scholarly journals Mechanism of hepatic glycogen synthase inactivation induced by Ca2+-mobilizing hormones. Studies using phospholipase C and phorbol myristate acetate

1986 ◽  
Vol 237 (1) ◽  
pp. 235-242 ◽  
Author(s):  
P F Blackmore ◽  
W G Strickland ◽  
S B Bocckino ◽  
J H Exton
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Glycogen Synthase ◽  
Partial Purification ◽  
Hepatic Glycogen ◽  
Kinase C ◽  
Low Concentrations ◽  
Dependent Inactivation

Incubation of hepatocytes with the protein kinase C activator and tumour promoter 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) produced a time- and concentration-dependent inactivation of glycogen synthase, but no change in phosphorylase. The same rate and extent of inactivation occurred in hepatocytes depleted of Ca2+ by treatment with the Ca2+ chelator EGTA. When hepatocytes were treated with the Ca2+-mobilizing hormone vasopressin (10 nM), the rate of glycogen synthase inactivation was similar to that observed with PMA (1 microM). Depletion of intracellular Ca2+ stores with EGTA abolished the ability of vasopressin to mobilize Ca2+ and activate phosphorylase without abolishing its ability to inactivate glycogen synthase and increase 1,2-diacylglycerol (DAG), the endogenous activator of protein kinase C. Protein kinase C, either in membranes or after partial purification, was shown to be activated in vitro by PMA in the presence of very low concentrations of Ca2+. Exogenous phospholipase C from Clostridium perfringens, at low concentrations, inactivated glycogen synthase and increased DAG without affecting cell Ca2+ or phosphorylase. It is proposed that the inactivation of glycogen synthase elicited by the Ca2+-mobilizing hormones is due, at least in part, to generation of DAG and activation of protein kinase C.

scholarly journals G-protein βγ subunits antagonize protein kinase C-dependent phosphorylation and inhibition of phospholipase C-β1

1997 ◽  
Vol 326 (3) ◽  
pp. 701-707 ◽  
Author(s):  
Irene LITOSCH
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
G Protein ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Negative Feedback Regulation ◽  
Kinase C ◽  
Stimulation Of

Protein kinase C (PKC) isoforms phosphorylated phospholipase C-β1 (PLC-β1) in vitro as follows: PKCα ≫ PKCϵ; not PKCζ. PLC-β3 was not phosphorylated by PKCα. G-protein βγ subunits inhibited the PKCα phosphorylation of PLC-β1 in a concentration-dependent manner. Half-maximal inhibition occurred with 500 nM βγ. G-protein βγ subunits also antagonized the PKCα-mediated inhibition of PLC-β1 enzymic activity. PKCα, in turn, inhibited the stimulation of PLC-β1 activity by βγ. There was little effect of PKCα on the stimulation of PLC-β1 by αq/11–guanosine 5′[γ-thio]triphosphate (GTP[S]). These findings demonstrate that G protein βγ subunits antagonize PKCα regulation of PLC-β1. Thus βγ subunits might have a role in modulating the negative feedback regulation of this signalling system by PKC.

scholarly journals Convergence of Multiple Signaling Cascades at Glycogen Synthase Kinase 3: Edg Receptor-Mediated Phosphorylation and Inactivation by Lysophosphatidic Acid through a Protein Kinase C-Dependent Intracellular Pathway

2002 ◽  
Vol 22 (7) ◽  
pp. 2099-2110 ◽  
Author(s):  
Xianjun Fang ◽  
Shuangxing Yu ◽  
Janos L. Tanyi ◽  
Yiling Lu ◽  
James R. Woodgett ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Lysophosphatidic Acid ◽  
Glycogen Synthase ◽  
Glycogen Synthase Kinase ◽  
Hek293 Cells ◽  
Glycogen Synthase Kinase 3 ◽  
Kinase C ◽  
Pkc Inhibitors

