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)

Glucose-induced inactivation of the gluconeogenetic enzymes fructose-1,6-biphosphatase, cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase was tested in yeast mutants defective in adenylate cyclase (cyr1 mutation) and in the cAMP-binding subunit of cAMP-dependent protein kinase (bcy 1 mutation). In the mutant AM7-11D (cyr1 mutation), glucose-induced cAMP overshoot was absent, and no significant inactivation of the gluconeogenetic enzymes was detected, thus supporting the role of cAMP in the process. Moreover, in the mutant AM9-8B (bcy1 mutation), no cAMP-dependent protein kinase activity was evidenced, and, in addition, a normal inactivation pattern was observed, thus indicating that other mechanisms evoked by glucose might be required in the process. In the double mutant AM7-11DR-4 (cyr1 bcy1 mutations), no inactivating effect was triggered by the sugar: this suggests that cAMP exerts some additional effect on the process, besides the activation of cAMP-dependent protein kinase. Furthermore, in AM7-11D, extracellular cAMP triggered about 50% of inactivation of fructose-1,6-bisphosphatase; this effect was largely reversed in acetate medium plus cycloheximide even after 150 min of incubation. However, an extensive and essentially irreversible inactivation was evidenced in the presence of glucose plus cAMP, whereas glucose alone was only slightly effective. Therefore, the reversible effect of cAMP, which probably corresponds to enzyme phosphorylation, seems to be required for the irreversible, probably proteolytic, glucose-stimulated inactivation of this enzyme. Cytoplasmic malate dehydrogenase and phosphoenolpyruvate carboxykinase in AM7-11D were also inactivated by cAMP, and much more by glucose plus cAMP, whereas glucose was practically ineffective. However, reversibility of the effect was not detected, and, in addition, no phosphorylation of phosphoenolpyruvate carboxykinase could be evidenced. Therefore, the sugar quite probably stimulates proteolysis of these enzymes, but the mechanism of cAMP in their degradation has still to be defined.
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PMID:Studies on glucose-induced inactivation of gluconeogenetic enzymes in adenylate cyclase and cAMP-dependent protein kinase yeast mutants. 609 42

A number of hormones and growth factors stimulate target cells through receptors which are coupled to second messenger pathways. The second messenger cAMP, for example, mediates a wide variety of cellular responses to hormonal signals, including changes in intermediary metabolism, cellular proliferation and cellular motility. In mammalian cells, all of these biological responses are triggered by the activation of the cAMP-dependent protein kinase A, a heterotetramer consisting of paired catalytic and regulatory subunits. Upon hormonal stimulation, cAMP binds tightly to the regulatory subunits, thereby liberating catalytic subunits and promoting the phosphorylation of cellular substrates. In the liver, cAMP functions as a starvation state signal, mediating hormonal cues from the pancreas and adrenal gland to stimulate glucose production. cAMP stimulates glucose production, in part, by regulating transcription of the gene for phosphoenolpyruvate carboxykinase (PEPCK), a rate-limiting enzyme in gluconeogenesis. Following hormonal stimulation, cAMP induces PEPCK gene expression 10-fold within 20-30 min. This induction appears to be independent of new protein synthesis.
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PMID:Regulation of somatostatin gene transcription by cAMP. 758 54

The phosphoenolpyruvate carboxykinase (PEPCK) gene is regulated at the transcriptional level by a variety of effectors in a tissue-specific fashion. In order to study the parameters involved in the tissue-specific hormonal regulation of the PEPCK gene, we have used a transient expression test in well-differentiated rat hepatoma cells as well as in dedifferentiated variants. In this test, the PEPCK promoter is induced by glucocorticoids in well-differentiated FGC4 cells, but not in H5 dedifferentiated variants, in spite of the presence in H5 cells of the glucocorticoid receptor. Study of the PEPCK promoter using electrophoretic mobility shift assays reveals binding sites for the liver-enriched transcription factors HNF1, vHNF1, HNF3, HNF4, and CAAT/enhancer binding protein members. Overexpression of the liver-enriched transcription factors absent in the dedifferentiated variants, such as HNF1 and HNF4, is not sufficient to restore glucocorticoid response of the PEPCK promoter in the variants. Moreover, systematic analysis of the PEPCK promoter reveals that the presence of a region covering a cAMP-responsive element (CRE1 at -80) and a CAAT box is necessary for full response of the PEPCK promoter to glucocorticoids in well-differentiated rat hepatoma cells. In a cotransfection test, overexpression of the regulatory subunit of protein kinase A (PKA), causing sequestering of PKA, abolishes the glucocorticoid response of the promoter in well-differentiated cells. On the other hand, in dedifferentiated variants, overexpression of the catalytic subunit of PKA restores the response to glucocorticoids. The action of PKA on the glucocorticoid response requires the presence of the CRE1 element and is promoter specific because it does not concern nonhepatic promoters such as the long terminal repeats of the mouse mammary tumor virus. These results suggest that the full response of the PEPCK promoter to glucocorticoids requires activation of another signal transduction pathway, the cAMP-mediated pathway.
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PMID:Response of the phosphoenolpyruvate carboxykinase gene to glucocorticoids depends on the integrity of the cAMP pathway. 781 33

