scholarly journals Identification of Regulatory Phosphorylation Sites in a Cell Volume– and Ste20 Kinase–dependent ClC Anion Channel

2008 ◽  
Vol 133 (1) ◽  
pp. 29-42 ◽  
Author(s):  
Rebecca A. Falin ◽  
Rebecca Morrison ◽  
Amy-Joan L. Ham ◽  
Kevin Strange
Keyword(s):  
Cell Volume ◽  
Intracellular Signaling ◽  
Anion Channel ◽  
Type I ◽  
Dependent Manner ◽  
Channel Activation ◽  
Gating Mechanism ◽  
Channel Inhibition ◽  
C Terminus ◽  
Regulatory Phosphorylation

Changes in phosphorylation regulate the activity of various ClC anion transport proteins. However, the physiological context under which such regulation occurs and the signaling cascades that mediate phosphorylation are poorly understood. We have exploited the genetic model organism Caenorhabditis elegans to characterize ClC regulatory mechanisms and signaling networks. CLH-3b is a ClC anion channel that is expressed in the worm oocyte and excretory cell. Channel activation occurs in response to oocyte meiotic maturation and swelling via serine/threonine dephosphorylation mediated by the type I phosphatases GLC-7α and GLC-7β. A Ste20 kinase, germinal center kinase (GCK)-3, binds to the cytoplasmic C terminus of CLH-3b and inhibits channel activity in a phosphorylation-dependent manner. Analysis of hyperpolarization-induced activation kinetics suggests that phosphorylation may inhibit the ClC fast gating mechanism. GCK-3 is an ortholog of mammalian SPAK and OSR1, kinases that bind to, phosphorylate, and regulate the cell volume–dependent activity of mammalian cation-Cl− cotransporters. Using mass spectrometry and patch clamp electrophysiology, we demonstrate here that CLH-3b is a target of regulatory phosphorylation. Concomitant phosphorylation of S742 and S747, which are located 70 and 75 amino acids downstream from the GCK-3 binding site, are required for kinase-mediated channel inhibition. In contrast, swelling-induced channel activation occurs with dephosphorylation of S747 alone. Replacement of both S742 and S747 with glutamate gives rise to kinase- and swelling-insensitive channels that exhibit activity and biophysical properties similar to those of wild-type CLH-3b inhibited by GCK-3. Our studies provide novel insights into ClC regulation and mechanisms of cell volume signaling, and provide the foundation for studies aimed at defining how conformational changes in the cytoplasmic C terminus alter ClC gating and function in response to intracellular signaling events.

scholarly journals Inhibitory Effects of Echinochrome A, Isolated from Shell of the Sea Urchin Anthocidaris crassispina, on Antigen-Stimulated Degranulation in Rat Basophilic Leukemia RBL-2H3 Cells through Suppression of Lyn Activation

2016 ◽  
Vol 11 (9) ◽  
pp. 1934578X1601100
Author(s):  
Tomohiro Itoh ◽  
Azusa Fujiwara ◽  
Masayuki Ninomiya ◽  
Toshimichi Maeda ◽  
Masashi Ando ◽  
...  
Keyword(s):  
Sea Urchin ◽  
Intracellular Signaling ◽  
Immunoglobulin E ◽  
Inhibitory Effect ◽  
Biologically Active ◽  
Type I ◽  
Dependent Manner ◽  
Echinochrome A ◽  
Basophilic Leukemia ◽  
Anthocidaris Crassispina

Echinochrome A (Echi-A) was isolated from the sea urchin Anthocidaris crassispina and its structure determined using 1D and 2D-NMR. In the present study, we examined the inhibitory effect of Echi-A on antigen-stimulated degranulation in rat basophilic leukemia RBL-2H3 cells, which were suppressed in a dose dependent manner. The antigens bind to the high affinity immunoglobulin E receptor, which is expressed on the surface of mast cells and basophils and activate intracellular signal transduction, resulting in the release of biologically active mediators such as histamine. In order to disclose the inhibitory mechanisms of degranulation by Echi-A, we examined the elevation in intracellular Ca2+ concentration ([Ca2+]i), production levels of intracellular reactive oxygen species (ROS) and early intracellular signaling events. Both elevation of [Ca2+]i and intracellular ROS production were markedly suppressed in cells treated with Echi-A. Echi-A also suppressed the activation of Lyn, Syk, and PLCγ1/2 in antigen-stimulated cells. These results indicated that inhibition of antigen-stimulated degranulation in RBL-2H3 cells by Echi-A is mainly due to the inactivation of Lyn/Syk/PLCγ signaling pathways. Our findings suggest that Echi-A could be a beneficial agent for alleviating the symptoms of type I allergy.

