Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In mesophyll cells (MC) of Digitaria sanguinalis, the C(4)-phosphoenolpyruvate carboxylase (C(4)-PEPC) initiating the photosynthetic pathway is controlled by a complex light-dependent phosphorylation process. We showed previously that the transduction cascade involves the phosphoinositide pathway and a Ca(2+)-dependent step, which precedes the upregulation of the PEPC kinase (PEPCk). We have now further characterized the cascade component requiring Ca(2+). A Ca(2+)-dependent protein kinase that shows several characteristics of the conventional type of mammalian protein kinase C (PKC) was detected in protein extracts from mesophyll cell protoplasts (MCPs). It catalyzed the in vitro phosphorylation of the C1-peptide PKC substrate and was markedly inhibited by a PKC-specific pseudosubstrate domain. However, it was only modestly activated by the phospholipids phosphatidylserine and lysophosphatidylcholine, while choline, oleyl acetylglycerol, phosphatidylinositol, and the phorbol ester phorbol 12-myristate 13-acetate did not show any effect. Nevertheless, its activity was found to be associated with a polypeptide of 75kDa that was recognized by a PKC antibody raised against the C-terminus of rabbit PKCbeta II. In addition, this protein kinase was also inhibited by the Ca(2+)-dependent protein kinase (CDPK)/PKC inhibitors W7, H7, and staurosporine. Surprisingly, it was found to be phosphorylated in dark-adapted MCPs, albeit to a low extent, and this did not change during protoplast induction by light. W7, H7, and staurosporine were shown to markedly inhibit C(4)-PEPC phosphorylation in light-treated MCPs. These results support the view that this protein kinase is a good candidate to represent the Ca(2+)-activated component of the C(4)-PEPC phosphorylation cascade.
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PMID:A Ca(2+)-dependent protein kinase with characteristics of protein kinase C in leaves and mesophyll cell protoplasts from Digitaria sanguinalis: possible involvement in the C(4)-phosphoenolpyruvate carboxylase phosphorylation cascade. 1473 23

Phosphatidic acid (PA) is emerging as an important lipid signalling molecule. In plants, it is implicated in various stress-signalling pathways and is formed in response to wounding, osmotic stress, cold stress, pathogen elicitors, Nod factors, ethylene and abscisic acid. How PA exerts its effects is still unknown, mainly because of the lack of characterized PA targets. In an approach to isolate such targets we have used PA-affinity chromatography. Several PA-binding proteins were present in the soluble fraction of tomato and Arabidopsis cells. Using mass spectrometric analysis, several of these proteins, including Hsp90, 14-3-3 proteins, an SnRK2 serine/threonine protein kinase and the PP2A regulatory subunit RCN1 could be identified. As an example, the binding of one major PA-binding protein, phosphoenolpyruvate carboxylase (PEPC), was characterized further. Competition experiments with different phospholipids confirmed specificity for PA. Hypo-osmotic treatment of the cells increased the amount of PEPC that bound the PA beads without increasing the absolute amount of PEPC. This suggests that PEPC's affinity for PA had increased. The work shows that PA-affinity chromatography/mass spectrometry is an effective way to isolate and identify PA-binding proteins from plants.
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PMID:Isolation and identification of phosphatidic acid targets from plants. 1527 72

In C4 photosynthesis, phosphoenolpyruvate carboxylase (PEPC) is the enzyme responsible for catalyzing the primary fixation of atmospheric CO2. The activity of PEPC is regulated diurnally by reversible phosphorylation. PEPC kinase (PEPCk), a protein kinase involved in this phosphorylation, is highly specific for PEPC and consists of only the core domain of protein kinase. Owing to its extremely low abundance in cells, analysis of its regulatory mechanism at the protein level has been difficult. Here we employed a transient expression system using maize mesophyll protoplasts. The PEPCk protein with a FLAG tag could be expressed correctly and detected with high sensitivity. Rapid degradation of PEPCk protein was confirmed and shown to be blocked by MG132, a 26S proteasome inhibitor. Furthermore, MG132 enhanced accumulation of PEPCk with increased molecular sizes at about 8 kDa intervals. Using anti-ubiquitin antibody, this increase was shown to be due to ubiquitination. This is the first report to show the involvement of the ubiquitin-proteasome pathway in PEPCk turnover. The occurrence of PEPCks with higher molecular sizes, which was noted previously with cell extracts from various plants, was also suggested to be due to ubiquitination of native PEPCk.
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PMID:The ubiquitin-proteasome pathway is involved in rapid degradation of phosphoenolpyruvate carboxylase kinase for C4 photosynthesis. 1569 55