ABSTRACT Lysophosphatidic acid (LPA) is a natural phospholipid with multiple biological functions. We show here that LPA induces phosphorylation and inactivation of glycogen synthase kinase 3 (GSK-3), a multifunctional serine/threonine kinase. The effect of LPA can be reconstituted by expression of Edg-4 or Edg-7 in cells lacking LPA responses. Compared to insulin, LPA stimulates only modest phosphatidylinositol 3-kinase (PI3K)-dependent activation of protein kinase B (PKB/Akt) that does not correlate with the magnitude of GSK-3 phosphorylation induced by LPA. PI3K inhibitors block insulin- but not LPA-induced GSK-3 phosphorylation. In contrast, the effect of LPA, but not that of insulin or platelet-derived growth factor (PDGF), is sensitive to protein kinase C (PKC) inhibitors. Downregulation of endogenous PKC activity selectively reduces LPA-mediated GSK-3 phosphorylation. Furthermore, several PKC isotypes phosphorylate GSK-3 in vitro and in vivo. To confirm a specific role for PKC in regulation of GSK-3, we further studied signaling properties of PDGF receptor β subunit (PDGFRβ) in HEK293 cells lacking endogenous PDGF receptors. In clones expressing a PDGFRβ mutant wherein the residues that couple to PI3K and other signaling functions are mutated with the link to phospholipase Cγ (PLCγ) left intact, PDGF is fully capable of stimulating GSK-3 phosphorylation. The process is sensitive to PKC inhibitors in contrast to the response through the wild-type PDGFRβ. Therefore, growth factors, such as PDGF, which control GSK-3 mainly through the PI3K-PKB/Akt module, possess the ability to regulate GSK-3 through an alternative, redundant PLCγ-PKC pathway. LPA and potentially other natural ligands primarily utilize a PKC-dependent pathway to modulate GSK-3.

scholarly journals Phosphorylation by protein kinase C decreases catalytic activity of avian phospholipase C-β

1999 ◽  
Vol 338 (2) ◽  
pp. 257-264 ◽  
Author(s):  
Theresa M. FILTZ ◽  
Michelle L. CUNNINGHAM ◽  
Kara J. STANIG ◽  
Andrew PATERSON ◽  
T. Kendall HARDEN
Keyword(s):  
Enzyme Activity ◽  
Protein Kinase C ◽  
Catalytic Activity ◽  
Protein Kinase ◽  
Phospholipase C ◽  
G Protein ◽  
Erythrocyte Membranes ◽  
Basal Activity ◽  
Kinase C

The potential role of protein kinase C (PKC)-promoted phosphorylation has been examined in the G-protein-regulated inositol lipid signalling pathway. Incubation of [32P]Pi-labelled turkey erythrocytes with either the P2Y1 receptor agonist 2-methylthioadenosine triphosphate (2MeSATP) or with PMA resulted in a marked increase in incorporation of 32P into the G-protein-activated phospholipase C PLC-βT. Purified PLC-βT also was phosphorylated by PKC in vitro to a stoichiometry (mean±S.E.M.) of 1.06±0.2 mol of phosphate/mol of PLC-βT. Phosphorylation by PKC was isoenzyme-specific because, under identical conditions, mammalian PLC-β2 also was phosphorylated to a stoichiometry near unity, whereas mammalian PLC-β1 was not phosphorylated by PKC. The effects of PKC-promoted phosphorylation on enzyme activity were assessed by reconstituting purified PLC-βT with turkey erythrocyte membranes devoid of endogenous PLC activity. Phosphorylation resulted in a decrease in basal activity, AlF4--stimulated activity, and activity stimulated by 2MeSATP plus guanosine 5´-[γ-thio]triphosphate in the reconstituted membranes. The decreases in enzyme activities were proportional to the extent of PKC-promoted phosphorylation. Catalytic activity assessed by using mixed detergent/phospholipid micelles also was decreased by up to 60% by phosphorylation. The effect of phosphorylation on Gqα-stimulated PLC-βT in reconstitution experiments with purified proteins was not greater than that observed on basal activity alone. Taken together, these results illustrate that PKC phosphorylates PLC-βT in vivo and to a physiologically relevant stoichiometry in vitro. Phosphorylation is accompanied by a concomitant loss of enzyme activity, reflected as a decrease in overall catalytic activity rather than as a specific modification of G-protein-regulated activity.

