructs targeted at the mRNA of this PEPC-PK. We generated several independent transgenic lines where PEPC is not phosphorylated in the light, demonstrating that this PEPC-PK is essential for the phosphorylation of PEPC in vivo. Malate sensitivity of PEPC extracted from these transgenic lines in the light was similar to the malate sensitivity of PEPC extracted from darkened wild-type leaves but greater than the malate sensitivity observed in PEPC extracted from wild-type leaves in the light, confirming the link between PEPC phosphorylation and the degree of malate inhibition. There were, however, no differences in the CO(2) and light response of CO(2) assimilation rates between wild-type plants and transgenic plants with low PEPC phosphorylation, showing that phosphorylation of PEPC in the light is not essential for efficient C(4) photosynthesis for plants grown under standard glasshouse conditions. This raises the intriguing question of what role this complexly regulated reversible phosphorylation of PEPC plays in C(4) photosynthesis.     

Light activation of maize phosphoenolpyruvate carboxylase protein-serine kinase activity is inhibited by mesophyll and bundle sheath-directed photosynthesis inhibitors

C₄ phosphoenolpyruvate carboxylase (PEPC) is post-translationally regulated by reversible phosphorylation of a specific N-terminal seryl residue in response to light/dark transitions of the parent leaf tissue. The protein-serine kinase (PEPC-PK) that phosphorylates/activates this mesophyll-cytoplasm target enzyme is slowly, but strikingly, activated by high light and inactivated in darkness in vivo by a mechanism involving cytoplasmic protein synthesis/degradation as a primary component. In this report, evidence is presented indicating that the inhibition of Calvin cycle activity by a variety of mesophyll (3-(3,4-dichlorophenyl)-1,1-dimethylurea, isocil, methyl viologen) and bundle sheath (dl-glyceraldehyde)-directed photosynthesis inhibitors blocks the light activation of maize (Zea mays L.) PEPC-PK and the ensuing regulatory phosphorylation of its target enzyme in vivo. Based on these and related observations, we propose that the Calvin cycle supplies the C₄ mesophyll cell with (a) a putative signal (e.g. phosphorylated metabolite, amino acid) that interacts with the cytoplasmic protein synthesis event to effect the light activation of PEPC-PK and the concomitant phosphorylation of PEPC, and (b) high levels of known positive effectors (e.g. triose-phosphate, glucose-6-phosphate) that interact directly with the carboxylase. The combined result of this complex regulatory cascade is to effectively desensitize PEPC to feedback inhibition by the millimolar levels of l-malate required for rapid diffusive transport to the bundle sheath during high rates of C₄ photosynthesis.     

A conserved 19-amino acid synthetic peptide from the carboxy terminus of phosphoenolpyruvate carboxylase inhibits the in vitro phosphorylation of the enzyme by the calcium-independent phosphoenolpyruvate carboxylase kinase

Higher plant phosphoenolpyruvate carboxylase (PEPC) is subject to in vivo phosphorylation of a regulatory serine located in the N-terminal domain of the protein. Studies using synthetic peptide substrates and mutated phosphorylation domain photosynthetic PEPC (C4 PEPC) suggested that the interaction of phosphoenolpyruvate carboxylase kinase (PEPCk) with its target was not restricted to this domain. However, no further information was available as to where PEPCk-C4 PEPC interactions take place. In this work, we have studied the possible interaction of the conserved 19-amino acid C-terminal sequence of sorghum (Sorghum vulgare Pers cv Tamaran) C4 PEPC with PEPCk. In reconstituted assays, a C-terminal synthetic peptide containing this sequence (peptide C19) was found to inhibit the phosphorylation reaction by the partially purified Ca2+-independent PEPCk (50% inhibition of initial activity = 230 microm). This effect was highly specific because peptide C19 did not alter C4 PEPC phosphorylation by either a partially purified sorghum leaf Ca2+-dependent protein kinase or the catalytic subunit of mammalian protein kinase A. In addition, the Ca2+-independent PEPCk was partially but significantly retained in affinity chromatography using a peptide C19 agarose column. Because peptide C15 (peptide C19 lacking the last four amino acids, QNTG) also inhibited C4 PEPC phosphorylation, it was concluded that the amino acid sequence downstream from the QNTG motif was responsible for the inhibitory effect. Specific antibodies raised against peptide C19 revealed that native C4 PEPC could be in two different conformational states. The results are discussed in relation with the reported crystal structure of the bacterial (Escherichia coli) and plant (maize [Zea mays]) enzymes.     