scholarly journals Two Distinct Notch1 Mutant Alleles Are Involved in the Induction of T-Cell Leukemia in c-myc Transgenic Mice

2000 ◽  
Vol 20 (11) ◽  
pp. 3831-3842 ◽  
Author(s):  
C. D. Hoemann ◽  
N. Beaulieu ◽  
L. Girard ◽  
N. Rebai ◽  
P. Jolicoeur
Keyword(s):  
Cell Surface ◽  
Transgenic Mice ◽  
Intracellular Signaling ◽  
Transmembrane Domain ◽  
Tumor Formation ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Extracellular Medium ◽  
Calcium Dependent

ABSTRACT We have previously characterized a large panel of provirus insertion Notch1 mutant alleles and their products arising in thymomas of MMTVD/myc transgenic mice. Here, we show that these Notch1 mutations represent two clearly distinct classes. In the first class (type I), proviral integrations were clustered just upstream of sequences encoding the transmembrane domain. Type I Notch1 alleles produced two types of mutantNotch1 RNA, one of which encoded the entire Notch1 cytoplasmic domain [N(IC)] and the other of which encoded a soluble ectodomain [N(EC)Mut] which, in contrast to the processed wild-type ectodomain [N(EC)WT], did not reside at the cell surface and became secreted in a temperature-dependent manner. A second, novel class of mutant Notch1 allele (type II) encoded a Notch1 receptor with the C-terminal PEST motif deleted (ΔCT). The type II Notch1ΔCT protein was expressed as a normally processed receptor [N(EC)WT and N(IC)ΔCT] at the cell surface, and its ectodomain was found to be shed into the extracellular medium in a temperature- and calcium-dependent manner. These data suggest that both type I and type II mutations generate two structurally distinct Notch1 N(EC) and N(IC) proteins that may participate in tumor formation, in collaboration with the c-myc oncogene, through distinct mechanisms. Constitutive type I N(IC) and type II N(IC)ΔCT expression may enhance Notch1 intracellular signaling, while secreted or shed type I N(EC)Mut and type II N(EC) proteins may differentially interact in an autocrine or paracrine fashion with ligands of Notch1 and affect their signaling.

Role of Grb2 in EGF-stimulated EGFR internalization

2002 ◽  
Vol 115 (9) ◽  
pp. 1791-1802 ◽  
Author(s):  
Tetsuo Yamazaki ◽  
Kristien Zaal ◽  
Dale Hailey ◽  
John Presley ◽  
Jennifer Lippincott-Schwartz ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intracellular Signaling ◽  
Growth Factor Receptor ◽  
Sh3 Domain ◽  
Membrane Receptors ◽  
Live Cells ◽  
Dependent Manner ◽  
C Terminus ◽  
Downstream Effectors ◽  
Transferrin Uptake

Grb2 is an adaptor molecule that couples membrane receptors such as the epidermal growth factor receptor (EGFR) to intracellular signaling pathways. To gain insight into the trafficking pathways followed by these molecules after activation by EGF, we visualized Grb2 and EGFR fused to GFP spectral variants in single live cells. In nonstimulated cells, Grb2-YFP was primarily localized diffusely in the cytoplasm, whereas EGFR-CFP was found on the plasma membrane and in endocytic structures localized in the perinuclear area. Within 1 minute of EGF stimulation, Grb2 redistributed to the plasma membrane where it bound EGFR-CFP in an SH2 dependent manner. The plasma membrane then began to dynamically ruffle, and Grb2-YFP and EGFR-CFP were found to internalize together in large macropinocytic structures. These structures were morphologically distinct from conventional, clathrin-derived endosomes and did not label with transferrin, AP-2 or clathrin heavy chain. Evidence that these structures did not require clathrin for internalization came from experiments showing that expression of the C-terminus of AP-180, which inhibited transferrin uptake, had no effect on EGF-induced internalization of EGFR. YFP-tagged Grb2 containing an inhibitory mutation in either N- or C-SH3 domain redistributed to the plasma membrane upon EGF stimulation, but the macropinocytic structures containing Grb2-YFP and EGFR-CFP did not translocate inward and appeared to remain tethered to the plasma membrane. This suggested that the Grb2 SH3 domain was responsible for coupling the membranes containing EGFR with downstream effectors involved in internalization of these membranes. Transferrin uptake was unaffected in the presence of all of the SH3 domain mutants, consistent with the EGF-stimulated EGFR internalization pathway being clathrin-independent. These results demonstrate a role for Grb2 in events associated with a macropinocytic internalization pathway for EGFR in activated cells.

