The Experts below are selected from a list of 255 Experts worldwide ranked by ideXlab platform
James R Woodgett - One of the best experts on this subject based on the ideXlab platform.
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Glycogen Synthase Kinase 3: A Kinase for All Pathways?
Current topics in developmental biology, 2016Co-Authors: Prital Patel, James R WoodgettAbstract:Glycogen synthase Kinase-3 (GSK-3) is an unusual Protein-Serine Kinase in that it is primarily regulated by inhibition and lies downstream of multiple cell signaling pathways. This raises a variety of questions in terms of its physiological role(s), how signaling specificity is maintained and why so many eggs have been placed into one basket. There are actually two baskets, as there are two isoforms, GSK-3α and β, that are highly related and largely redundant. Their many substrates range from regulators of cellular metabolism to molecules that control growth and differentiation. In this chapter, we review the characteristics of GSK-3, update progress in understanding the Kinase, and try to answer some of the questions raised by its unusual properties. Indeed, the Kinase may trigger transformation in our thinking of how cellular signals are organized and controlled.
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Judging a Protein by more than its name: GSK-3.
Science's STKE : signal transduction knowledge environment, 2001Co-Authors: James R WoodgettAbstract:As knowledge of cellular signal transduction has accumulated, general truisms have emerged, including the notion that signaling Proteins are usually activated by stimuli and that they, in turn, mediate the actions of specific agonists. Glycogen synthase Kinase-3 (GSK-3) is an unusual Protein-Serine Kinase that bucks these conventions. This evolutionarily conserved Protein Kinase is active in resting cells and is inhibited in response to activation of several distinct pathways, including those acting by elevation of 3′ phosphorylated phosphatidylinositol lipids and adenosine 3′-5′-monophosphate (cAMP). In addition, GSK-3 is distinctly regulated by, and is a core component of, the Wnt pathway. This review describes the unique characteristics of this decidedly oddball Protein Kinase in terms of its diverse biological functions, plethora of targets, role in several human diseases, and consequential potential as a therapeutic target.
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Modulation of cellular apoptotic potential: contributions to oncogenesis
Oncogene, 1999Co-Authors: Vuk Stambolic, Tak W Mak, James R WoodgettAbstract:The importance of apoptosis as a natural means to eliminate unwanted or damaged cells has been realized over the past decade. Many components required to exercise programmed cell death have been identified and shown to pre-exist in most, if not all, cells. Such ubiquity requires that apoptosis be tightly controlled and suggests the propensity of cells to trigger the cellular death machinery can be regulated. Recently, several signaling pathways have been demonstrated to impact the apoptotic potential of cells, most notably the phosphatidylinositol 3′ Kinase (PI3′K) pathway. The 3′ phosphorylated lipid products generated by this enzyme promote activation of a Protein-Serine Kinase, PKB/AKT, which is necessary and sufficient to confer cell PI3′K-dependent survival signals. The relevance of this pathway to human cancer was revealed by the recent finding that the product of the PTEN tumor suppressor gene acts to antagonize PI3′K. This review focuses on the regulation and mechanisms by which PKB activation protects cells and the oncologic consequences of dysregulation of the pathway.
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Regulation of the Protein Kinase Activity of ShaggyZeste-white3 by Components of the Wingless Pathway in Drosophila Cells and Embryos
The Journal of biological chemistry, 1999Co-Authors: Laurent Ruel, Vuk Stambolic, Adnan Ali, Armen S. Manoukian, James R WoodgettAbstract:The Protein-Serine Kinase Shaggy(Zeste-white3) (Sgg(Zw3)) is the Drosophila homolog of mammalian glycogen synthase Kinase-3 and has been genetically implicated in signal transduction pathways necessary for the establishment of patterning. Sgg(Zw3) is a putative component of the Wingless (Wg) pathway, and epistasis analyses suggest that Sgg(Zw3) function is repressed by Wg signaling. Here, we have investigated the biochemical consequences of Wg signaling with respect to the Sgg(Zw3) Protein Kinase in two types of Drosophila cell lines and in embryos. Our results demonstrate that Sgg(Zw3) activity is inhibited following exposure of cells to Wg Protein and by expression of downstream components of Wg signaling, Drosophila frizzled 2 and dishevelled. Wg-dependent inactivation of Sgg(Zw3) is accompanied by Serine phosphorylation. We also show that the level of Sgg(Zw3) activity regulates the stability of Armadillo Protein and modulates the level of phosphorylation of D-Axin and Armadillo. Together, these results provide direct biochemical evidence in support of the genetic model of Wg signaling and provide a model for dissecting the molecular interactions between the signaling Proteins.
