scholarly journals Translocation of protein kinase C-γ and ε -Direct visualization in living cells using fusion protein with green fluorescent protein

1997 ◽  
Vol 73 ◽  
pp. 69
Author(s):  
Norio Sakai ◽  
Keiko Sasaki ◽  
Chie Hasegawa ◽  
Mutsumi Ohkura ◽  
Kiyoshi Sumioka ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Green Fluorescent Protein ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Direct Visualization ◽  
Kinase C ◽  
Green Fluorescent

scholarly journals Direct Visualization of the Translocation of the γ-Subspecies of Protein Kinase C in Living Cells Using Fusion Proteins with Green Fluorescent Protein

1997 ◽  
Vol 139 (6) ◽  
pp. 1465-1476 ◽  
Author(s):  
Norio Sakai ◽  
Keiko Sasaki ◽  
Natsu Ikegaki ◽  
Yasuhito Shirai ◽  
Yoshitaka Ono ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Green Fluorescent Protein ◽  
Nmda Receptor ◽  
Fusion Protein ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Kinase C ◽  
Gfp Fusion Protein ◽  
Green Fluorescent

We expressed the γ-subspecies of protein kinase C (γ-PKC) fused with green fluorescent protein (GFP) in various cell lines and observed the movement of this fusion protein in living cells under a confocal laser scanning fluorescent microscope. γ-PKC–GFP fusion protein had enzymological properties very similar to that of native γ-PKC. The fluorescence of γ-PKC– GFP was observed throughout the cytoplasm in transiently transfected COS-7 cells. Stimulation by an active phorbol ester (12-O-tetradecanoylphorbol 13-acetate [TPA]) but not by an inactive phorbol ester (4α-phorbol 12, 13-didecanoate) induced a significant translocation of γ-PKC–GFP from cytoplasm to the plasma membrane. A23187, a Ca2+ ionophore, induced a more rapid translocation of γ-PKC–GFP than TPA. The A23187-induced translocation was abolished by elimination of extracellular and intracellular Ca2+. TPA- induced translocation of γ-PKC–GFP was unidirected, while Ca2+ ionophore–induced translocation was reversible; that is, γ-PKC–GFP translocated to the membrane returned to the cytosol and finally accumulated as patchy dots on the plasma membrane. To investigate the significance of C1 and C2 domains of γ-PKC in translocation, we expressed mutant γ-PKC–GFP fusion protein in which the two cysteine rich regions in the C1 region were disrupted (designated as BS 238) or the C2 region was deleted (BS 239). BS 238 mutant was translocated by Ca2+ ionophore but not by TPA. In contrast, BS 239 mutant was translocated by TPA but not by Ca2+ ionophore. To examine the translocation of γ-PKC–GFP under physiological conditions, we expressed it in NG-108 cells, N-methyl-d-aspartate (NMDA) receptor–transfected COS-7 cells, or CHO cells expressing metabotropic glutamate receptor 1 (CHO/mGluR1 cells). In NG-108 cells , K+ depolarization induced rapid translocation of γ-PKC–GFP. In NMDA receptor–transfected COS-7 cells, application of NMDA plus glycine also translocated γ-PKC–GFP. Furthermore, rapid translocation and sequential retranslocation of γ-PKC–GFP were observed in CHO/ mGluR1 cells on stimulation with the receptor. Neither cytochalasin D nor colchicine affected the translocation of γ-PKC–GFP, indicating that translocation of γ-PKC was independent of actin and microtubule. γ-PKC–GFP fusion protein is a useful tool for investigating the molecular mechanism of γ-PKC translocation and the role of γ-PKC in the central nervous system.

scholarly journals Three Distinct Mechanisms for Translocation and Activation of the δ Subspecies of Protein Kinase C

1998 ◽  
Vol 18 (9) ◽  
pp. 5263-5271 ◽  
Author(s):  
Shiho Ohmori ◽  
Yasuhito Shirai ◽  
Norio Sakai ◽  
Motoko Fujii ◽  
Hiroaki Konishi ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Fusion Protein ◽  
Kinase Activity ◽  
Fluorescent Protein ◽  
Simultaneous Treatment ◽  
Kinase C ◽  
Green Fluorescent ◽  
Number Of Cells