Activation of maxi-anion channel by protein tyrosine dephosphorylation

2009 ◽  
Vol 297 (4) ◽  
pp. C990-C1000 ◽  
Author(s):  
Abduqodir H. Toychiev ◽  
Ravshan Z. Sabirov ◽  
Nobuyaki Takahashi ◽  
Yuhko Ando-Akatsuka ◽  
Hongtao Liu ◽  
...  
Keyword(s):  
Single Channel ◽  
Tyrosine Phosphatase ◽  
Anion Channel ◽  
Activation Mechanism ◽  
Receptor Protein ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Channel Activation ◽  
Phosphatase Inhibitors ◽  
Protein Tyrosine

The maxi-anion channel with a large single-channel conductance of >300 pS, and unknown molecular identity, is functionally expressed in a large variety of cell types. The channel is activated by a number of experimental maneuvers such as exposing cells to hypotonic or ischemic stress. The most effective and consistent method of activating it is patch membrane excision. However, the activation mechanism of the maxi-anion channel remains poorly understood at present. In the present study, involvement of phosphorylation/dephosphorylation in excision-induced activation was examined. In mouse mammary fibroblastic C127 cells, activity of the channel was suppressed by intracellular application of Mg-ATP, but not Mg-5′-adenylylimidodiphosphate (AMP-PNP), in a concentration-dependent manner. When a cocktail of broad-spectrum tyrosine phosphatase inhibitors was applied, channel activation was completely abolished, whereas inhibitors of serine/threonine protein phosphatases had no effect. On the other hand, protein tyrosine kinase inhibitors brought the channel out of an inactivated state. In mouse adult skin fibroblasts (MAFs) in primary culture, similar maxi-anion channels were found to be activated on membrane excision, in a manner sensitive to tyrosine phosphatase inhibitors. In MAFs isolated from animals deficient in receptor protein tyrosine phosphatase (RPTP)ζ, activation of the maxi-anion channel was significantly slower and less prominent compared with that observed in wild-type MAFs; however, channel activation was restored by transfection of the RPTPζ gene. Thus it is concluded that activation of the maxi-anion channel involves protein dephosphorylation mediated by protein tyrosine phosphatases that include RPTPζ in mouse fibroblasts, but not in C127 cells.

scholarly journals GCK-3, a Newly Identified Ste20 Kinase, Binds To and Regulates the Activity of a Cell Cycle–dependent ClC Anion Channel

2005 ◽  
Vol 125 (2) ◽  
pp. 113-125 ◽  
Author(s):  
Jerod Denton ◽  
Keith Nehrke ◽  
Xiaoyan Yin ◽  
Rebecca Morrison ◽  
Kevin Strange
Keyword(s):  
Cell Cycle ◽  
Signaling Pathways ◽  
Cell Volume ◽  
Specific Binding ◽  
Anion Channel ◽  
Fluid Secretion ◽  
Hek293 Cells ◽  
Cell Swelling ◽  
Binding Motif ◽  
Dependent Manner

CLH-3b is a Caenorhabditis elegans ClC anion channel that is expressed in the worm oocyte. The channel is activated during oocyte meiotic maturation and in response to cell swelling by serine/threonine dephosphorylation events mediated by the type 1 phosphatases GLC-7α and GLC-7β. We have now identified a new member of the Ste20 kinase superfamily, GCK-3, that interacts with the CLH-3b COOH terminus via a specific binding motif. GCK-3 inhibits CLH-3b in a phosphorylation-dependent manner when the two proteins are coexpressed in HEK293 cells. clh-3 and gck-3 are expressed predominantly in the C. elegans oocyte and the fluid-secreting excretory cell. Knockdown of gck-3 expression constitutively activates CLH-3b in nonmaturing worm oocytes. We conclude that GCK-3 functions in cell cycle– and cell volume–regulated signaling pathways that control CLH-3b activity. GCK-3 inactivates CLH-3b by phosphorylating the channel and/or associated regulatory proteins. Our studies provide new insight into physiologically relevant signaling pathways that control ClC channel activity and suggest novel mechanisms for coupling cell volume changes to cell cycle events and for coordinately regulating ion channels and transporters that control cellular Cl− content, cell volume, and epithelial fluid secretion.

scholarly journals Drosophila Bestrophin-1 Chloride Current Is Dually Regulated by Calcium and Cell Volume

