The Lipophilicity of Phorbol Esters as a Critical Factor in Determining the Pattern of Translocation of Protein Kinase C δ Fused to Green Fluorescent Protein

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.

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820 Translocation of protein kinase C subspecies in living cells — Visualization using fusion protein with green fluorescent protein (GFP)

Neuroscience Research ◽

10.1016/s0168-0102(97)90282-0 ◽

1997 ◽

Vol 28 ◽

pp. S106

Author(s):

Norio Sakai ◽

Naoya Murakami ◽

Yasuhito Shirai ◽

Naoaki Saito

Keyword(s):

Protein Kinase C ◽

Green Fluorescent Protein ◽

Protein Kinase ◽

Fusion Protein ◽

Fluorescent Protein ◽

Living Cells ◽

Kinase C ◽

Green Fluorescent

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

Molecular Biology of the Cell ◽

10.1091/mbc.e04-12-1100 ◽

2006 ◽

Vol 17 (2) ◽

pp. 799-813 ◽

Cited By ~ 35

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

The Journal of Cell Biology ◽

10.1083/jcb.139.6.1465 ◽

1997 ◽

Vol 139 (6) ◽

pp. 1465-1476 ◽

Cited By ~ 167

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.

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RACK1 Regulates Integrin-mediated Adhesion, Protrusion, and Chemotactic Cell Migration via Its Src-binding Site

Molecular Biology of the Cell ◽

10.1091/mbc.e02-03-0142 ◽

2003 ◽

Vol 14 (2) ◽

pp. 658-669 ◽

Cited By ~ 100

Author(s):

Elisabeth A. Cox ◽

David Bennin ◽

Ashley T. Doan ◽

Timothy O'Toole ◽

Anna Huttenlocher

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Binding Site ◽

Fluorescent Protein ◽

Focal Adhesions ◽

Expression Cloning ◽

Chemotactic Migration ◽

Cell Protrusion ◽

Kinase C ◽

Green Fluorescent

Mammalian cDNA expression cloning was used to identify novel regulators of integrin-mediated cell-substratum adhesions. Using a focal adhesion morphology screen, we identified a cDNA with homology to a receptor for activated protein kinase C (RACK1) that induced a loss of central focal adhesions and stress fibers in CHO-K1 cells. The identified cDNA was a C-terminal truncated form of RACK1 that had one of the putative protein kinase C binding sites but lacked the region proposed to bind the β integrin cytoplasmic domain and the tyrosine kinase Src. To investigate the role of RACK1 during cell spreading and migration, we tagged RACK1, a C-terminal truncated RACK1 and a point mutant that does not bind Src (RACK Y246F) with green fluorescent protein and expressed them in CHO-K1 cells. We found that RACK1 regulates the organization of focal adhesions and that it localizes to a subset of nascent focal complexes in areas of protrusion that contain paxillin but not vinculin. We also found that RACK1 regulates cell protrusion and chemotactic migration through its Src binding site. Together, these findings suggest that RACK1 regulates adhesion, protrusion, and chemotactic migration through its interaction with Src.

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Rice black-streaked dwarf virus P10 suppresses protein kinase C in insect vector through changing the subcellular localization of LsRACK1

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2018.0315 ◽

2019 ◽

Vol 374 (1767) ◽

pp. 20180315 ◽

Cited By ~ 3

Author(s):

Lina Lu ◽

Qi Wang ◽

Deqing Huang ◽

Qiufang Xu ◽

Xueping Zhou ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Subcellular Localization ◽

Fluorescent Protein ◽

Brown Planthopper ◽

Subcellular Fractionation ◽

Dwarf Virus ◽

Insect Vector ◽

Kinase C ◽

Green Fluorescent

Rice black-streaked dwarf virus (RBSDV) was known to be transmitted by the small brown planthopper (SBPH) in a persistent, circulative and propagative manner in nature. Here, we show that RBSDV major outer capsid protein (also known as P10) suppresses the protein kinase C (PKC) activity of SBPH through interacting with the receptor for activated protein kinase C 1 (LsRACK1). The N terminal of P10 (amino acids (aa) 1–270) and C terminal of LsRACK1 (aa 268–315) were mapped as crucial for the interaction. Confocal microscopy and subcellular fractionation showed that RBSDV P10 fused to enhanced green fluorescent protein formed vesicular structures associated with endoplasmic reticulum (ER) membranes in Spodoptera frugiperda nine cells. Our results also indicated that RBSDV P10 retargeted the initial subcellular localization of LsRACK1 from cytoplasm and cell membrane to ER and affected the function of LsRACKs to activate PKC. Inhibition of RACK1 by double stranded RNA-induced gene silencing significantly promoted the replication of RBSDV in SBPH. In addition, the PKC pathway participates in the antivirus innate immune response of SBPH. This study highlights that RACK1 negatively regulates the accumulation of RBSDV in SBPH through activating the PKC signalling pathway, and RBSDV P10 changes the subcellular localization of LsRACK1 and affects its function to activate PKC. This article is part of the theme issue ‘Biotic signalling sheds light on smart pest management’.

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