Treatment of the common ice plant (Mesembryanthemum crystallinum) with high salinity caused the well-documented increase in phosphoenolpyruvate 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 (phosphoenolpyruvate) 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 C4 enzyme from maize (Arch. Biochem. Biophys., 1993, 304, 496-502, and 307, 416-419).
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PMID:Salt induction and the partial purification/characterization of phosphoenolpyruvate carboxylase protein-serine kinase from an inducible crassulacean-acid-metabolism (CAM) plant, Mesembryanthemum crystallinum L. 794 3

The influence of pH on the in vitro activity and regulatory properties of Sorghum leaf C4 phosphoenolpyruvate carboxylase (PEPC) was investigated with respect to the phosphorylation status of the enzyme. In vitro protein phosphorylation was achieved using the catalytic subunit of a cAMP-dependent protein kinase (PKA) and a recombinant, immunopurified PEPC (0.9 mol of covalent Pi/mol PEPC subunit). Between pH 6.8 and 8, velocity and IC50 for L-malate increased for both the nonphosphorylated and the phosphorylated forms. With respect to the nonphosphorylated PEPC, the phospho-PEPC always gave high values for these kinetic parameters at the pH range investigated, especially between pH 7 and 7.3. The phosphorylation-induced stimulation of PEPC activity was four- to fivefold at pH 7.1 and approximately twofold at pH 7.3. The IC50 for L-malate showed a two- to threefold increase at pH 7.3, but varied less at pH 7.1 upon PEPC phosphorylation. Thus, phosphorylation of PEPC caused a predominant V effect or a mixed (V/IC50) effect at pH 7.1 or 7.3, respectively. This was also observed with the enzyme from desalted crude protein extracts from dark or light-adapted Sorghum leaves and leaf-derived mesophyll protoplasts illuminated in the presence of methylamine, a compound known to increase cytosolic pH (pHc). At pH 7.3, desensitization to L-malate of phospho-PEPC was due to an enhanced ability of PEP to compete with the inhibitor. The positive effector glucose-6P acted similarly to phosphorylation; however, a combination of both factors (glucose-6P and phosphorylation) led to a much larger increase in the IC50 for L-malate than that observed by a single factor.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The effect of pH on the covalent and metabolic control of C4 phosphoenolpyruvate carboxylase from Sorghum leaf. 798 87

We have previously shown that insulin is less effective in inducing expression of several genes in H4 hepatoma cells with reduced functional protein kinase-C (PKC) activity. However, other reports suggest that insulin regulation of gene transcription is not PKC dependent. Insulin and phorbol 12-myristate 13-acetate (PMA) rapidly inhibit transcription of the tyrosine aminotransferase and albumin genes. Prolonged PMA pretreatment, to desensitize cells to PMA, resulted in a loss of insulin ability to inhibit albumin transcription. Insulin was still able to inhibit tyrosine aminotransferase transcription, but less than in non-PMA-pretreated cells, and there was also a slight decrease in the ability of insulin to inhibit phosphoenolpyruvate carboxykinase transcription. We previously demonstrated decreased responsiveness of PMA-induced gene expression in insulin-desensitized cells. In the present work, using insulin-desensitized H4 cells (insulin pretreatment for 24 h), subsequent treatment with PMA did not alter phosphoenolpyruvate carboxykinase transcription rates, whereas PMA did inhibit tyrosine aminotransferase transcription rates to an extent similar to observed in nonpretreated cells. Unexpectedly, there was a significant increase in albumin transcription after PMA addition to insulin-pretreated cells. These findings support our hypothesis that the role of PKC in the regulation of gene expression by insulin varies for different insulin-regulated genes.
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PMID:Evidence for diverse roles of protein kinase-C in the inhibition of gene expression by insulin: the tyrosine aminotransferase, albumin, and phosphoenolpyruvate carboxykinase genes. 798 15

We report that the small tumor (small-t) antigen of simian virus 40 (SV40) forms complexes with nuclear protein phosphatase 2A (PP2A) and regulates the phosphorylation and transcriptional transactivation function of the cyclic AMP (cAMP)-regulatory element binding protein (CREB). PP2A coimmunoprecipitated with small t from nuclear extracts from HepG2 cells expressing small t or from rat liver nuclear extracts to which recombinant small t was added. Protein phosphatase 1 was not detected in small-t immunoprecipitates. In HepG2 cells expressing small t, dibutyryl-cAMP (Bt2cAMP) stimulated the phosphorylation of CREB 65-fold, whereas CREB phosphorylation was stimulated only 5- to 8-fold by Bt2cAMP in cells not expressing small t. Small t also inhibited the dephosphorylation of cAMP-dependent protein kinase (PKA)-phosphorylated CREB in rat liver nuclear extracts. In cells expressing small t, Bt2cAMP-stimulated transcription from the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter was enhanced over the level of transcription from the PEPCK promoter in cells not expressing small t. Small t also enhanced Bt2cAMP-stimulated transcription from a Gal4-responsive promoter in cells expressing a chimeric protein containing the Gal4 DNA-binding domain linked to the CREB transactivation domain. However, small t did not stimulate transcription either from a 5' deletion mutant of the PEPCK promoter that is not able to bind CREB or from the Gal4-responsive promoter in the absence of the Gal4-CREB protein. These data suggest that small t enhances Bt2cAMP-stimulated gene transcription by inhibiting the dephosphorylation of PKA-phosphorylated CREB by nuclear PP2A. These findings support previous observations that nuclear PP2A is the primary phosphatase that dephosphorylates PKA-phosphorylated CREB.
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PMID:Simian virus 40 small tumor antigen inhibits dephosphorylation of protein kinase A-phosphorylated CREB and regulates CREB transcriptional stimulation. 806 21

