Differential Role of PKC Isoforms in GnRH and Phorbol 12-Myristate 13-Acetate Activation of Extracellular Signal-Regulated Kinase and Jun N-Terminal Kinase

Endocrinology ◽  
2010 ◽  
Vol 151 (10) ◽  
pp. 4894-4907 ◽  
Author(s):  
Masha Dobkin-Bekman ◽  
Liat Rahamim Ben-Navi ◽  
Boris Shterntal ◽  
Ludmila Sviridonov ◽  
Fiorenza Przedecki ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Preliminary Evidence ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Pkc Isoforms ◽  
Green Fluorescent ◽  
Jnk Activation ◽  
Erk2 Activation

GnRH is the first key hormone of reproduction. The role of protein kinase C (PKC) isoforms in GnRH-stimulated MAPK [ERK and Jun N-terminal kinase (JNK)] was examined in the αT3-1 and LβT2 gonadotrope cells. Incubation of the cells with GnRH resulted in a protracted activation of ERK1/2 and a slower and more transient activation of JNK1/2. Gonadotropes express conventional PKCα and conventional PKCβII, novel PKCδ, novel PKCε, and novel PKCθ, and atypical PKC-ι/λ. The use of green fluorescent protein-PKC constructs revealed that GnRH induced rapid translocation of PKCα and PKCβII to the plasma membrane, followed by their redistribution to the cytosol. PKCδ and PKCε localized to the cytoplasm and Golgi, followed by the rapid redistribution by GnRH of PKCδ to the perinuclear zone and of PKCε to the plasma membrane. Interestingly, PKCα, PKCβII, and PKCε translocation to the plasma membrane was more pronounced and more prolonged in phorbol-12-myristate-13-acetate (PMA) than in GnRH-treated cells. The use of selective inhibitors and dominant-negative plasmids for the various PKCs has revealed that PKCβII, PKCδ, and PKCε mediate ERK2 activation by GnRH, whereas PKCα, PKCβII, PKCδ, and PKCε mediate ERK2 activation by PMA. Also, PKCα, PKCβII, PKCδ, and PKCε are involved in GnRH and PMA stimulation of JNK1 in a cell-context-dependent manner. We present preliminary evidence that persistent vs. transient redistribution of selected PKCs or redistribution of a given PKC to the perinuclear zone vs. the plasma membrane may dictate its selective role in ERK or JNK activation. Thus, we have described the contribution of selective PKCs to ERK and JNK activation by GnRH.

scholarly journals Parathyroid Hormone Receptor Recycling: Role of Receptor Dephosphorylation and β-Arrestin

2002 ◽  
Vol 16 (12) ◽  
pp. 2720-2732 ◽  
Author(s):  
Stephanie Chauvin ◽  
Margaret Bencsik ◽  
Tom Bambino ◽  
Robert A. Nissenson
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Receptor Recycling ◽  
Binding Assays ◽  
Parathyroid Hormone Receptor ◽  
293 Cells ◽  
Dominant Negative Form ◽  
Green Fluorescent

Abstract The recovery of PTH receptor (PTHR) function after acute homologous receptor desensitization and down-regulation in bone and kidney cells has been attributed to receptor recycling. To determine the role of receptor dephosphorylation in PTHR recycling, we performed morphological and functional assays on human embryonic kidney 293 cells stably expressing wild-type (wt) or mutant PTHRs. Confocal microscopy and ligand binding assays revealed that the wt PTHR is rapidly recycled back to the plasma membrane after removal of the agonist. Receptors that were engineered to either lack the sites of phosphorylation or to resemble constitutively phosphorylated receptors were able to recycle back to the plasma membrane with the same kinetics as the wt PTHR. The PTHR was found to be dephosphorylated by an enzyme apparently distinct from protein phosphatases 1 or 2A. The PTHR and β-arrestin-2-green fluorescent protein (GFP) were found to stably colocalize during PTHR internalization, whereas after agonist removal and during receptor recycling, the colocalization slowly disappeared. Experiments using phosphorylation-deficient PTHRs and a dominant-negative form of β-arrestin showed that β-arrestin does not regulate the efficiency of PTHR recycling. These studies indicate that, unlike many G protein-coupled receptors, PTHR recycling does not require receptor dephosphorylation or its dissociation from β-arrestin.