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Reconstitution of novel signalling cascades responding to cellular stresses.
Philosophical transactions of the Royal Society of London. Series B Biological sciences, 1996Co-Authors: James R Woodgett, John M. Kyriakis, Joseph Avruch, Leonard I. Zon, Brent W. Zanke, Dennis J. TempletonAbstract:Mammalian cells respond to their immediate environment by inducing signal transduction cascades that regulate metabolism, secretion and gene expression. Several of these signalling pathways are structurally and organizationally related insofar as they require activation of a Protein-Serine Kinase via it's phosphorylation on tyrosine and threonine; the archetype being mitogen-activated Protein Kinase (MAPK) which responds primarily to mitogenic stimuli via Ras. In contrast, two more recently identified cascades are responsive to cellular stresses such as heat, inflammatory cytokines, ischaemia and metabolic poisons. The recent identification of the components of these pathways has allowed manipulation of the stress-responsive pathways and evaluation of their physiological roles. These studies reveal a high degree of independence between the pathways not apparent from in vitro studies. Manipulation of the pathways in vivo will likely result in novel therapies for inflammatory disease and reperfusion injury.
Charles Grose - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation by the Varicella-Zoster Virus ORF47 Protein Serine Kinase Determines whether Endocytosed Viral gE Traffics to the trans-Golgi Network or Recycles to the Cell Membrane
Journal of virology, 2002Co-Authors: T. K. Kenyon, Jeffrey I. Cohen, Charles GroseAbstract:Like all alphaherpesviruses, varicella-zoster virus (VZV) infection proceeds by both cell-cell spread and virion production. Virions are enveloped within vacuoles located near the trans-Golgi network (TGN), while in cell-cell spread, surface glycoProteins fuse cells into syncytia. In this report, we delineate a potential role for Serine/threonine phosphorylation of the cytoplasmic tail of the predominant VZV glycoProtein, gE, in these processes. The fact that VZV gE (formerly called gpI) is phosphorylated has been documented (E. A. Montalvo and C. Grose, Proc. Natl. Acad. Sci. USA 83:8967-8971, 1986), although respective roles of viral and cellular Protein Kinases have never been delineated. VZV ORF47 is a viral Serine Protein Kinase that recognized a consensus sequence similar to that of casein Kinase II (CKII). During open reading frame 47 (ORF47)-specific in vitro Kinase assays, ORF47 phosphorylated four residues in the cytoplasmic tail of VZV gE (S593, S595, T596, and T598), thus modifying the known phosphofurin acidic cluster sorting Protein 1 domain. CKII phosphorylated gE predominantly on the two threonine residues. In wild-type-virus-infected cells, where ORF47-mediated phosphorylation predominated, gE endocytosed and relocalized to the TGN. In cells infected with a VZV ORF47-null mutant, internalized VZV gE recycled to the plasma membrane and did not localize to the TGN. The mutant virus also formed larger syncytia than the wild-type virus, linking CKII-mediated gE phosphorylation with increased cell-cell spread. Thus, ORF47 and CKII behaved as "team players" in the phosphorylation of VZV gE. Taken together, the results showed that phosphorylation of VZV gE by ORF47 or CKII determined whether VZV infection proceeded toward a pathway likely involved with either virion production or cell-cell spread.
