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)

The gamma 2 subunit of the GABA receptor (GABAA-R) is alternatively spliced. The long variant (gamma 2L) contains eight additional amino acids that possess a consensus sequence site for protein phosphorylation. Previous studies have demonstrated that a peptide or fusion protein containing these eight amino acids is a substrate for protein kinase C (PKC), but not cyclic AMP-dependent protein kinase A (PKA)-stimulated phosphorylation. We have examined the ability of PKA, PKC, and Ca2+/calmodulin-dependent protein kinase (CAM kinase II) to phosphorylate a synthetic peptide corresponding to residues 336-351 of the intracellular loop of the gamma 2L subunit and inclusive of the alternatively spliced phosphorylation consensus sequence site. PKC and CAM kinase II produced significant phosphorylation of this peptide, but PKA was ineffective. The Km values for PKC- and CAM kinase II-stimulated phosphorylation of this peptide were 102 and 35 microM, respectively. Maximal velocities of 678 and 278 nmol of phosphate/min/mg were achieved by PKC and CAM kinase II, respectively. The phosphorylation site in the eight-amino-acid insert of the gamma 2L subunit has been shown to be necessary for ethanol potentiation of the GABAA-R. Thus, our results suggest that PKC, CAM kinase II, or both may play a role in the effects of ethanol on GABAergic function.
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PMID:Ca2+/calmodulin-dependent protein kinase II and protein kinase C phosphorylate a synthetic peptide corresponding to a sequence that is specific for the gamma 2L subunit of the GABAA receptor. 839 May 66

Phosphorylation of pure cytochrome P4502E1 (CYP2E1) was achieved in vitro using Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), protein kinase C (PKC) and cAMP-dependent protein kinase (PKA). The stoichiometry and time-course of phosphorylation were determined. CaM kinase II was the most efficient enzyme capable of catalyzing this phosphorylation reaction: the maximum incorporation of 32PO4 was 0.8 mol/mol CYP2E1 in 20 min. PKA phosphorylated a maximum of 0.7 mol of 32PO4/mol of cytochrome within 60 min. The phosphorylation by PKC reached a maximum of 0.19 mol of 32PO4/mol of cytochrome and this occurred within a few minutes of incubation. Limited digestion by S. aureus V8 protease (SAP) of CYP2E1, which had been phosphorylated by either PKA and PKC, yielded a single major phosphopeptide with an M(r) of approximately 18,000. Limited digestion of CYP2E1, that had been phosphorylated by CaM kinase II, yielded phosphorylated polypeptides with M(r) of approximately 18,000 and 15,000. These results raise the possibility that these three kinases may be involved in the regulation of CYP2E1.
Alcohol Alcohol 1993 Jul
PMID:Phosphorylation of cytochrome P4502E1 (CYP2E1) by calmodulin dependent protein kinase, protein kinase C and cAMP dependent protein kinase. 839 26

PCK1 encoding phosphoenolpyruvate carboxykinase is transcriptionally regulated by two upstream activating elements. By screening for mutants that failed to derepress a UAS2PCK1-CYC1-lacZ reporter gene we isolated the new recessive derepression mutation cat5. The CAT5 gene encodes a protein of 272 amino acids showing a 42% identity to the ZC395.2 gene product of Caenorhabditis elegans whose function is unknown. Deletion of CAT5 caused a complete loss of glucose derepression affecting gluconeogenic key enzymes. Respiration, but not mitochondrial cytochrome c oxidase activity, was also affected. CAT5 expression is 5- to 6-fold repressed by glucose, and CAT5 transcriptional activation was dependent on CAT1 (SNF1), CAT8 and CAT5 itself. The CAT5 gene is necessary for UAS1PCK1 and UAS2PCK1 protein binding since a carbon source-specific interaction was no longer detectable in cat5 mutants. Glucose derepression of gluconeogenesis depends on the active Cat1 (Snf1) protein kinase and the Cat8 zinc cluster activator. Mig1p-independent overexpression of CAT8 did not stimulate activation of gluconeogenic promoters in cat1 and in cat5 mutants. Since Cat8p multicopy expression suppresses the ethanol growth deficiency in cat1 (snf1) mutants, these results indicate that activation of Cat8p by the Cat1p (Snf1p) kinase and the Cat5p protein might be necessary for release from glucose repression.
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PMID:CAT5, a new gene necessary for derepression of gluconeogenic enzymes in Saccharomyces cerevisiae. 855 31