Regulation of phosphoenolpyruvate carboxylase phosphorylation by metabolites and abscisic acid during the development and germination of barley seeds

During barley (Hordeum vulgare) seed development, phosphoenolpyruvate carboxylase (PEPC) activity increased and PEPC-specific antibodies revealed housekeeping (103-kD) and inducible (108-kD) subunits. Bacterial-type PEPC fragments were immunologically detected in denatured protein extracts from dry and imbibed conditions; however, on nondenaturing gels, the activity of the recently reported octameric PEPC (in castor [Ricinus communis] oil seeds) was not detected. The phosphorylation state of the PEPC, as judged by l-malate 50% inhibition of initial activity values, phosphoprotein chromatography, and immunodetection of the phosphorylated N terminus, was found to be high between 8 and 18 d postanthesis (DPA) and during imbibition. In contrast, the enzyme appeared to be in a low phosphorylation state from 20 DPA up to dry seed. The time course of 32/36-kD, Ca(2+)-independent PEPC kinase activity exhibited a substantial increase after 30 DPA that did not coincide with the PEPC phosphorylation profile. This kinase was found to be inhibited by l-malate and not by putative protein inhibitors, and the PEPC phosphorylation status correlated with high glucose-6-phosphate to malate ratios, thereby suggesting an in vivo metabolic control of the kinase. PEPC phosphorylation was also regulated by photosynthate supply at 11 DPA. In addition, when fed exogenously to imbibing seeds, abscisic acid significantly increased PEPC kinase activity. This was further enhanced by the cytosolic protein synthesis inhibitor cycloheximide but blocked by protease inhibitors, thereby suggesting that the phytohormone acts on the stability of the kinase. We propose that a similar abscisic acid-dependent effect may contribute to produce the increase in PEPC kinase activity during desiccation stages.     

Salt stress increases the Ca2+-independent phosphoenolpyruvate carboxylase kinase activity in Sorghum leaves

In C4 plants, the photosynthetic enzyme phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) is subjected to a phosphorylation process via the light-dependent up-regulation of a Ca2+-independent PEPCase-kinase. The present work aimed to study the effect of salt stress on PEPCase phosphorylation in Sorghum vulgare Pers. leaves. The growth of salt-treated plants was reduced compared with that of the control plants. PEPCase activity modestly increased (around 20-40%) whereas PEPCase phosphorylation was markedly enhanced, on a protein basis, in extracts from illuminated leaves. The enhanced protein kinase activity was found to display a low molecular mass in the range 32-35 kDa, to be independent of Ca2+ and to be up-regulated by light. Furthermore, up-regulation was blocked in vivo by the cytosolic protein synthesis inhibitor cycloheximide. Collectively, these data demonstrated that salinity stress altered the Ca2+-independent PEPCase-kinase, presumably by increasing the mesophyll content of the enzyme. Potassium chloride, but not abscisic acid, mimicked the effect of NaCl on PEPCase-kinase activity.     

In Vivo Regulatory Phosphorylation of Soybean Nodule Phosphoenolpyruvate Carboxylase

In this report we provide evidence that cytosolic phosphoenolpyruvate carboxylase (PEPC) in soybean (Glycine max L.) root nodules is regulated in vivo by a seryl-phosphorylation cycle, as with the C4, Crassulacean acid metabolism, and C3 leaf isoforms. Pretreatment of parent plants by stem girdling for 5 or 14 h caused a significant decrease in the apparent phosphorylation state of nodule PEPC, as indicated by the 50% inhibition constant (L-malate) and specific activity values assayed at suboptimal conditions, whereas short-term darkness alone was without effect. However, extended (26 h) darkness led to the formation of a relatively dephosphorylated nodule PEPC, an effect that was reversed by illuminating the darkened plants for 3 h. This reversal of the apparent phosphorylation state in the light was prevented by concomitant stem girdling. In contrast, the optimal activity of nodule PEPC and its protein level showed little or no change in all pretreated plants. These results suggest that the phosphorylation state of PEPC in soybean root nodules is possibly modulated by photosynthate transported recently from the shoots. In situ [32P]orthophosphate labeling, immunoprecipitation, and phosphoamino acid analyses confirmed directly that PEPC in detached intact soybean nodules is phosphorylated on a serine residue(s).     