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Varicella-Zoster Virus ORF47 Protein Serine Kinase: Characterization of a Cloned, Biologically Active Phosphotransferase and Two Viral Substrates, ORF62 and ORF63
Journal of virology, 2001Co-Authors: T. K. Kenyon, J. Lynch, John Hay, William T. Ruyechan, Charles GroseAbstract:Varicella-zoster virus (VZV) codes for a Protein Serine Kinase called ORF47; the herpes simplex virus (HSV) homolog is UL13. No recombinant alphaherpesvirus Serine Kinase has been biologically active in vitro. We discovered that preservation of the intrinsic Kinase activity of recombinant VZV ORF47 required unusually stringent in vitro conditions, including physiological concentrations of polyamines. In this assay, ORF47 phosphorylated two VZV regulatory Proteins: the ORF62 Protein (homolog of HSV ICP4) and the ORF63 Protein (homolog of HSV ICP22). Of interest, ORF47 Kinase also coprecipitated ORF63 Protein from the Kinase assay supernatant.
Eva-maria Mandelkow - One of the best experts on this subject based on the ideXlab platform.
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Microtubule-associated Protein/Microtubule Affinity-regulating Kinase (p110mark) A NOVEL Protein Kinase THAT REGULATES TAU-MICROTUBULE INTERACTIONS AND DYNAMIC INSTABILITY BY PHOSPHORYLATION AT THE ALZHEIMER-SPECIFIC SITE Serine 262
The Journal of biological chemistry, 1995Co-Authors: Gerard Drewes, B. Trinczek, Susanne Illenberger, Jacek Biernat, Gerold Schmitt-ulms, Helmut E. Meyer, Eva-maria MandelkowAbstract:Aberrant phosphorylation of the microtubule-associated Protein tau is one of the pathological features of neuronal degeneration in Alzheimer's disease. The phosphorylation of Ser-262 within the microtubule binding region of tau is of particular interest because so far it is observed only in Alzheimer's disease (Hasegawa, M., Morishima-Kawashima, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y. (1992) J. Biol. Chem. 26, 17047-17054) and because phosphorylation of this site alone dramatically reduces the affinity for microtubules in vitro (Biernat, J., Gustke, N., Drewes, G., Mandelkow, E.-M., and Mandelkow, E. (1993) Neuron 11, 153-163). Here we describe the purification and characterization of a Protein-Serine Kinase from brain tissue with an apparent molecular mass of 110 kDa on SDS gels. This Kinase specifically phosphorylates tau on its KIGS or KCGS motifs in the repeat domain, whereas no significant phosphorylation outside this region was detected. Phosphorylation occurs mainly on Ser-262 located in the first repeat. This largely abolishes tau's binding to microtubules and makes them dynamically unstable, in contrast to other Protein Kinases that phosphorylate tau at or near the repeat domain. The data suggest a role for this novel Kinase in cellular events involving rearrangement of the microtuble-associated Proteins/microtubule arrays and their pathological degeneration in Alzheimer's disease.
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microtubule associated Protein microtubule affinity regulating Kinase p110mark a novel Protein Kinase that regulates tau microtubule interactions and dynamic instability by phosphorylation at the alzheimer specific site Serine 262
Journal of Biological Chemistry, 1995Co-Authors: Gerard Drewes, B. Trinczek, Susanne Illenberger, Jacek Biernat, Helmut E. Meyer, Eva-maria Mandelkow, Gerold SchmittulmsAbstract:Aberrant phosphorylation of the microtubule-associated Protein tau is one of the pathological features of neuronal degeneration in Alzheimer's disease. The phosphorylation of Ser-262 within the microtubule binding region of tau is of particular interest because so far it is observed only in Alzheimer's disease (Hasegawa, M., Morishima-Kawashima, M., Takio, K., Suzuki, M., Titani, K., and Ihara, Y. (1992) J. Biol. Chem. 26, 17047-17054) and because phosphorylation of this site alone dramatically reduces the affinity for microtubules in vitro (Biernat, J., Gustke, N., Drewes, G., Mandelkow, E.-M., and Mandelkow, E. (1993) Neuron 11, 153-163). Here we describe the purification and characterization of a Protein-Serine Kinase from brain tissue with an apparent molecular mass of 110 kDa on SDS gels. This Kinase specifically phosphorylates tau on its KIGS or KCGS motifs in the repeat domain, whereas no significant phosphorylation outside this region was detected. Phosphorylation occurs mainly on Ser-262 located in the first repeat. This largely abolishes tau's binding to microtubules and makes them dynamically unstable, in contrast to other Protein Kinases that phosphorylate tau at or near the repeat domain. The data suggest a role for this novel Kinase in cellular events involving rearrangement of the microtuble-associated Proteins/microtubule arrays and their pathological degeneration in Alzheimer's disease.