GRP78, a molecular chaperone expressed in the endoplasmic reticulum, is a "glucose-regulated protein" induced by stress responses that deplete glucose or intracisternal calcium or otherwise disrupt glycoprotein trafficking. Previously we showed that chronic ethanol exposure increases the expression of GRP78. To further understand the mechanism underlying ethanol regulation of GRP78 expression, we studied the interaction between ethanol and classical modulators of GRP78 expression in NG108-15 neuroblastoma x glioma cells. We found that, in addition to increasing basal levels of GRP78 mRNA ("induction"), ethanol produced greater than additive increases in the induction of GRP78 mRNA by the "classical" GRP inducers A23187, brefeldin A, and thapsigargin ("potentiation"). Both the ethanol induction and potentiation responses modulated grp78 gene transcription as determined by stable transfection analyses with the rat grp78 promoter. Ethanol potentiated the action of all classical inducers of grp78 transcription that were studied. In contrast, co-treatment with the classical GRP inducers thapsigargin and tunicamycin produced only simple additive increases in grp78 promoter activity. Transient transfection studies with deletion mutants of the rat grp78 promoter showed that cis-acting promoter sequences required for ethanol induction differ from those mediating responses to classical GRP inducers. Furthermore, linker-scanning mutations of the grp78 promoter suggested that the ethanol potentiation response required a cis-acting promoter element different from those involved in induction by ethanol or classical inducing agents. While the ethanol induction response required 16-24 h to be detectable, ethanol potentiation of thapsigargin occurred within 6 h. The potentiation response also decayed rapidly after ethanol removal. In addition, the protein kinase A inhibitor Rp-cAMPS and protein phosphatase inhibitor okadaic acid both increased ethanol potentiation of thapsigargin while Sp-cAMPS, an activator of protein kinase A, decreased ethanol potentiation. Taken together, our findings suggest two mechanisms by which ethanol regulates grp78 transcription, both differing from the action of classical GRP inducers such as thapsigargin. One mechanism (potentiation) involves a protein phosphorylation cascade and potentiates the action of classical GRP inducers. In contrast, GRP78 induction by ethanol involves promoter sequences and a mechanistic pathway separate from that of the ethanol potentiation response or classical GRP78 inducers. These studies show that ethanol produces a novel and complex regulation of grp78 transcription which could be of particular importance during neuronal exposure to GRP-inducing stressors as might occur with central nervous system injury.
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PMID:Interaction of ethanol with inducers of glucose-regulated stress proteins. Ethanol potentiates inducers of grp78 transcription. 857 45

The effects of chronic ethanol exposure on the stress-like protein adenotin were investigated using the radioligand [3H]-5'-N-ethylcarboxamidoadenosine ([3H]NECA). A 4-day exposure to 150 mM ethanol increased both the KD and the density of [3H]NECA binding sites. These changes were not due to residual ethanol as the acute addition of ethanol did not alter [3H]NECA binding. Chronic ethanol exposure of A126-1B2-1 cells, which are a mutant PC 12 cell line deficient in protein kinase A (PKA), increased the cellular density of adenotin, but did not affect the KD for the radioligand. Conversely, when PC 12 cells were exposed to 10 microM forskolin for either 2 or 4 days, the cellular density of adenotin was not altered, but the affinity of adenotin for [3H]NECA was reduced significantly. An increase in KD was not observed after a 1-hr exposure of PC 12 cells to forskolin, indicating that the reduction in affinity for the radioligand was not due simply to a PKA-mediated phosphorylation of adenotin. The present study demonstrated that chronic ethanol regulates adenotin through two different mechanisms. The ethanol-induced increase in the density of adenotin does not involve PKA, while the reduction in affinity ap pears to involve a cAMP-dependent mechanism.
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PMID:Regulation of the stress-like protein adenotin in PC 12 cells by ethanol exposure. 861 87