2007 ◽  
Vol 130 (5) ◽  
pp. 513-524 ◽  
Author(s):  
Li-Ting Chien ◽  
H. Criss Hartzell
Keyword(s):  
Cell Volume ◽  
Anion Channel ◽  
Cell Swelling ◽  
Macular Dystrophy ◽  
Dependent Manner ◽  
S2 Cells ◽  
New Family ◽  
Drosophila S2 Cells ◽  
Cl Channels ◽  
Dose Dependent

Mutations in the human bestrophin-1 (hBest1) gene are responsible for Best vitelliform macular dystrophy, however the mechanisms leading to retinal degeneration have not yet been determined because the function of the bestrophin protein is not fully understood. Bestrophins have been proposed to comprise a new family of Cl− channels that are activated by Ca2+. While the regulation of bestrophin currents has focused on intracellular Ca2+, little is known about other pathways/mechanisms that may also regulate bestrophin currents. Here we show that Cl− currents in Drosophila S2 cells, that we have previously shown are mediated by bestrophins, are dually regulated by Ca2+ and cell volume. The bestrophin Cl− currents were activated in a dose-dependent manner by osmotic pressure differences between the internal and external solutions. The increase in the current was accompanied by cell swelling. The volume-regulated Cl− current was abolished by treating cells with each of four different RNAi constructs that reduced dBest1 expression. The volume-regulated current was rescued by transfecting with dBest1. Furthermore, cells not expressing dBest1 were severely depressed in their ability to regulate their cell volume. Volume regulation and Ca2+ regulation can occur independently of one another: the volume-regulated current was activated in the complete absence of Ca2+ and the Ca2+-activated current was activated independently of alterations in cell volume. These two pathways of bestrophin channel activation can interact; intracellular Ca2+ potentiates the magnitude of the current activated by changes in cell volume. We conclude that in addition to being regulated by intracellular Ca2+, Drosophila bestrophins are also novel members of the volume-regulated anion channel (VRAC) family that are necessary for cell volume homeostasis.

The Integrin α2β1 (GPIa/IIa)-I-Domain Inhibits Platelet-Collagen Interaction

1997 ◽  
Vol 77 (05) ◽  
pp. 0981-0985 ◽  
Author(s):  
H Depraetere ◽  
C Wille ◽  
Y Gansemans ◽  
P Stanssens ◽  
M Lauwereys ◽  
...  
Keyword(s):  
Platelet Aggregation ◽  
Collagen Type ◽  
Stop Codon ◽  
Collagen Type I ◽  
Type I ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
C Terminus ◽  
A Domains ◽  
Domain I

SummaryThe integrin α2β1 is a major cellular receptor for collagen. The α2subunit contains an ± 200 amino acids inserted domain (I-domain) in the N-terminal region. A certain degree of homology exists between the I-domains found in integrins, collagen and the A-domains of vWF.The α2-I-domain encoding region (aa residues D145 to S334) was obtained by RT-PCR from mRNA of non stimulated human PBL’s. The primers were designed to introduce the necessary restriction sites for cloning of the DNA fragment in frame downstream of the malE gene, as well as a stop codon after the last triplet. The resulting construct pMAL-c2-α2-I allows the expression of the I-domain, fused to the C-terminus of maltose binding protein (mal). The α2-I-mal is purified from the bacterial extract by affinity chromatography on an amylose column.The purified α2-I-mal has been characterized by ELISA’s. The animal bound to immobilised collagen type I in a concentration dependent manner and could be blocked by the functional monoclonal anti-α2β1 antibody 6F1.The interaction of α2-I-mal with collagen furthermore is Mg2+- dependent since the binding was inhibited in the presence of 10 mM EDTA or 10 mM Ca2+ but sustained in the presence of 10 mM Mg2+.Finally, α2-I-mal itself was able to inhibit adhesion of washed platelets to collagen immobilised on a microtiterplate in a dose-dependent manner (α2-I-mal IC50:0.7 μ M) as well as platelet aggregation induced by collagen type I (α2-I-mal IC50: 0.7 μM).With these results we could confirm that the α2-I-domain represents the collagen-binding site of α2β1 and we furthermore could indicate that this domain is able to prevent platelet adhesion to collagen and collagen-induced platelet aggregation, pointing to the primordial role of α2-I-mal and hence of α2β1 in platelet-collagen interaction.

scholarly journals The C terminus of talin links integrins to cell cycle progression

2011 ◽  
Vol 195 (3) ◽  
pp. 499-513 ◽  
Author(s):  
Pengbo Wang ◽  
Christoph Ballestrem ◽  
Charles H. Streuli
Keyword(s):  
Cell Cycle ◽  
Cell Fate ◽  
Focal Adhesion ◽  
Intracellular Signaling ◽  
Cell Cycle Progression ◽  
Mammary Epithelial Cells ◽  
Dependent Manner ◽  
Cycle Progression ◽  
Cell Fate Decisions ◽  
C Terminus