Pierre Gadal - One of the best experts on this subject based on the ideXlab platform.
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Molecular basis of plant adaptation to light. Example of two enzymes of the C4 photosynthesis cycle
Comptes Rendus de l'Académie des Sciences - Series III - Sciences de la Vie, 1998Co-Authors: Pierre Gadal, Jeannoel Pierre, Jean Vidal, Szeherazada Rydz, Eric Ruelland, Myroslawa Miginiac-maslowAbstract:Abstract The light-dependent mechanisms responsible for the regulation of two enzymes of the C4 photosynthetic cycle, phosphoenolpyruvate carboxylase and NADP-dependent malate dehydrogenase in Sorghum are presented as an illustration of adaptative mechanisms of plants to light. Emphasis is put on the organization of the signaltransduction chains which upregulate both enzymes at transcriptional and post-translational levels. For both enzymes, small gene families include a photosynthesis-related gene, the expression of which is light-triggered. The light-controlled phosphorylation of C4 PEPC implies cross-talk between photosynthetic cell types, cytosolic pH and calcium changes, and the upregulation of a specific Protein-Serine Kinase. This post-translational modification strongly influences its regulation by photosynthesis-related metabolites. NADP-MDH activity is controlled by a chloroplastic redox cascade involving thioredoxin as the ultimate relay. The significance of these regulatory circuits is discussed in relation with the flexibility of the metabolism in the context of the C4 photosynthesis.
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the light dependent transduction pathway controlling the regulatory phosphorylation of c4 phosphoenolpyruvate carboxylase in protoplasts from digitaria sanguinalis
The Plant Cell, 1996Co-Authors: Nathalie Giglioliguivarch, Jeannoel Pierre, Raymond Chollet, Jean Vidal, Spencer Brown, Pierre GadalAbstract:Phosphoenolpyruvate carboxylase (PEPC) was characterized in extracts from C4 mesophyll protoplasts isolated from Digitaria sanguinalis leaves and shown to display the structural, functional, and regulatory properties typical of a C4 PEPC. In situ increases in the apparent phosphorylation state of the enzyme and the activity of its Ca2+-independent Protein-Serine Kinase were induced by light plus NH4Cl or methylamine. The photosynthesis-related metabolite 3-phosphoglycerate (3-PGA) was used as a substitute for the weak base in these experiments. The early effects of light plus the weak base or 3-PGA treatment were alkalinization of protoplast cytosolic pH, shown by fluorescence cytometry, and calcium mobilization from vacuoles, as suggested by the use of the calcium channel blockers TMB-8 and verapamil. The increases in PEPC Kinase activity and the apparent phosphorylation state of PEPC also were blocked in situ by the electron transport and ATP synthesis inhibitors DCMU and gramicidin, respectively, the calcium/calmodulin antagonists W7, W5, and compound 48/80, and the cytosolic Protein synthesis inhibitor cycloheximide. These results suggest that the production of ATP and/or NADPH by the illuminated mesophyll chloroplast is required for the activation of the transduction pathway, which presumably includes an upstream Ca2+-dependent Protein Kinase and a cytosolic Protein synthesis event. The collective data support the view that the C4 PEPC light transduction pathway is contained entirely within the mesophyll cell and imply cross-talk between the mesophyll and bundle sheath cells in the form of the photosynthetic metabolite 3-PGA.