Previous studies demonstrated that the cysteine-rich amino-terminal domain of Raf-1 kinase interacts selectively with phosphatidylserine (Ghosh, S., Xie, W. Q., Quest, A. F. G., Mabrouk, G. M., Strum, J. C., and Bell, R. M. (1994) J. Biol. Chem. 269, 10000-10007). Further analysis showed that full-length Raf-1 bound to both phosphatidylserine and phosphatidic acid (PA). Specifically, a carboxyl-terminal domain of Raf-1 kinase (RafC; residues 295 648 of human Raf-1) interacted strongly with phosphatidic acid. The binding of RafC to PA displayed positive cooperativity with Hill numbers between 3.3 and 6.2; the apparent Kd ranged from 4.9 +/- 0.6 to 7.8 +/- 0.9 mol % PA. The interaction of RafC with PA displayed a pH dependence distinct from the interaction between the cysteine-rich domain of Raf-1 and PA. Also, the RafC-PA interaction was unaffected at high ionic strength. Of all the lipids tested, only PA and cardiolipin exhibited high affinity binding; other acidic lipids were either ineffective or weakly effective. By deletion mutagenesis, the PA binding site within RafC was narrowed down to a 35-amino acid segment between residues 389 and 423. RafC did not bind phosphatidyl alcohols; also, inhibition of PA formation in Madin-Darby canine kidney cells by treatment with 1% ethanol significantly reduced the translocation of Raf-1 from the cytosol to the membrane following stimulation with 12-O-tetradecanoylphorbol-13-acetate. These results suggest a potential role of the lipid second messenger, PA, in the regulation of translocation and subsequent activation of Raf-1 in vivo.
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PMID:Raf-1 kinase possesses distinct binding domains for phosphatidylserine and phosphatidic acid. Phosphatidic acid regulates the translocation of Raf-1 in 12-O-tetradecanoylphorbol-13-acetate-stimulated Madin-Darby canine kidney cells. 862 48

Adenosine mediates some of the acute and chronic effects of ethanol in neural cells. In cultured NG108-15 cells, ethanol inhibits adenosine uptake via a specific facilitative nucleoside transporter leading to an increase in extracellular adenosine, activation of adenosine A2 receptors and increases in intracellular cyclic AMP (cAMP). After chronic ethanol exposure, an adaptive decrease in receptor-stimulated cAMP levels occurs. Additionally, the transporter becomes insensitive to rechallenge with ethanol and adenosine uptake is not inhibited. cAMP levels are decreased in cells chronically exposed to ethanol and we show here that cAMP-dependent kinase (PKA) activity in cellular homogenates also is decreased. Therefore, decreased cAMP-dependent phosphorylation may be responsible for loss of ethanol sensitivity. To test this hypothesis, NG108-15 cells were treated with agents that alter PKA activity and the ethanol sensitivity of adenosine transport was measured. In naive cells, decreasing PKA activity with the cAMP antagonist, Rp-adenosine-3',5'-cyclic phosphorothioate, resulted in ethanol-insensitive adenosine uptake. This effect was blocked by the phosphatase inhibitor, okadaic acid. These results suggest that loss of ethanol sensitivity is correlated with decreased PKA activity. Therefore, stimulating PKA activity in chronically treated cells should restore sensitivity of adenosine uptake to inhibition by ethanol. Indeed, the cAMP agonist, Sp-adenosine-3',5'-cyclic phosphorothioate, restored ethanol sensitivity of transport in cells treated chronically with ethanol. Our results suggest that ethanol sensitivity of adenosine transport is regulated by PKA and protein phosphatase activities in NG108-15 cells. Moreover, the effects of chronic ethanol exposure on adenosine transport can be reversed by activating PKA.
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PMID:Activation of cyclic AMP-dependent protein kinase reverses tolerance of a nucleoside transporter to ethanol. 863 98

To investigate the intracellular pathways leading to ETOH-induced apoptosis, thymocytes and splenic T and B cells were cultured 16 h with or without ETOH and different stimuli, and apoptotic cell death was determined. At concentrations of 0.4%-2% in culture, ETOH induced apoptosis in all three types of cells, but it had a more profound effect on thymocytes and B cells as compared with its effect on T cells. In thymocytes, ETOH-induced apoptosis was abrogated by chelation of extracellular calcium with EGTA, and inhibition of protein synthesis with CHX, or of PKC with H7 but not of PKA with HA 1004. ETOH potentiated the apoptosis of thymocytes induced with the calcium ionophore A23187 and suboptimal doses of PMA, but it had negligible effect on dAMP- and PGE2-induced apoptosis of thymocytes. In contrast to findings in thymocytes, the ETOH-induced apoptosis of T and B cells was almost completely abrogated by PMA, but not by H7 or CHX. In spleen cells, calcium chelation with EGTA triggered apoptosis. ETOH significantly inhibited EGTA-induced apoptosis of B cells but had little effect on EGTA-induced apoptosis of T cells. IL-4 reduced the ETOH-induced apoptosis of B and T cells, but it was not effective in the prevention of apoptosis of thymocytes. Inhibition of the calcium-dependent neutral protease calpain I did not rescue cells from apoptosis. Moreover, treatment with CI-I potentiated ETOH-induced apoptosis in T cells. These results suggest that both thymocytes and splenic T and B cells have relevant apoptotic pathways that can be induced by ETOH, but the mechanisms of ETOH-induced apoptosis differ in these cells.
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PMID:Different pathways of in vitro ethanol-induced apoptosis in thymocytes and splenic T and B lymphocytes. 865 90