scholarly journals Photorelease of Diacylglycerol Increases the Amplitude and Duration of Protein Kinase C-BetaII Relocation in cyto

2017 ◽  
Author(s):  
Joachim Goedhart ◽  
Theodorus W.J. Gadella
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Quantum Yield ◽  
Phospholipase C ◽  
Simultaneous Presence ◽  
Fluorescent Compound ◽  
Kinase C ◽  
Pkc Isoforms ◽  
Pkc Signaling

ABSTRACTDiacylglycerol (DAG) is a lipid second messenger produced by receptor stimulated phospholipase C and is capable of activating several PKC isoforms. Classical PKC isoforms require simultaneous presence of calcium and DAG for activation and relocation to membranes. The aim of this study was to synthesize a photolabile precursor of DAG and examine the effect of an immediate increase of the signaling lipid on PKC relocation. Caged DAG was synthesized using a photoreleasable 7-diethyl-aminocoumarin (DEACM) group. Photolysis was monitored in vitro by an increase in coumarin fluorescence from which an uncaging quantum yield of 1.1% was determined. This quantum yield proved ideal for efficient uncaging at high UV power while allowing localization of the fluorescent compound at low UV power. Taking advantage of the coumarin fluorescence, it was demonstrated that DEACM-DiC8 accumulated inside cells. Effects of DAG photorelease on periodic relocations of PKCbetaII, induced by histamine, UTP or EGF, were studied. Photorelease of DAG in cyto immediately increased the amplitude and duration of relocation events, regardless of the agonist used. Together, the results demonstrate the usefulness of caged DAG for dissecting PKC signaling and suggest that DAG levels are limiting during signaling.

scholarly journals Sphingosine enhances platelet aggregation through an increase in phospholipase C activity by a protein kinase C-independent mechanism

1992 ◽  
Vol 282 (1) ◽  
pp. 243-247 ◽  
Author(s):  
T Hashizume ◽  
T Sato ◽  
T Fujii
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Platelet Activating Factor ◽  
Membrane Phospholipids ◽  
Stimulus Response ◽  
Protein Kinase C Inhibitors ◽  
Kinase C ◽  
Low Concentrations ◽  
Positive Modulator

Sphingosine (a potent inhibitor of protein kinase C) at 5-10 microM, which are concentrations lower than those that inhibit this enzyme activity, enhanced the aggregation of rabbit platelets induced by low concentrations of U46619, platelet-activating factor, thrombin and arachidonic acid, whereas H-7 and staurosporine, other protein kinase C inhibitors, failed to do so. Of the sphingosine analogues which also inhibit protein kinase C, psychosine and lyso-GM3 did not show such an enhancing effect. Sphingosine promoted both Ins(1,4,5)P3 formation and an increase in the cytoplasmic free Ca2+ concentration in response to all the agonists used. Furthermore, the hydrolytic action of exogenously added phospholipase C (from Clostridium perfringens) on platelet membrane phospholipids was dose-dependently enhanced by pretreatment of the platelets with sphingosine. These results imply that sphingosine, at relatively low concentrations, brings about hyperaggregability of the platelets by the agonists employed, probably owing to enhancement of the phospholipase C activity. Such an effect appears to be induced by a mechanism independent of protein kinase C inhibition. We suggest that sphingosine might act as a positive modulator for the stimulus-response coupling in the platelets.

scholarly journals The role of protein kinase C in the inactivation of hepatic glycogen synthase by calcium-mobilizing agonists