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Phosphoenolpyruvate carboxylase: structure, regulation and evolution
Plant Science, 1994Co-Authors: Loïc Lepiniec, Raymond Chollet, Jean Vidal, Pierre Gadal, Claude CrétinAbstract:Abstract Plant phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC) is encoded by a small multigene family in which the expression of each member is controlled individually by exogenous (light, environmental) and/or endogenous (hormonal and developmental) stimuli. The involvement of putative trans-actig factors and consensus cis-elements of promoters in the specific transcriptional regulation of the PEPC genes is discussed. At the post-translational level, the regulatory strategy of the plant enzyme is mainly to offset the negative effect of the feedback inhibitor, L-malate, the end-product of the oxaloacetate reduction. All plant PEPC-forms are under positive and negative allosteric control by metabolite effectors and possess a consensus phosphorylation site containing a target Serine residue near their N-terminus (e.g. Ser8 in C4 PEPC from sorghum). In C4 and Crassulacean acid metabolism (CAM) plants, a complex signal-transduction chain activates a Ca2+-independent Protein-Serine Kinase responsible for regulatory phosphorylation of PEPC. A more thorough understanding of the functional and regulatory properties of the bacterial and C4 enzymes has emerged by exploiting recombinant Proteins and site-directed mutagenesis. In these newly opened areas, PEPC offers one of the best characterized paradigms of plant signaling. Finally, some emerging ideas on the evolution and phylogenetic relationships of the various PEPC isoforms are presented.
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Production and properties of recombinant C3-type phosphoenolpyruvate carboxylase from Sorghum vulgare: in vitro phosphorylation by leaf and root PyrPC Protein Serine Kinases.
Biochemical and biophysical research communications, 1993Co-Authors: Valérie Pacquit, Jean Vidal, S. Santi, Claude Crétin, Van Le Bui, Pierre GadalAbstract:In this work, the C3-type form of Sorghum phosphoenolpyruvate carboxylase (PyrPC) was produced in PyrPC-deficient strains of Escherichia coli transformed by a plasmid bearing the corresponding full-length cDNA (CPR1). The full-sized Protein was purified to homogeneity by immunoaffinity chromatography. Some functional and regulatory properties were described; notably, the immunopurified PyrPC could be phosphorylated in reconstituted assay by 1) both a mammalian PKA and the PyrPC Protein Serine Kinase purified from Sorghum leaves and 2) a novel Protein Kinase affinity-purified from Sorghum roots. In all cases phosphorylation was accompanied by a marked reduction in its malate sensitivity.
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Regulatory Phosphorylation of C4 Phosphoenolpyruvate Carboxylase (A Cardinal Event Influencing the Photosynthesis Rate in Sorghum and Maize).
Plant physiology, 1993Co-Authors: Naïma Bakrim, Jean Vidal, Pierre Gadal, Jean-louis Prioul, E. Deleens, J.-p. Rocher, Martine Arrio-dupont, Raymond CholletAbstract: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.
Raymond Chollet - One of the best experts on this subject based on the ideXlab platform.
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Structural Requirements for Phosphorylation of C4-Leaf Phosphoenolpyruvate Carboxylase by its Highly Regulated Protein-Serine Kinase. A Comparative Study with Synthetic-Peptide Substrates and Mutant Target Proteins
Functional Plant Biology, 1997Co-Authors: Valérie Pacquit, Jean Vidal, Jin-an Jiao, Stephen M. G. Duff, Gururaj B. Maralihalli, Gautam Sarath, Shirley A. Condon, Raymond CholletAbstract:A family of synthetic peptides modelled after the highly conserved, N-terminal phosphorylation domain of C4 phosphoenolpyruvate carboxylase (PEPC) and a complementary set of recombinant mutant target Proteins were exploited to investigate the local structural requirements for phosphorylation of this cytosolic C4 enzyme by its Ca2+-independent Protein Kinase. The only peptide homolog examined that was significantly phosphorylated by maize (Zea mays L.) leaf PEPC-Kinase spanned the P−5 through P+16 region surrounding the target Serine residue in maize PEPC (fifth through sixteenth residues on the N- and C-terminal sides, respectively, of the phosphorylatable Serine at position ‘P’). However, its apparent Km value was 200-times that of intact C4 PEPC. The results from the related site-directed mutagenesis experiments with the recombinant sorghum (Sorghum vulgare) C4 -enzyme indicated that alteration of several highly conserved residues flanking the target Serine with non-conservative Ala substitutions at the P−4, P−3, and P+10 positions had only modest effects, if any, on its in-vitro phosphorylation by maize PEPC-Kinase. These collective findings implicate a secondary site(s) of interaction in C4 PEPC, removed from the N-terminal phosphorylation domain, that is also an important recognition element for its low abundance, highly regulated Protein-Serine/threonine Kinase.