Insulin is a key hormone that controls glucose homeostasis. In liver, insulin suppresses gluconeogenesis by inhibiting the transcriptions of phosphoenolpyruvate carboxylase (PEPCK) and glucose-6-phosphatase (G6Pase) genes. In insulin resistance and type II diabetes there is an elevation of hepatic gluconeogenesis, which contributes to hyperglycemia. To search for novel genes that negatively regulate insulin signaling in controlling metabolic pathways, we screened a cDNA library derived from the white adipose tissue of ob/ob mice using a reporter system comprised of the PEPCK promoter placed upstream of the alkaline phosphatase gene. The mitogen-activated dual specificity protein kinase phosphatase 3 (MKP-3) was identified as a candidate gene that antagonized insulin suppression on PEPCK gene transcription from this screen. In this study, we showed that MKP-3 was expressed in insulin-responsive tissues and that its expression was markedly elevated in the livers of insulin-resistant obese mice. In addition, MKP-3 can activate PEPCK promoter in synergy with dexamethasone in hepatoma cells. Furthermore, ectopic expression of MKP-3 in hepatoma cells by adenoviral infection increased the expression of PEPCK and G6Pase genes and led to elevated glucose production. Taken together, our data strongly suggests that MKP-3 plays a role in regulating gluconeogenic gene expression and hepatic gluconeogenesis. Therefore, dysregulation of MKP-3 expression and/or function in liver may contribute to the pathogenesis of insulin resistance and type II diabetes.
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PMID:Dual specificity MAPK phosphatase 3 activates PEPCK gene transcription and increases gluconeogenesis in rat hepatoma cells. 1612 24

The properties of phosphoenolpyruvate carboxylase (PEPC) were studied, with respect to calcium (Ca2+), in leaves of Amaranthus hypochondriacus, a C4 plant. Experiments were conducted in vitro (by adding Ca2+ during enzyme assay) or in vivo (by feeding Ca2+ to intact leaves through petiole). Inclusion of 10 microM Ca2+ during assay marginally increased (<30%) malate sensitivity of PEPC in extracts from dark-adapted leaves. The effect of Ca2+ was marginal on PEPC in extracts from illuminated leaves. Upon applying a low concentration of Ca2+ to leaves, the PEPC activity in leaves increased by 1.5-fold, while inhibition by malate decreased markedly. The light activation of PEPC in Ca2+-fed leaves was slightly higher than in the absence of Ca2+-ethyleneglycol-bis-(beta-aminoethyl ether) N,N,N',N'-tetra acetic acid (EGTA). To assess further the role of Ca2+, 5 mM EGTA (Ca2+ chelator) was either added during the enzyme assay or fed to leaves through petiole. EGTA had no effect on PEPC, when added during enzyme assay. Upon feeding EGTA, the PEPC activity in the dark-adapted leaf extracts increased by 30%, and the effect on malate sensitivity was marginal. However, there was a decrease in PEPC activity in illuminated extracts, resulting in a marked decrease in the extent of light activation of PEPC. The extent of phosphorylation of PEPC was much higher in Ca2+ or Ca2+-EGTA-fed leaves than in the control, but EGTA decreased the light-induced phosphorylation. Our results suggest that optimal alone concentration of Ca2+ is essential for PEPC in leaves of A. hypochondriacus, particularly in vivo. We suggest that Ca2+ regulates PEPC, at an upstream level, such as transcription, by modulating PEPC-protein kinase, thus facilitating the light activation of PEPC.
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PMID:Modulation of phosphoenolpyruvate carboxylase in vivo by Ca2+ in Amaranthus hypochondriacus, a NAD-ME type C4 plant: possible involvement of Ca2+ in up-regulation of PEPC-protein kinase in vivo. 1625 67