1988 ◽  
Vol 251 (1) ◽  
pp. 47-53 ◽  
Author(s):  
B Bouscarel ◽  
K Meurer ◽  
C Decker ◽  
J H Exton
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Glycogen Synthase ◽  
Glycogen Synthesis ◽  
Hepatic Glycogen ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Kinase C ◽  
Protein Kinase C Activity ◽  
Atp Inhibition

The regulation of glycogen synthase by Ca2+-mobilizing hormones was studied by using rat liver parenchymal cells in primary culture. Long-term exposure of hepatocytes to 4 beta-phorbol 12-myristate 13-acetate (TPA) resulted in a decrease in vasopressin or ATP inhibition of glycogen synthesis and glycogen synthase activity, without any change in the activation of glycogen phosphorylase. In contrast, treatment with TPA did not diminish the effects of glucagon, isoprenaline or A23187 on glycogen synthase or phosphorylase. TPA treatment for 18 h did not change specific [3H]vasopressin binding, but abolished protein kinase C activity in a concentration-dependent manner. The effects of TPA to decrease protein kinase C activity and to reverse the inactivation of glycogen synthase by vasopressin were well correlated and were mimicked by mezerein, but not by 4 alpha-phorbol. However, 1 microM-TPA totally inhibited protein kinase C activity, but reversed only 60% of the vasopressin effect on glycogen synthase. It is therefore concluded that Ca2+-mobilizing hormones inhibit glycogen synthase partly, but not wholly, through a mechanism involving protein kinase C.

scholarly journals Protein Kinase C: A Putative New Target for the Control of Human Medullary Thyroid Carcinoma Cell Proliferation in Vitro

Endocrinology ◽  
2012 ◽  
Vol 153 (5) ◽  
pp. 2088-2098 ◽  
Author(s):  
Daniela Molè ◽  
Erica Gentilin ◽  
Teresa Gagliano ◽  
Federico Tagliati ◽  
Marta Bondanelli ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Thyroid Carcinoma ◽  
Medullary Thyroid Carcinoma ◽  
Glycogen Synthase ◽  
Primary Cultures ◽  
Medullary Thyroid ◽  
Kinase C

We investigate the role of protein kinase C (PKC) in the control of medullary thyroid carcinoma (MTC) cell proliferation by a PKC inhibitor, Enzastaurin, in human MTC primary cultures and in the TT cell line. We found that PKC inhibition reduces cell proliferation by inducing caspase-mediated apoptosis and blocks the stimulatory effect of IGF-I on calcitonin secretion. Enzastaurin reduces PKCβII (Thr500) phosphorylation, indicating a direct involvement of this isoform as well as the phosphorylated levels of Akt (Ser 473) and glycogen synthase kinase (Ser9), PKC pathway downstream targets and pharmacodynamic markers for PKC inhibition. PKCβII and PKCδ enzyme isoforms expression and localization were investigated. These data indicate that in vitro PKC is involved in the control of human MTC proliferation and survival by modulating apoptosis, with a mechanism that implicates PKCβII inhibition and translocation in different subcellular compartments. Targeting PKC may represent a useful therapeutic approach for controlling MTC proliferation.

scholarly journals Defective coupling of apical PTH receptors to phospholipase C prevents internalization of the Na+-phosphate cotransporter NaPi-IIa in Nherf1-deficient mice

2007 ◽  
Vol 292 (2) ◽  
pp. C927-C934 ◽  
Author(s):  
Paola Capuano ◽  
Desa Bacic ◽  
Marcel Roos ◽  
Serge M. Gisler ◽  
Gerti Stange ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Proximal Tubule ◽  
Phospholipase C ◽  
Wild Type ◽  
Phosphate Reabsorption ◽  
Kinase C ◽  
Deficient Mice ◽  
Stimulation Of