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the light dependent transduction pathway controlling the regulatory phosphorylation of c4 phosphoenolpyruvate carboxylase in protoplasts from digitaria sanguinalis
The Plant Cell, 1996Co-Authors: Nathalie Giglioliguivarch, Jeannoel Pierre, Raymond Chollet, Jean Vidal, Spencer Brown, Pierre GadalAbstract:Phosphoenolpyruvate carboxylase (PEPC) was characterized in extracts from C4 mesophyll protoplasts isolated from Digitaria sanguinalis leaves and shown to display the structural, functional, and regulatory properties typical of a C4 PEPC. In situ increases in the apparent phosphorylation state of the enzyme and the activity of its Ca2+-independent Protein-Serine Kinase were induced by light plus NH4Cl or methylamine. The photosynthesis-related metabolite 3-phosphoglycerate (3-PGA) was used as a substitute for the weak base in these experiments. The early effects of light plus the weak base or 3-PGA treatment were alkalinization of protoplast cytosolic pH, shown by fluorescence cytometry, and calcium mobilization from vacuoles, as suggested by the use of the calcium channel blockers TMB-8 and verapamil. The increases in PEPC Kinase activity and the apparent phosphorylation state of PEPC also were blocked in situ by the electron transport and ATP synthesis inhibitors DCMU and gramicidin, respectively, the calcium/calmodulin antagonists W7, W5, and compound 48/80, and the cytosolic Protein synthesis inhibitor cycloheximide. These results suggest that the production of ATP and/or NADPH by the illuminated mesophyll chloroplast is required for the activation of the transduction pathway, which presumably includes an upstream Ca2+-dependent Protein Kinase and a cytosolic Protein synthesis event. The collective data support the view that the C4 PEPC light transduction pathway is contained entirely within the mesophyll cell and imply cross-talk between the mesophyll and bundle sheath cells in the form of the photosynthetic metabolite 3-PGA.
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salt induction and the partial purification characterization of phosphoenolpyruvate carboxylase Protein Serine Kinase from an inducible crassulacean acid metabolism cam plant mesembryanthemum crystallinum l
Archives of Biochemistry and Biophysics, 1994Co-Authors: Raymond CholletAbstract:Abstract Treatment of the common ice plant ( Mesembryanthemum crystallinum ) with high salinity caused the well-documented increase in phospho enol pyruvate carboxylase (PEPC) Protein and a concomitant rise in the activity of a Ca 2+ -independent PEPC-Kinase (PEPC-PK). When the plants were irrigated with 0.5 M NaCl, PEPC Protein level and PEPC-PK activity started to increase after 2 days of treatment and continued to rise for the next 8 days, attaining about a 14- and 8-fold total increase, respectively. This salt-induced PEPC-Kinase activity was detected only in leaves harvested from the stressed plants at night. This highly regulated Protein Kinase was partially purified about 3500-fold from these darkened, salt-stressed plants by sequential fast-Protein liquid chromatography on phenyl-Sepharose, blue dextran-agarose, and Superdex 75. The gel-filtration data indicated that the native PEPC-Kinase has a molecular weight around 33,000. Complementary analysis by denaturing electrophoresis and subsequent in situ renaturation and assay of PEPC-Kinase activity revealed two major PEPC-PK polypeptides with approximate molecular masses of 39 and 32 kDa. The partially purified M. crystallinum PEPC-Kinase readily phosphorylated PEPCs purified from maize, M. crystallinum , and tobacco leaves and a recombinant sorghum enzyme. In contrast, this Ca 2+ -independent Protein Kinase phosphorylated neither a recombinant sorghum mutant PEPC in which the target residue (Ser-8) was changed by site-directed mutagenesis to Asp nor histone III-S, casein, and bovine serum albumin. The optimal pH for PEPC-PK activity was pH 8.0 and this activity was affected by both the substrate (phospho enol pyruvate) and the negative allosteric effector (L-malate) of PEPC in a pH-dependent manner. Overall, the molecular properties of this highly regulated PEPC-Kinase from M. crystallinum are strikingly similar to those reported recently by this laboratory for the reversibly light-activated C 4 enzyme from maize ( Arch. Biochem. Biophys. , 1993, 304, 496-502, and 307, 416-419).