ABSTRACT We expressed δ subspecies of protein kinase C (δ-PKC) fused with green fluorescent protein (GFP) in CHO-K1 cells and observed the movement of this fusion protein in living cells after three different stimulations. The δ-PKC–GFP fusion protein had enzymological characteristics very similar to those of the native δ-PKC and was present throughout the cytoplasm in CHO-K1 cells. ATP at 1 mM caused a transient translocation of δ-PKC–GFP to the plasma membrane approximately 30 s after the stimulation and a sequent retranslocation to the cytoplasm within 3 min. A tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA; 1 μM), induced a slower translocation of δ-PKC–GFP, and the translocation was unidirectional. Concomitantly, the kinase activity of δ-PKC–GFP was increased by these two stimulations, when the kinase activity of the immunoprecipitated δ-PKC–GFP was measured in vitro in the absence of PKC activators such as phosphatidylserine and diacylglycerol. Hydrogen peroxide (H2O2; 5 mM) failed to translocate δ-PKC–GFP but increased its kinase activity more than threefold. δ-PKC–GFP was strongly tyrosine phosphorylated when treated with H2O2 but was tyrosine phosphorylated not at all by ATP stimulation and only slightly by TPA treatment. Both TPA and ATP induced the translocation of δ-PKC–GFP even after treatment with H2O2. Simultaneous treatment with TPA and H2O2 further activated δ-PKC–GFP up to more than fivefold. TPA treatment of cells overexpressing δ-PKC–GFP led to an increase in the number of cells in G2/M phase and of dikaryons, while stimulation with H2O2 increased the number of cells in S phase and induced no significant change in cell morphology. These results indicate that at least three different mechanisms are involved in the translocation and activation of δ-PKC.

scholarly journals Involvement of Tetrodotoxin-Resistant Na+ Current and Protein Kinase C in the Action of Growth Hormone (GH)-Releasing Hormone on Primary Cultured Somatotropes from GH-Green Fluorescent Protein Transgenic Mice

Endocrinology ◽  
2008 ◽  
Vol 149 (9) ◽  
pp. 4726-4735 ◽  
Author(s):  
Seung-Kwon Yang ◽  
Kun Wang ◽  
Helena Parkington ◽  
Chen Chen
Keyword(s):  
Protein Kinase C ◽  
Green Fluorescent Protein ◽  
Protein Kinase ◽  
Fluorescent Protein ◽  
Phosphodiesterase Inhibitor ◽  
Na Channels ◽  
Na Current ◽  
Gh Secretion ◽  
Kinase C ◽  
Green Fluorescent

GHRH depolarizes the membrane of somatotropes, leading to an increase in intracellular free Ca2+ concentration and GH secretion. Na+ channels mediate the rapid depolarization during the initial phase of the action potential, and this regulates Ca2+ influx and GH secretion. GHRH increases a tetrodotoxin-sensitive somatotrope Na+ current that is mediated by cAMP. TTX-resistant (TTX-R) Na+ channels are abundant in sensory neurons and cardiac myocytes, but their occurrence and/or function in somatotropes has not been investigated. Here we demonstrate expression of TTX-R Na+ channels and a TTX-R Na+ current, using patch-clamp method, in green fluorescent protein-GH transgenic mouse somatotropes. GHRH (100nm) increased the TTX-R Na+ current in a reversible manner. The GHRH-induced increase in TTX-R Na+ current was not affected by the cAMP antagonist Rp-cAMP or protein kinase A inhibitors KT5720 or H89. The TTX-R current was increased by 8-bromoadenosine-cAMP (cAMP analog), forskolin (adenylyl-cyclase activator), and 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor), but the additional, GHRH-induced increase in TTX-R Na+ currents was not affected. U-73122 (phospholipase C inhibitor) and protein kinase C (PKC) inhibitors, Gö-6983 and chelerythrine, blocked the effect of GHRH. PKC activators, phorbol dibutyrate and phorbol myristate acetate, increased the TTX-R Na+ current, but GHRH had no further effect on the current. Na+-free extracellular medium significantly reduced GHRH-stimulated GH secretion. We conclude that GHRH-induced increase in the TTX-R Na+ current in mouse somatotropes is mediated by the PKC system. An increase in the TTX-R Na+ current may contribute to the GHRH-induced exocytosis of GH granules from mouse somatotropes.

scholarly journals Green Fluorescent Protein (GFP)-tagged Cysteine-rich Domains from Protein Kinase C as Fluorescent Indicators for Diacylglycerol Signaling in Living Cells

1998 ◽  
Vol 140 (3) ◽  
pp. 485-498 ◽  
Author(s):  
Elena Oancea ◽  
Mary N. Teruel ◽  
Andrew F.G. Quest ◽  
Tobias Meyer
Keyword(s):  
Plasma Membrane ◽  
Protein Kinase C ◽  
Green Fluorescent Protein ◽  
Phorbol Ester ◽  
Lipid Membranes ◽  
Fluorescent Protein ◽  
Living Cells ◽  
Membrane Translocation ◽  
Kinase C ◽  
Green Fluorescent