The regulation of carbon partitioning between carbohydrates (principally sucrose) and amino acids has been only poorly characterized in higher plants. The hypothesis that the pathway of sucrose and amino acid biosynthesis compete for carbon skeletons and energy is widely accepted. In this review, we suggest a mechanism involving the regulation of cytosolic protein kinases whereby the flow of carbon is regulated at the level of partitioning between the pathways of carbohydrate and nitrogen metabolism via the covalent modulation of component enzymes. The addition of nitrate to wheat seedlings (Triticum aestivum) grown in the absence of exogenous nitrogen has a dramatic, if transient, impact on sucrose formation and on the activities of sucrose phosphate synthase (which is inactivated) and phosphoenolpyruvate carboxylase (which is activated). The activities of these two enzymes are modulated by protein phosphorylation in response to the addition of nitrate, but they respond in an inverse fashion. Sucrose phosphate synthase in inactivated and phosphoenolpyruvate carboxylase is activated. Nitrate functions as a signal metabolite activating the cytosolic protein kinase, thereby modulating the activities of at least two of the key enzymes in assimilate partitioning and redirecting the flow of carbon away from sucrose biosynthesis toward amino acid synthesis.
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PMID:Nitrate activation of cytosolic protein kinases diverts photosynthetic carbon from sucrose to amino Acid biosynthesis: basis for a new concept. 1665 3

Autoradiography of total soluble maize (Zea mays) leaf proteins incubated with (32)P-labeled adenylates and separated by denaturing electrophoresis revealed that many polypeptides were phosphorylated in vitro by endogenous protein kinase(s). The most intense band was at 94 to 100 kilodaltons and was observed when using either [gamma-(32)P]ATP or [beta-(32)P]ADP as the phosphate donor. This band was comprised of the subunits of both pyruvate, Pi dikinase (PPDK) and phosphoenolpyruvate carboxylase (PEPCase). PPDK activity was previously shown to be dark/light-regulated via a novel ADP-dependent phosphorylation/Pi-dependent dephosphorylation of a threonyl residue. The identity of the acid-stable 94 to 100 kilodalton band phosphorylated by ATP was established unequivocally as PEPCase by two-dimensional gel electrophoresis and immunoblotting. The phosphorylated amino acid was a serine residue, as determined by two-dimensional thin-layer electrophoresis. While the in vitro phosphorylation of PEPCase from illuminated maize leaves by an endogenous protein kinase resulted in a partial inactivation ( approximately 25%) of the enzyme when assayed at pH 7 and subsaturating levels of PEP, effector modulation by l-malate and glucose-6-phosphate was relatively unaffected. Changes in the aggregation state of maize PEPCase (homotetrameric native structure) were studied by nondenaturing electrophoresis and immunoblotting. Enzyme from leaves of illuminated plants dissociated upon dilution, whereas the protein from darkened tissue did not dissociate, thus indicating a physical difference between the enzyme from light- versus dark-adapted maize plants.
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PMID:In vitro phosphorylation of maize leaf phosphoenolpyruvate carboxylase. 1666 42

ATP-dependent polycation-stimulated phosphorylation of highly purified phytochrome preparations from etiolated Avena seedlings has been reported previously (Y-S Wong, H-C Cheng, DA Walsh, JC Lagarias [1986] J Biol Chem 261: 12089-12097). In this study, we present a more detailed description of the properties of this protein kinase based on the analysis of over 30 different Avena phytochrome preparations. ATP-dependent phosphorylation of phytochrome was strongly stimulated by a wide range of polycationic molecules, including synthetic and natural polypeptides as well as nonpeptide cationic polymers. Many of the compounds known to stimulate other known protein kinases (i.e., cyclic nucleotides, Ca(2+), calmodulin, diacylglycerol, phospholipids) were either inhibitory or nonstimulatory. Among the polycations, histone H1, polylysine, and polybrene were the most effective, giving average stimulations of four- to sevenfold. Polycation-stimulated protein phosphorylation was inhibited by elevated ionic strength; of the salts examined, magnesium pyrophosphate was a particularly potent inhibitor of the kinase activity. MgATP was preferred as the phosphoryl donor to either MgGTP or magnesium pyrophosphate. The K(m) for MgATP was estimated to be 30 micromolar when histone H1 was used as a protein substrate. The Pr form of phytochrome was always a better substrate than the Pfr form regardless of the polycation present. Polylysine-stimulated, phytochrome(preparation)-dependent phosphorylation of purified maize phosphoenolpyruvate carboxylase was observed, as well as phosphorylation of a number of polypeptides in crude soluble protein extracts from etiolated Avena seedlings.
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PMID:Properties of a polycation-stimulated protein kinase associated with purified Avena phytochrome. 1666 91