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Salt Induction and the Partial Purification/Characterization of Phosphoenolpyruvate Carboxylase Protein-Serine Kinase from an Inducible Crassulacean-Acid-Metabolism (CAM) Plant, Mesembryanthemum crystallinum L.
Archives of biochemistry and biophysics, 1994Co-Authors: Raymond CholletAbstract:Abstract Treatment of the common ice plant ( Mesembryanthemum crystallinum ) with high salinity caused the well-documented increase in phospho enol pyruvate carboxylase (PEPC) Protein and a concomitant rise in the activity of a Ca 2+ -independent PEPC-Kinase (PEPC-PK). When the plants were irrigated with 0.5 M NaCl, PEPC Protein level and PEPC-PK activity started to increase after 2 days of treatment and continued to rise for the next 8 days, attaining about a 14- and 8-fold total increase, respectively. This salt-induced PEPC-Kinase activity was detected only in leaves harvested from the stressed plants at night. This highly regulated Protein Kinase was partially purified about 3500-fold from these darkened, salt-stressed plants by sequential fast-Protein liquid chromatography on phenyl-Sepharose, blue dextran-agarose, and Superdex 75. The gel-filtration data indicated that the native PEPC-Kinase has a molecular weight around 33,000. Complementary analysis by denaturing electrophoresis and subsequent in situ renaturation and assay of PEPC-Kinase activity revealed two major PEPC-PK polypeptides with approximate molecular masses of 39 and 32 kDa. The partially purified M. crystallinum PEPC-Kinase readily phosphorylated PEPCs purified from maize, M. crystallinum , and tobacco leaves and a recombinant sorghum enzyme. In contrast, this Ca 2+ -independent Protein Kinase phosphorylated neither a recombinant sorghum mutant PEPC in which the target residue (Ser-8) was changed by site-directed mutagenesis to Asp nor histone III-S, casein, and bovine serum albumin. The optimal pH for PEPC-PK activity was pH 8.0 and this activity was affected by both the substrate (phospho enol pyruvate) and the negative allosteric effector (L-malate) of PEPC in a pH-dependent manner. Overall, the molecular properties of this highly regulated PEPC-Kinase from M. crystallinum are strikingly similar to those reported recently by this laboratory for the reversibly light-activated C 4 enzyme from maize ( Arch. Biochem. Biophys. , 1993, 304, 496-502, and 307, 416-419).
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Phosphoenolpyruvate carboxylase: structure, regulation and evolution
Plant Science, 1994Co-Authors: Loïc Lepiniec, Raymond Chollet, Jean Vidal, Pierre Gadal, Claude CrétinAbstract:Abstract Plant phosphoenolpyruvate carboxylase (EC 4.1.1.31; PEPC) is encoded by a small multigene family in which the expression of each member is controlled individually by exogenous (light, environmental) and/or endogenous (hormonal and developmental) stimuli. The involvement of putative trans-actig factors and consensus cis-elements of promoters in the specific transcriptional regulation of the PEPC genes is discussed. At the post-translational level, the regulatory strategy of the plant enzyme is mainly to offset the negative effect of the feedback inhibitor, L-malate, the end-product of the oxaloacetate reduction. All plant PEPC-forms are under positive and negative allosteric control by metabolite effectors and possess a consensus phosphorylation site containing a target Serine residue near their N-terminus (e.g. Ser8 in C4 PEPC from sorghum). In C4 and Crassulacean acid metabolism (CAM) plants, a complex signal-transduction chain activates a Ca2+-independent Protein-Serine Kinase responsible for regulatory phosphorylation of PEPC. A more thorough understanding of the functional and regulatory properties of the bacterial and C4 enzymes has emerged by exploiting recombinant Proteins and site-directed mutagenesis. In these newly opened areas, PEPC offers one of the best characterized paradigms of plant signaling. Finally, some emerging ideas on the evolution and phylogenetic relationships of the various PEPC isoforms are presented.