Integrins are cell adhesion receptors that sense the extracellular matrix (ECM) environment. One of their functions is to regulate cell fate decisions, although the question of how integrins initiate intracellular signaling is not fully resolved. In this paper, we examine the role of talin, an adapter protein at cell–matrix attachment sites, in outside-in signaling. We used lentiviral small hairpin ribonucleic acid to deplete talin in mammary epithelial cells. These cells still attached to the ECM in an integrin-dependent manner and spread. They had a normal actin cytoskeleton, but vinculin, paxillin, focal adhesion kinase (FAK), and integrin-linked kinase were not recruited to adhesion sites. Talin-deficient cells showed proliferation defects, and reexpressing a tail portion of the talin rod, but not its head domain, restored integrin-mediated FAK phosphorylation, suppressed p21 expression, and rescued cell cycle. Thus, talin recruits and activates focal adhesion proteins required for proliferation via the C terminus of its rod domain. Our study reveals a new function for talin, which is to link integrin adhesions with cell cycle progression.

scholarly journals Viperin inhibits hepatitis C virus replication by interfering with binding of NS5A to host protein hVAP-33

2012 ◽  
Vol 93 (1) ◽  
pp. 83-92 ◽  
Author(s):  
Shanshan Wang ◽  
Xianfang Wu ◽  
Tingting Pan ◽  
Wuhui Song ◽  
Yaohui Wang ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Microscopic Analysis ◽  
Underlying Mechanism ◽  
Host Protein ◽  
Type I ◽  
Dependent Manner ◽  
C Terminus ◽  
Dose Dependent

Viperin is a type-I and -II interferon-inducible intracytoplasmic protein that mediates antiviral activity against several viruses. A previous study has reported that viperin could limit hepatitis C virus (HCV) replication in vitro. However, the underlying mechanism remains elusive. In the present study, we found that overexpression of viperin could inhibit HCV replication in a dose-dependent manner in both the replicon and HCVcc systems. Furthermore, through co-immunoprecipitation and laser confocal microscopic analysis, viperin was found to interact with the host protein hVAP-33. Mutagenesis analysis demonstrated that the anti-HCV activity of viperin was located to its C terminus, which was required for the interaction with the C-terminal domain of hVAP-33. Competitive co-immunoprecipitation analysis showed that viperin could interact competitively with hVAP-33, and could therefore interfere with its interactions with HCV NS5A. In summary, these findings suggest a novel mechanism by which viperin inhibits HCV replication, possibly through binding to host protein hVAP-33 and interfering with its interaction with NS5A.

scholarly journals Selective and strain-specific NFAT4 activation by the Toxoplasma gondii polymorphic dense granule protein GRA6

2014 ◽  
Vol 211 (10) ◽  
pp. 2013-2032 ◽  
Author(s):  
Ji Su Ma ◽  
Miwa Sasai ◽  
Jun Ohshima ◽  
Youngae Lee ◽  
Hironori Bando ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Dense Granule ◽  
Type I ◽  
Dependent Manner ◽  
Activated T Cells ◽  
Host Immune Responses ◽  
Dense Granules ◽  
Granule Protein ◽  
C Terminus ◽  
Toxoplasma Gondii Infection

Toxoplasma gondii infection results in co-option and subversion of host cellular signaling pathways. This process involves discharge of T. gondii effector molecules from parasite secretory organelles such as rhoptries and dense granules. We report that the T. gondii polymorphic dense granule protein GRA6 regulates activation of the host transcription factor nuclear factor of activated T cells 4 (NFAT4). GRA6 overexpression robustly and selectively activated NFAT4 via calcium modulating ligand (CAMLG). Infection with wild-type (WT) but not GRA6-deficient parasites induced NFAT4 activation. Moreover, GRA6-deficient parasites failed to exhibit full virulence in local infection, and the treatment of WT mice with an NFAT inhibitor mitigated virulence of WT parasites. Notably, NFAT4-deficient mice displayed prolonged survival, decreased recruitment of CD11b+ Ly6G+ cells to the site of infection, and impaired expression of chemokines such as Cxcl2 and Ccl2. In addition, infection with type I parasites culminated in significantly higher NFAT4 activation than type II parasites due to a polymorphism in the C terminus of GRA6. Collectively, our data suggest that GRA6-dependent NFAT4 activation is required for T. gondii manipulation of host immune responses to maximize the parasite virulence in a strain-dependent manner.

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