Cysteine-rich domains (Cys-domains) are ∼50–amino acid–long protein domains that complex two zinc ions and include a consensus sequence with six cysteine and two histidine residues. In vitro studies have shown that Cys-domains from several protein kinase C (PKC) isoforms and a number of other signaling proteins bind lipid membranes in the presence of diacylglycerol or phorbol ester. Here we examine the second messenger functions of diacylglycerol in living cells by monitoring the membrane translocation of the green fluorescent protein (GFP)-tagged first Cys-domain of PKC-γ (Cys1–GFP). Strikingly, stimulation of G-protein or tyrosine kinase–coupled receptors induced a transient translocation of cytosolic Cys1–GFP to the plasma membrane. The plasma membrane translocation was mimicked by addition of the diacylglycerol analogue DiC8 or the phorbol ester, phorbol myristate acetate (PMA). Photobleaching recovery studies showed that PMA nearly immobilized Cys1–GFP in the membrane, whereas DiC8 left Cys1–GFP diffusible within the membrane. Addition of a smaller and more hydrophilic phorbol ester, phorbol dibuterate (PDBu), localized Cys1–GFP preferentially to the plasma and nuclear membranes. This selective membrane localization was lost in the presence of arachidonic acid. GFP-tagged Cys1Cys2-domains and full-length PKC-γ also translocated from the cytosol to the plasma membrane in response to receptor or PMA stimuli, whereas significant plasma membrane translocation of Cys2–GFP was only observed in response to PMA addition. These studies introduce GFP-tagged Cys-domains as fluorescent diacylglycerol indicators and show that in living cells the individual Cys-domains can trigger a diacylglycerol or phorbol ester–mediated translocation of proteins to selective lipid membranes.

scholarly journals Targeting of Protein Kinase C-ϵ during Fcγ Receptor-dependent Phagocytosis Requires the ϵC1B Domain and Phospholipase C-γ1

2006 ◽  
Vol 17 (2) ◽  
pp. 799-813 ◽  
Author(s):  
Keylon L. Cheeseman ◽  
Takehiko Ueyama ◽  
Tanya M. Michaud ◽  
Kaori Kashiwagi ◽  
Demin Wang ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Phospholipase C ◽  
Phospholipase D ◽  
Fluorescent Protein ◽  
Wild Type ◽  
Fcγ Receptor ◽  
Kinase C ◽  
Green Fluorescent

Protein kinase C-ϵ (PKC-ϵ) translocates to phagosomes and promotes uptake of IgG-opsonized targets. To identify the regions responsible for this concentration, green fluorescent protein (GFP)-protein kinase C-ϵ mutants were tracked during phagocytosis and in response to exogenous lipids. Deletion of the diacylglycerol (DAG)-binding ϵC1 and ϵC1B domains, or the ϵC1B point mutant ϵC259G, decreased accumulation at phagosomes and membrane translocation in response to exogenous DAG. Quantitation of GFP revealed that ϵC259G, ϵC1, and ϵC1B accumulation at phagosomes was significantly less than that of intact PKC-ϵ. Also, the DAG antagonist 1-hexadecyl-2-acetyl glycerol (EI-150) blocked PKC-ϵ translocation. Thus, DAG binding to ϵC1B is necessary for PKC-ϵ translocation. The role of phospholipase D (PLD), phosphatidylinositol-specific phospholipase C (PI-PLC)-γ1, and PI-PLC-γ2 in PKC-ϵ accumulation was assessed. Although GFP-PLD2 localized to phagosomes and enhanced phagocytosis, PLD inhibition did not alter target ingestion or PKC-ϵ localization. In contrast, the PI-PLC inhibitor U73122 decreased both phagocytosis and PKC-ϵ accumulation. Although expression of PI-PLC-γ2 is higher than that of PI-PLC-γ1, PI-PLC-γ1 but not PI-PLC-γ2 consistently concentrated at phagosomes. Macrophages from PI-PLC-γ2-/-mice were similar to wild-type macrophages in their rate and extent of phagocytosis, their accumulation of PKC-ϵ at the phagosome, and their sensitivity to U73122. This implicates PI-PLC-γ1 as the enzyme that supports PKC-ϵ localization and phagocytosis. That PI-PLC-γ1 was transiently tyrosine phosphorylated in nascent phagosomes is consistent with this conclusion. Together, these results support a model in which PI-PLC-γ1 provides DAG that binds to ϵC1B, facilitating PKC-ϵ localization to phagosomes for efficient IgG-mediated phagocytosis.

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