Madin-Darby canine kidney (MDCK) cells stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) in the presence of ethanol synthesize phosphatidylethanol (PEt) instead of phosphatidic acid (PA) and diglyceride (DG). We have used ethanol to block the production of phospholipase D (PLD)-derived PA and DG (from PA hydrolysis) to study their role in signal transduction. In MDCK cells, TPA-stimulated prostaglandin E2 (PGE2) synthesis was inhibited by ethanol at concentrations which inhibit PA and DG formation. In addition, TPA elicited a prolonged increase in PGE2 synthesis that is dependent upon continuous activation of PLD. The TPA-stimulated translocation of protein kinase Calpha (PKCalpha) from cytosol to membrane was unaffected by ethanol. This suggests that PLD-derived products act downstream of PKC in TPA-stimulated prostaglandin synthesis. The calcium ionophore, A23187, did not activate PLD, and PGE2 synthesis in response to A23187 was unaffected by ethanol. TPA increased prostaglandin endoperoxide H synthase (PGHS) activity and increased the amount of immunodetectable prostaglandin endoperoxide H synthase 2 (PGHS-2). A23187 did not induce PGHS-2 and A23187-stimulated PGE2 synthesis appears to be due to the constitutively expressed PGHS-1. Blocking the formation of PLD-derived products, PA and DG, inhibited the induction of PGHS-2 by TPA. These results indicate that prolonged PGE2 synthesis in response to TPA is due to the continuous induction of PGHS-2, which is dependent upon PLD activation. In contrast, induction of PGHS-2 by epidermal growth factor was not affected by ethanol. Epidermal growth factor did not induce PKCalpha translocation nor activate PLD. Taken together, these data suggest that PLD-derived PA or DG act as second messengers in the induction of PGHS-2 by PKC-dependent pathways. The demonstration that inhibition of TPA-induced PA formation inhibits Raf-1 translocation in MDCK cells (Ghosh, S., Strum, J. C., Sciorra, V. A., Daniel, L. W. , and Bell, R. M. (1996) J. Biol. Chem. 271, 8472-8480) suggests that PA is the active PLD metabolite in TPA-stimulated signaling.
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PMID:Phospholipase D-derived products in the regulation of 12-O-tetradecanoylphorbol-13-acetate-stimulated prostaglandin synthesis in madin-darby canine kidney cells. 866 19

We have demonstrated previously that chronic administration of morphine, cocaine, or ethanol produces some common biochemical adaptations in the ventral tegmental area (VTA) and nucleus accumbens (NAc), components of the mesolimbic dopamine system implicated in the reinforcing and locomotor activating properties of these drugs of abuse. Because this neural pathway is also regulated by stress, and because stress has been shown to influence an animal's behavioral responses to drugs of abuse, it was of interest to determine whether repeated exposure to stress results in similar biochemical adaptations. By use of immunoblot analysis, we show here that a course of chronic "unpredictable" stress, like chronic drug exposure, increased levels of immunoreactivity of tyrosine hydroxylase and glial fibrillary acidic protein and decreased levels of immunoreactivity of neurofilament proteins in the VTA. Chronic unpredictable stress also increased levels of cyclic AMP-dependent protein kinase activity and decreased levels of immunoreactivity of the G protein subunit, Gi alpha, in the NAc. These effects required long-term exposure to stress and were in most cases not seen in the substantia nigra and caudate-putamen, components of the nigrostriatal dopamine system studied for comparison. The biochemical effects of chronic stress in the VTA and NAc differed among three strains of rat studied. Fischer 344 rats were the most responsive in that they exhibited all of the aforementioned adaptations, whereas Lewis rats were the least responsive in that they exhibited none of these adaptations; Sprague-Dawley rats exhibited an intermediate number of responses. Taken together, the results of the present study demonstrate that chronic exposure to stress results in biochemical adaptations in the mesolimbic dopamine system that resemble the chronic actions of several drugs of abuse. These adaptations could contribute to the convergent behavioral effects induced by treatments that are mediated via the VTA-NAc pathway.
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PMID:Biochemical adaptations in the mesolimbic dopamine system in response to repeated stress. 872 55


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