Ser9 phosphorylation of mitochondrial GSK-3β is a primary mechanism of cardiomyocyte protection by erythropoietin against oxidant-induced apoptosis

2008 ◽  
Vol 295 (5) ◽  
pp. H2079-H2086 ◽  
Author(s):  
Katsuhiko Ohori ◽  
Tetsuji Miura ◽  
Masaya Tanno ◽  
Takayuki Miki ◽  
Takahiro Sato ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Oxidant Stress ◽  
Dependent Manner ◽  
Phosphatidylinositol 3 Kinase ◽  
H9c2 Cardiomyocytes ◽  
Gsk 3Β ◽  
Induced Apoptosis ◽  
Green Fluorescent ◽  
Interfering Rna

The aim of this study was to determine the role of GSK-3β in cardiomyocyte protection afforded by erythropoietin (EPO) against oxidant stress-induced apoptosis. Treatment with EPO (10 units/ml) induced Ser473 phosphorylation of Akt and Ser9 phosphorylation of GSK-3β and significantly reduced the proportion of apoptotic H9c2 cardiomyocytes after exposure to H2O2 from 38.3 ± 2.7% to 26.0 ± 2.9%. This protection was not detected in cells transfected with constitutively active GSK-3β (S9A), which lacks Ser9 for inhibitory phosphorylation. The antiapoptotic effect of EPO was mimicked completely by GSK-3β knockdown using small interfering RNA and partly by the transfection with kinase-deficient GSK-3β (K85R). The level of colocalization of intracellular GSK-3β with mitochondria assessed by enhanced green fluorescent protein-tagged GSK-3β or immunocytochemistry was not altered by EPO treatment. However, EPO increased the level of Ser9-phospho-GSK-3β colocalized with mitochondria by 50% in a phosphatidylinositol 3-kinase-dependent manner. Mitochondrial translocation of Bcl-2-associated X protein (BAX) after exposure to H2O2 was inhibited by EPO pretreatment and by GSK-3β knockdown. These results suggest that the suppression of GSK-3β activity by Akt-mediated Ser9 phosphorylation in the mitochondria affords cardiomyocytes tolerance against oxidant-induced apoptosis, possibly by inhibiting the access of BAX to the mitochondria.

scholarly journals Lamin A/C Binding Protein LAP2α Is Required for Nuclear Anchorage of Retinoblastoma Protein

2002 ◽  
Vol 13 (12) ◽  
pp. 4401-4413 ◽  
Author(s):  
Ewa Markiewicz ◽  
Thomas Dechat ◽  
Roland Foisner ◽  
Roy. A Quinlan ◽  
Christopher J. Hutchison
Keyword(s):  
Tissue Culture ◽  
Fluorescent Protein ◽  
Solid Phase ◽  
Retinoblastoma Protein ◽  
Dominant Negative ◽  
Lamin A ◽  
Dependent Manner ◽  
Culture Cells ◽  
Tissue Culture Cells ◽  
Green Fluorescent

The phosphorylation-dependent anchorage of retinoblastoma protein Rb in the nucleus is essential for its function. We show that its pocket C domain is both necessary and sufficient for nuclear anchorage by transiently expressing green fluorescent protein (GFP) chimeras of Rb fragments in tissue culture cells and by extracting the cells with hypotonic solutions. Solid phase binding assays using glutathioneS-transferase-fusion of Rb pockets A, B, and C revealed a direct association of lamin C exclusively to pocket C. Lamina-associated polypeptide (LAP) 2α, a binding partner of lamins A/C, bound strongly to pocket C and weakly to pocket B. When LAP2α was immunoprecipitated from soluble nuclear fractions, lamins A/C and hypophosphorylated Rb were coprecipitated efficiently. Similarly, immunoprecipitation of expressed GFP-Rb fragments by using anti-GFP antibodies coprecipitated LAP2α, provided that pocket C was present in the GFP chimeras. On redistribution of endogenous lamin A/C and LAP2α into nuclear aggregates by overexpressing dominant negative lamin mutants in tissue culture cells, Rb was also sequestered into these aggregates. In primary skin fibroblasts, LAP2α is expressed in a growth-dependent manner. Anchorage of hypophosphorylated Rb in the nucleus was weakened significantly in the absence of LAP2α. Together, these data suggest that hypophosphorylated Rb is anchored in the nucleus by the interaction of pocket C with LAP2α–lamin A/C complexes.

scholarly journals Ligand-induced Trafficking of the Sphingosine-1-phosphate Receptor EDG-1