Platelet aggregation induced by alpha 2-adrenoceptor and protein kinase C activation. A novel synergism.

Adrenaline or UK 14304 (a specific alpha 2-adrenoceptor agonist) and phorbol ester (phorbol 12,13-dibutyrate; PdBu) or bioactive diacylglycerols (sn-1,2-dioctanoylglycerol; DiC8) synergistically induced platelet aggregation and ATP secretion. The effect on aggregation was more pronounced than the effect on secretion, and it was observed in aspirinized, platelet-rich plasma or suspensions of washed aspirinized platelets containing ADP scavengers. No prior shape change was found. In the presence of adrenaline, DiC8 induced reversible aggregation and PdBu evoked irreversible aggregation that correlated with the different kinetics of DiC8- and PdBu-induced protein kinase C activation. Adrenaline and UK 14304 did not induce or enhance phosphorylation induced by DiC8 or PdBu of myosin light chain (20 kDa), the substrate of protein kinase C (47 kDa), or a 38 kDa protein. Immunoprecipitation studies using a Gcommon alpha antiserum or a Gi alpha antiserum showed that Gi alpha is not phosphorylated after exposure of platelets to PdBu or PdBu plus adrenaline. Adrenaline, PdBu or adrenaline plus PdBu did not cause stimulation of phospholipase C as reflected in production of [32P]phosphatidic acid. Adrenaline caused a small increase of Ca2+ in the platelet cytosol of platelets loaded with Indo-1; this effect was also observed in the absence of extracellular Ca2+. However, under conditions of maximal aggregation induced by adrenaline plus PdBu, no increase of cytosolic Ca2+ was observed. Platelet aggregation induced by PdBu plus adrenaline was not inhibited by a high intracellular concentration of the calcium chelator Quin-2. These experiments indicate that alpha 2-adrenoceptor agonists, known to interact with Gi, and protein kinase C activators synergistically induced platelet aggregation through a novel mechanism. The synergism occurs distally to Gi protein activation and protein kinase C-dependent protein phosphorylation and does not involve phospholipase C activation or Ca2+ mobilization.     

A Ca(2+)-dependent protein kinase phosphorylates phosphoenolpyruvate carboxylase in maize.

In C4 plants the activity of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) is regulated by phosphorylation/dephosphorylation which is mediated by light/dark signals. The study using protein kinase inhibitors showed that the inhibition pattern of maize PEPC-protein kinase (PEPC-PK) is similar to that of myosin light chain kinase, a Ca(2+)-calmodulin-dependent PK. The kinase activity was also inhibited by EGTA and the inhibition was relieved by Ca2+. These results suggest that PEPC-PK is Ca(2+)-dependent in contrast with previous observations by other research groups.     

Regulatory phosphorylation of phosphoenolpyruvate carboxylase in protoplasts from Sorghum mesophyll cells and the role of pH and Ca2+ as possible components of the light-transduction pathway.

The light-dependent phosphorylation of the photosynthetic phosphoenolpyruvate carboxylase (PyrPC) was shown to occur in protoplasts from Sorghum mesophyll cells. It was accompanied by an increase in PyrPC protein-serine-kinase activity and conferred the target-specific functional properties, i.e. an increase in Vmax and apparent Ki for L-malate, as previously found with the whole leaf. The light-dependent regulatory phosphorylation of PyrPC was (a) specifically promoted by the weak bases NH4Cl and methylamine (agents which increase cytosolic pH), but not by KNO3, (b) inhibited by the cytosolic protein-synthesis inhibitor, cycloheximide, thus confirming that protein turnover is a component of the signal-transduction cascade, as reported in [4], (c) found to moderately decrease in the presence of EGTA and to be strongly depressed when the Ca(2+)-selective ionophore A23187 was added to the incubation medium together with EGTA. Addition of Ca2+, but not of Mg2+, to the Ca(2+)-depleted protoplasts partially, but significantly, relieved the inhibition. Calcium deprivation apparently affected the in-situ light-activation of the PyrPC protein kinase. These data indicated that both Ca2+ and an increase in cytosolic pH are required for the induction of PyrPC protein kinase activity/PyrPC phosphorylation in illuminated protoplasts from Sorghum mesophyll cells.     