Transcription of the phosphoenolpyruvate carboxykinase (PEPCK) and PEPCK-chloramphenicol acetyltransferase (CAT) genes is induced by cAMP and glucocorticoids and is inhibited by insulin in H4IIE cells, as it is in liver. In contrast, PEPCK-CAT expression in HepG2 cells is not affected by insulin but is induced by cAMP, which in turn is repressed by glucocorticoids. Mutations were introduced into well defined transcription factor binding sites to investigate possible interactions between the cAMP regulatory element (CRE) binding protein (CREB) and glucocorticoid response unit (GRU) binding proteins. H4IIE rat hepatoma cells were transfected with PEPCK-CAT plasmids with or without an expression vector for protein kinase A (PKA). Glucocorticoid-induced CAT activity was dependent upon the GRU and was decreased in plasmids lacking the CRE. To determine the direct effects of CREB, the DNA binding and dimerization domain of GAL4 was substituted for that of CREB (CRG), and the PEPCK CRE was replaced with a GAL4 binding site (G4PEPCK-CAT). CRG elevated basal and glucocorticoid-induced activities of G4PEPCK-CAT equally and restored responsiveness to PKA. The basal activity of CRG was not diminished by concomitant treatment with PKA plus its inhibitor peptide, PKI, or by mutation of the PKA phosphorylation. Deletion of C-terminal regions of the CREB activation domain from CRG diminished basal activation without affecting induction by PKA. The glucocorticoid-induced level of CAT activity decreased in proportion to the reduced ability of CREB to activate basal transcription. Induction by glucocorticoid, in the absence or presence of PKA, was not affected by CRG, indicating that interaction of GRU-bound factors with CREB is not required for glucocorticoid induction of PEPCK. These results indicate that CREB is directly involved in basal and PKA-induced expression of PEPCK, and that CREB supports glucocorticoid-induced PEPCK expression through its positive effect on basal transcription.
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PMID:Involvement of 3',5'-cyclic adenosine monophosphate regulatory element binding protein (CREB) in both basal and hormone-mediated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene. 811 62

The minimal promoter/transcription factor requirements for induction of phosphoenolpyruvate carboxykinase (PEPCK) transcription by cAMP-activated protein kinase A (PKA) and inhibition of this induction by insulin were investigated. H4 hepatoma cells were treated with or without insulin following cotransfection with chloramphenicol acetyltransferase reporter genes and expression vectors coding for the cAMP response element-binding protein (CREB) activation domain fused to the GAL4 DNA binding domain (CRG) and the catalytic subunit of PKA. Mutation of the PEPCK CRE to a GAL4 binding site (G4-PEPCK) within the fully responsive PEPCK promoter (-600/+69) made induction by PKA dependent upon cotransfection of CRG and this induction by CRG+PKA was inhibited by insulin. Mutation of the insulin regulatory sequence (delta IRS-G4-PEPCK) did not prevent induction by cAMP or inhibition by insulin. Fusion of GAL4 binding sites to the PEPCK TATA region (-40/+1, G4-PT) allowed induction by CRG+PKA and inhibition by insulin. However, inhibition by insulin was not observed when the CREB activation domain in CRG was replaced with the activation domain of VP16 (G4-VP16) or when the PEPCK TATA region was replaced with TATA regions from other genes. Our results indicate that the minimal requirements for induction of PEPCK by PKA and inhibition by insulin include: 1) the CREB activation domain, 2) the PEPCK TATA sequence, and 3) insulin-responsive hepatoma cells. These data suggest that specific factors interacting with both the PEPCK TATA region and the CREB activation domain are required for insulin inhibition of PKA-induced transcription.
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PMID:Inhibition by insulin of protein kinase A-induced transcription of the phosphoenolpyruvate carboxykinase gene. Mediation by the activation domain of cAMP response element-binding protein (CREB) and factors bound to the TATA box. 818 41

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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PMID:Production and properties of recombinant C3-type phosphoenolpyruvate carboxylase from Sorghum vulgare: in vitro phosphorylation by leaf and root PyrPC protein serine kinases. 828 Jan 59


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