1999 ◽  
Vol 10 (4) ◽  
pp. 1179-1190 ◽  
Author(s):  
Catherine H. Liu ◽  
Shobha Thangada ◽  
Menq-Jer Lee ◽  
James R. Van Brocklyn ◽  
Sarah Spiegel ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescent Protein ◽  
Receptor Internalization ◽  
Lipid Mediator ◽  
Dependent Manner ◽  
High Affinity ◽  
Affinity Ligand ◽  
Sphingosine 1 Phosphate ◽  
High Affinity Receptor ◽  
Green Fluorescent

The endothelial-derived G-protein–coupled receptor EDG-1 is a high-affinity receptor for the bioactive lipid mediator sphingosine-1-phosphate (SPP). In the present study, we constructed the EDG-1–green fluorescent protein (GFP) chimera to examine the dynamics and subcellular localization of SPP–EDG-1 interaction. SPP binds to EDG-1–GFP and transduces intracellular signals in a manner indistinguishable from that seen with the wild-type receptor. Human embryonic kidney 293 cells stably transfected with the EDG-1–GFP cDNA expressed the receptor primarily on the plasma membrane. Exogenous SPP treatment, in a dose-dependent manner, induced receptor translocation to perinuclear vesicles with a τ1/2of ∼15 min. The EDG-1–GFP–containing vesicles are distinct from mitochondria but colocalize in part with endocytic vesicles and lysosomes. Neither the low-affinity agonist lysophosphatidic acid nor other sphingolipids, ceramide, ceramide-1-phosphate, or sphingosylphosphorylcholine, influenced receptor trafficking. Receptor internalization was completely inhibited by truncation of the C terminus. After SPP washout, EDG-1–GFP recycles back to the plasma membrane with a τ1/2of ∼30 min. We conclude that the high-affinity ligand SPP specifically induces the reversible trafficking of EDG-1 via the endosomal pathway and that the C-terminal intracellular domain of the receptor is critical for this process.

scholarly journals Intracellular Trafficking of a Palmitoylated Membrane-Associated Protein Component of Enveloped Vaccinia Virus

2003 ◽  
Vol 77 (16) ◽  
pp. 9008-9019 ◽  
Author(s):  
Matloob Husain ◽  
Bernard Moss
Keyword(s):  
Plasma Membrane ◽  
Vaccinia Virus ◽  
Intracellular Trafficking ◽  
Plasma Membranes ◽  
Secretory Pathway ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Coated Pits ◽  
Early Endosomes ◽  
Green Fluorescent

ABSTRACT The F13L protein of vaccinia virus, an essential and abundant palmitoylated peripheral membrane component of intra- and extracellular enveloped virions, associates with Golgi, endosomal, and plasma membranes in the presence or absence of other viral proteins. In the present study, the trafficking of a fully functional F13L-green fluorescent protein (GFP) chimera in transfected and productively infected cells was analyzed using specific markers and inhibitors. We found that Sar1H79G, a trans-dominant-negative protein inhibitor of cargo transport from the endoplasmic reticulum, had no apparent effect on the intracellular distribution of F13L-GFP, suggesting that the initial membrane localization occurs at a downstream compartment of the secretory pathway. Recycling of F13L-GFP from the plasma membrane was demonstrated by partial colocalization with FM4-64, a fluorescent membrane marker of endocytosis. Punctate F13L-GFP fluorescence overlapped with clathrin and Texas red-conjugated transferrin, suggesting that endocytosis occurred via clathrin-coated pits. The inhibitory effects of chlorpromazine and trans-dominant-negative forms of dynamin and Eps15 protein on the recycling of F13L-GFP provided further evidence for clathrin-mediated endocytosis. In addition, the F13L protein was specifically coimmunoprecipitated with α-adaptin, a component of the AP-2 complex that interacts with Eps15. Nocodazole and wortmannin perturbed the intracellular trafficking of F13L-GFP, consistent with its entry into late and early endosomes through the secretory and endocytic pathways, respectively. The recycling pathway described here provides a mechanism for the reutilization of the F13L protein following its deposition in the plasma membrane during the exocytosis of enveloped virions.

scholarly journals Leukotriene D4 induces association of active RhoA with phospholipase C-γ1 in intestinal epithelial cells