Comparison of substrate recognition by protein kinase C (type III) between rat liver cytosolic and particulate fractions

1. Phosphorylation of rat liver endogenous substrates by protein kinase C (type III) was compared between cytosolic and particulate (mitochondria, microsomes and plasma membrane) fractions. 2. The rate and the maximum level of protein phosphorylation were several-fold higher in particulate fractions than in cytosolic fraction. 3. Protein phosphorylation in cytosolic fraction was dependent on both Ca2+ and phospholipid, but only Ca2+ was necessary in phosphorylation of particulate fractions. 4. These results suggest that protein kinase C (type III) has much more target proteins in particulate fractions rather than in cytosolic fraction and Ca2+ was important regulator in particulate protein phosphorylation.     

Coordinate regulation of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase by light and CO2 during C4 photosynthesis

The aim of this study was to investigate the relationship between the phosphorylation and activation states of phosphoenolpyruvate carboxykinase (PEPCK) and to investigate how the phosphorylation states of PEPCK and phosphoenolpyruvate carboxylase (PEPC) are coordinated in response to light intensity and CO(2) concentration during photosynthesis in leaves of the C(4) plant Guinea grass (Panicum maximum). There was a linear, reciprocal relationship between the phosphorylation state of PEPCK and its activation state, determined in a selective assay that distinguishes phosphorylated from nonphosphorylated forms of the enzyme. At high photon flux density and high CO(2) (750 microL L(-1)), PEPC was maximally phosphorylated and PEPCK maximally dephosphorylated within 1 h of illumination. The phosphorylation state of both enzymes did not saturate until high light intensities (about 1,400 micromol quanta m(-2) s(-1)) were reached. After illumination at lower light intensities and CO(2) concentrations, the overall change in phosphorylation state was smaller and it took longer for the change in phosphorylation state to occur. Phosphorylation states of PEPC and PEPCK showed a strikingly similar, but inverse, pattern in relation to changes in light and CO(2). The protein phosphatase inhibitor, okadaic acid, promoted the phosphorylation of both enzymes. The protein synthesis inhibitor, cycloheximide, blocked dark phosphorylation of PEPCK. The data show that PEPC and PEPCK phosphorylation states are closely coordinated in vivo, despite being located in the mesophyll and bundle sheath cells, respectively.     

Protein phosphorylation and secretion in digitonin-permeabilized adrenal chromaffin cells. Effects of micromolar Ca2+, phorbol esters, and diacylglycerol

The effects of phorbol esters, dioctanoylglycerol (DiC8), and micromolar Ca2+ on protein phosphorylation and catecholamine secretion in digitonin-treated chromaffin cells were investigated. [gamma-32P]ATP was used as a substrate for phosphorylation in the permeabilized cells. 12-O-Tetradecanoylphorbol-13-acetate (TPA) enhanced Ca2+-dependent catecholamine secretion from digitonin-permeabilized cells. The enhancement required MgATP. Only those phorbol esters which activate protein kinase C in vitro enhanced both catecholamine secretion and protein phosphorylation. DiC8, which activates protein kinase C in vitro and mimics phorbol ester effects in situ, also enhanced both catecholamine secretion and protein phosphorylation. Preincubation of intact cells with TPA or DiC8 was necessary for maximal effects on both catecholamine secretion and protein phosphorylation in subsequently digitonin-treated chromaffin cells. The TPA-induced enhancement of protein phosphorylation was almost entirely Ca2+-independent, whereas DiC8-induced enhancement of protein phosphorylation was mainly Ca2+-dependent. Micromolar Ca2+ alone also enhanced the phosphorylation of a large number of proteins. Most of the proteins phosphorylated in response to TPA or potentiated by DiC8 in combination with Ca2+ were also phosphorylated by micromolar Ca2+ in the absence of exogenous protein kinase C activators. In intact cells, 1,1-dimethyl-4-phenylpiperazinium (DMPP) induced Ca2+-dependent phosphorylation of at least 17 proteins which were detected by two-dimensional gel electrophoresis. All of the proteins phosphorylated upon incubation with 1,1-dimethyl-4-phenylpiperazinium were phosphorylated upon incubation with micromolar Ca2+ in digitonin-treated cells. These results demonstrate that TPA- or DiC8-enhanced Ca2+-dependent catecholamine secretion is associated with enhanced protein phosphorylation which is probably mediated by protein kinase C and that activation of protein kinase C modulates catecholamine secretion from digitonin-treated chromaffin cells.