Reversible seryl-phosphorylation contributes to the light/dark regulation of C(4)-leaf phosphoenolpyruvate carboxylase (PEPC) activity in vivo. The specific regulatory residue that, upon in vitro phosphorylation by a maize-leaf protein-serine kinase(s), leads to an increase in catalytic activity and a decrease in malate-sensitivity of the target enzyme has been recently identified as Ser-15 in (32)P-phosphorylated/activated dark-form maize PEPC (J-A Jiao, R Chollet [1990] Arch Biochem Biophys 283: 300-305). In order to ascertain whether this N-terminal seryl residue is, indeed, the in vivo regulatory phosphorylation site, [(32)P]phosphopeptides were isolated and purified from in vivo(32)P-labeled maize and sorghum leaf PEPC and subjected to automated Edman degradation analysis. The results show that purified light-form maize PEPC contains 14-fold more (32)P-radioactivity than the corresponding dark-form enzyme on an equal protein basis and, more notably, only a single N-terminal serine residue (Ser-15 in maize PEPC and its structural homolog, Ser-8, in the sorghum enzyme) was found to be (32)P-phosphorylated in the light or dark. These in vivo observations, combined with the results from our previous in vitro phosphorylation studies (J-A Jiao, R Chollet [1989] Arch Biochem Biophys 269: 526-535; [1990] Arch Biochem Biophys 283: 300-305), demonstrate that an N-terminal seryl residue in C(4) PEPC is, indeed, the regulatory site that undergoes light/dark changes in phosphorylation-status and, thus, plays a major, if not cardinal role in the light-induced changes in catalytic and regulatory properties of this cytoplasmic C(4)-photosynthesis enzyme in vivo.
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PMID:In vivo regulatory phosphorylation site in c(4)-leaf phosphoenolpyruvate carboxylase from maize and sorghum. 1666 68

Phosphoenolpyruvate carboxylase (PEPcase) activity was studied in excised leaves of wheat (Triticum aestivum L.) in the dark and in the light, in presence of either N-free (low-NO(3) (-) leaves) or 40 millimolar KNO(3) (high-NO(3) (-) leaves) nutrient solutions. PEPcase activity increased to 2.7-fold higher than that measured in dark-adapted tissue (control) during the first 60 minutes and continued to increase more slowly to 3.8-fold that of the control. This level was reached after 200 minutes exposure of the leaves to light and high NO(3) (-). In contrast, the lower rate of increase recorded for low-NO(3) (-) leaves ceased after 60 minutes of exposure to light at 2.3-fold the control level. The short-term NO(3) (-) effect increased linearly with the level of NO(3) (-) uptake. In immunoprecipitation experiments, the antibody concentration for PEPcase precipitation increased with the protein extracts from the different treatments in the order: control, illuminated low-NO(3) (-) leaves, illuminated high-NO(3) (-) leaves. This order also applied with regard to a decreasing sensitivity to malate and an increasing stimulation by okadaic acid (an inhibitor of P-protein phosphatases). Following these studies, (32)P labeling experiments were carried out in vivo. These showed that the light-induced change in the properties of the PEPcase was due to an alteration in the phosphorylation state of the protein and that this effect was enhanced in high-NO(3) (-) conditions. Based on the responses of PEPcase and sucrose phosphate synthase in wheat leaves to light and NO(3) (-), an interpretation of the role of NO(3) (-) as either an inhibitor of P-protein phosphatase(s) or activator of protein kinase(s) is inferred. In the presence of NO(3) (-), the phosphorylation state of both PEPcase and sucrose phosphate synthase is increased. This causes activation of the former enzyme and inhibition of the latter. We suggest that NO(3) (-) modulates the relative protein kinase/protein phosphatase ratio to favor increased phosphorylation of both enzymes in order to redirect carbon flow away from sucrose synthesis and toward amino acid synthesis.
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PMID:Effect of Light and NO(3) on Wheat Leaf Phosphoenolpyruvate Carboxylase Activity: Evidence for Covalent Modulation of the C(3) Enzyme. 1666 73


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