2002 ◽  
Vol 365 (1) ◽  
pp. 157-163 ◽  
Author(s):  
Charles Kumar THODETI ◽  
Ramin MASSOUMI ◽  
Lene BINDSLEV ◽  
Anita SJÖLANDER
Keyword(s):  
Plasma Membrane ◽  
Epithelial Cells ◽  
Phospholipase C ◽  
Fluorescent Protein ◽  
Intestinal Epithelial Cells ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Leukotriene D4 ◽  
Green Fluorescent ◽  
Constitutively Active

It has been previously suggested that leukotriene-induced Ca2+ signalling is mediated through a Rho-dependent process, but neither direct activation of Rho nor a mechanism underlying such signalling has been reported. Accordingly, we used the Rhotekin binding assay to assess RhoA activation in intestinal epithelial cells and observed that RhoA was activated by leukotriene D4 (LTD4). We also found that, within 15s, activation of RhoA by LTD4 led to an increased association of RhoA with G-protein βγ (Gβγ) and phospholipase C-γ1 (PLC-γ1) in the plasma membrane, as evidenced by the results of co-immunoprecipitation, glutathione S-transferase (GST) pulldown assays, and confocal microscopy. Amounts of RhoA increased in both Gβ and PLC-γ1 immunoprecipitates within 15s of LTD4 treatment. An interaction between RhoA, Gβγ and PLC-γ1 is supported by our finding that a GST fusion protein of constitutively active RhoA (GST-RhoAV14) precipitated Gβγ and PLC-γ1 from cell lysates in an agonist-dependent manner. Such an association is also substantiated by our confocal immunofluorescence results, which revealed that LTD4 induction increased co-localization of constitutively active RhoA and PLC-γ1 to the plasma membrane of cells transfected with enhanced green fluorescent protein L63RhoA. Furthermore, microinjection of neutralizing RhoA antibodies, but not control antibodies, significantly reduced LTD4-induced Ca2+ mobilization. Our results are the first to demonstrate a LTD4-induced activation of RhoA and more importantly its association with PLC-γ1, which are essential for the PLC-γ1-mediated calcium mobilization.

scholarly journals Dynamin Is Required for GnRH Signaling to L-Type Calcium Channels and Activation of ERK

Endocrinology ◽  
2015 ◽  
Vol 157 (2) ◽  
pp. 831-843 ◽  
Author(s):  
Brian S. Edwards ◽  
An K. Dang ◽  
Dilyara A. Murtazina ◽  
Melissa G. Dozier ◽  
Jennifer D. Whitesell ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Actin Cytoskeleton ◽  
Fluorescent Protein ◽  
Leading Edge ◽  
Dominant Negative ◽  
Actin Binding ◽  
Erk Phosphorylation ◽  
Green Fluorescent ◽  
Ca2 Channels

Abstract We have shown that GnRH-mediated engagement of the cytoskeleton induces cell movement and is necessary for ERK activation. It also has previously been established that a dominant negative form of the mechano-GTPase dynamin (K44A) attenuates GnRH activation of ERK. At present, it is not clear at what level these cellular events might be linked. To explore this, we used live cell imaging in the gonadotrope-derived αT3–1 cell line to determine that dynamin-green fluorescent protein accumulated in GnRH-induced lamellipodia and plasma membrane protrusions. Coincident with translocation of dynamin-green fluorescent protein to the plasma membrane, we demonstrated that dynamin colocalizes with the actin cytoskeleton and the actin binding protein, cortactin at the leading edge of the plasma membrane. We next wanted to assess the physiological significance of these findings by inhibiting dynamin GTPase activity using dynasore. We find that dynasore suppresses activation of ERK, but not c-Jun N-terminal kinase, after exposure to GnRH agonist. Furthermore, exposure of αT3–1 cells to dynasore inhibited GnRH-induced cyto-architectural rearrangements. Recently it has been discovered that GnRH induced Ca2+ influx via the L-type Ca2+ channels requires an intact cytoskeleton to mediate ERK phosphorylation. Interestingly, not only does dynasore attenuate GnRH-mediated actin reorganization, it also suppresses Ca2+ influx through L-type Ca2+ channels visualized in living cells using total internal reflection fluorescence microscopy. Collectively, our data suggest that GnRH-induced membrane remodeling events are mediated in part by the association of dynamin and cortactin engaging the actin cytoskeleton, which then regulates Ca2+ influx via L-type channels to facilitate ERK phosphorylation.

scholarly journals p120-Catenin Regulates Clathrin-dependent Endocytosis of VE-Cadherin