Phosphate reabsorption in the renal proximal tubule occurs mostly via the type IIa Na+-phosphate cotransporter (NaPi-IIa) in the brush border membrane (BBM). The activity and localization of NaPi-IIa are regulated, among other factors, by parathyroid hormone (PTH). NaPi-IIa interacts in vitro via its last three COOH-terminal amino acids with the PDZ protein Na+/H+-exchanger isoform 3 regulatory factor (NHERF)-1 (NHERF1). Renal phosphate reabsorption in Nherf1-deficient mice is altered, and NaPi-IIa expression in the BBM is reduced. In addition, it has been proposed that NHERF1 and NHERF2 are important for the coupling of PTH receptors (PTHRs) to phospholipase C (PLC) and the activation of the protein kinase C pathway. We tested the role of NHERF1 in the regulation of NaPi-IIa by PTH in Nherf1-deficient mice. Immunohistochemistry and Western blotting demonstrated that stimulation of apical and basolateral receptors with PTH-(1–34) led to internalization of NaPi-IIa in wild-type and Nherf1-deficient mice. Stimulation of only apical receptors with PTH-(3–34) failed to induce internalization in Nherf1-deficient mice. Expression and localization of apical PTHRs were similar in wild-type and Nherf1-deficient mice. Activation of the protein kinase C- and A-dependent pathways with 1,2-dioctanoyl- sn-glycerol or 8-bromo-cAMP induced normal internalization of NaPi-IIa in wild-type, as well as Nherf1-deficient, mice. Stimulation of PLC activity due to apical PTHRs was impaired in Nherf1-deficient mice. These data suggest that NHERF1 in the proximal tubule is important for PTH-induced internalization of NaPi-IIa and, specifically, couples the apical PTHR to PLC.

Protein kinase C and phospholipase C in adenosine receptor-mediated relaxation in coronary artery

1991 ◽  
Vol 261 (6) ◽  
pp. H1848-H1854 ◽  
Author(s):  
D. J. Cushing ◽  
S. R. Makujina ◽  
M. H. Sabouni ◽  
S. J. Mustafa
Keyword(s):  
Protein Kinase C ◽  
Coronary Artery ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Adenosine Receptor ◽  
Isometric Force ◽  
Indirect Evidence ◽  
Porcine Coronary Artery ◽  
Kinase C

The effect of adenosine, 2-chloroadenosine (CAD), and 5'-(N-ethylcarboxamido)-adenosine (NECA) on the contraction produced by phorbol 12,13-dibutyrate (PDB) was investigated in porcine coronary artery in vitro to determine whether adenosine receptor-mediated relaxation was linked to protein kinase C. Also, the coronary relaxation produced by adenosine and NECA in KCl-contracted coronary rings was investigated before and after treatment with the phospholipase C inhibitor neomycin to examine a possible link between phospholipase C and adenosine receptor-mediated relaxation. Ring segments of coronary artery were suspended in organ baths for measurement of isometric force. PDB (10 nM-1 microM) caused concentration-dependent contraction, and this response was significantly attenuated by pretreatment with the protein kinase C inhibitor staurosporine (200 nM) but not 1-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (10 microM). Treatment of rings with either adenosine, CAD, or NECA (100 microM) significantly attenuated the PDB-induced contraction, whereas treatment with either sodium nitroprusside (SNP; 1 microM) or isoproterenol (Isop; 1 microM) did not affect the contraction produced by PDB. The attenuation of the PDB-induced contraction by adenosine and its analogues was blocked by prior treatment of the coronary rings with 8-phenyltheophylline (10 microM). In a separate series of experiments, pretreatment of rings with the phospholipase C inhibitor neomycin (1 mM) resulted in a significant attenuation of the relaxing response to both adenosine and NECA while having no significant effect on the relaxation-response to SNP or Isop. These results provide indirect evidence that adenosine receptor-mediated relaxation in porcine coronary artery may be linked to modulation of protein kinase C and phospholipase C.

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