2005 ◽  
Vol 16 (11) ◽  
pp. 5141-5151 ◽  
Author(s):  
Kanyan Xiao ◽  
Jennifer Garner ◽  
Kathleen M. Buckley ◽  
Peter A. Vincent ◽  
Christine M. Chiasson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Dependent Manner ◽  
Lysosomal Degradation ◽  
P120 Catenin ◽  
Binding Partner ◽  
Endocytic Machinery ◽  
Green Fluorescent ◽  
Retention Signal

VE-cadherin is an adhesion molecule critical to vascular barrier function and angiogenesis. VE-cadherin expression levels are regulated by p120 catenin, which prevents lysosomal degradation of cadherins by unknown mechanisms. To test whether the VE-cadherin cytoplasmic domain mediates endocytosis, and to elucidate the nature of the endocytic machinery involved, the VE-cadherin tail was fused to the interleukin (IL)-2 receptor (IL-2R) extracellular domain. Internalization assays demonstrated that the VE-cadherin tail dramatically increased endocytosis of the IL-2R in a clathrin-dependent manner. Interestingly, p120 inhibited VE-cadherin endocytosis via a mechanism that required direct interactions between p120 and the VE-cadherin cytoplasmic tail. However, p120 did not inhibit transferrin internalization, demonstrating that p120 selectively regulates cadherin internalization rather than globally inhibiting clathrin-dependent endocytosis. Finally, cell surface labeling experiments in cells expressing green fluorescent protein-tagged p120 indicated that the VE-cadherin–p120 complex dissociates upon internalization. These results support a model in which the VE-cadherin tail mediates interactions with clathrin-dependent endocytic machinery, and this endocytic processing is inhibited by p120 binding to the cadherin tail. These findings suggest a novel mechanism by which a cytoplasmic binding partner for a transmembrane receptor can serve as a selective plasma membrane retention signal, thereby modulating the availability of the protein for endo-lysosomal processing.

scholarly journals Coxiella burnetii Localizes in a Rab7-Labeled Compartment with Autophagic Characteristics

2002 ◽  
Vol 70 (10) ◽  
pp. 5816-5821 ◽  
Author(s):  
Walter Berón ◽  
Maximiliano G. Gutierrez ◽  
Michel Rabinovitch ◽  
Maria I. Colombo
Keyword(s):  
Phase Ii ◽  
Coxiella Burnetii ◽  
Fluorescent Protein ◽  
Q Fever ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Wild Type ◽  
Green Fluorescent

ABSTRACT The obligate intracellular bacterium Coxiella burnetii, the agent of Q fever in humans and of coxiellosis in other animals, survives and replicates within large, acidified, phagolysosome-like vacuoles known to fuse homo- and heterotypically with other vesicles. To further characterize these vacuoles, HeLa cells were infected with C. burnetii phase II; 48 h later, bacteria-containing vacuoles were labeled by LysoTracker, a marker of acidic compartments, and accumulated monodansylcadaverine and displayed protein LC3, both markers of autophagic vacuoles. Furthermore, 3-methyladenine and wortmannin, agents known to inhibit early stages in the autophagic process, each blocked Coxiella vacuole formation. These autophagosomal features suggest that Coxiella vacuoles interact with the autophagic pathway. The localization and role of wild-type and mutated Rab5 and Rab7, markers of early and late endosomes, respectively, were also examined to determine the role of these small GTPases in the trafficking of C. burnetii phase II. Green fluorescent protein (GFP)-Rab5 and GFP-Rab7 constructs were overexpressed and visualized by fluorescence microscopy. Coxiella-containing large vacuoles were labeled with wild-type Rab7 (Rab7wt) and with GTPase-deficient mutant Rab7Q67L, whereas no colocalization was observed with the dominant-negative mutant Rab7T22N. The vacuoles were also decorated by GFP-Rab5Q79L but not by GFP-Rab5wt. These results suggest that Rab7 participates in the biogenesis of the parasitophorous vacuoles.

scholarly journals Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

1999 ◽  
Vol 339 (2) ◽  
pp. 299-307 ◽  
Author(s):  
Arthur L. KRUCKEBERG ◽  
Ling YE ◽  
Jan A. BERDEN ◽  
Karel van DAM
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Green Fluorescent Protein ◽  
Glucose Transport ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Hexose Transporter ◽  
High Concentrations ◽  
Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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