Role of protein phosphatases in the activation of CFTR (ABCC7) by genistein and bromotetramisole

2000 ◽  
Vol 279 (1) ◽  
pp. C108-C119 ◽  
Author(s):  
Jiexin Luo ◽  
Tang Zhu ◽  
Alexandra Evagelidis ◽  
Mary D. Pato ◽  
John W. Hanrahan
Keyword(s):  
Protein Phosphatase ◽  
Chloride Channels ◽  
Protein Phosphatases ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Airway Epithelial Cells ◽  
Transmembrane Conductance Regulator ◽  
Airway Epithelial ◽  
Ovary Cells

Genistein and bromotetramisole (Br-t) strongly activate cystic fibrosis transmembrane conductance regulator (CFTR; ABCC7) chloride channels on Chinese hamster ovary cells and human airway epithelial cells. We have examined the possible role of phosphatases in stimulation by these drugs using patch-clamp and biochemical methods. Genistein inhibited the spontaneous rundown of channel activity that occurs after membrane patches are excised from cAMP-stimulated cells but had no effect on purified protein phosphatase type 1 (PP1), PP2A, PP2B, PP2C, or endogenous phosphatases when assayed as [32P]PO4release from prelabeled casein, recombinant GST-R domain fusion protein, or immunoprecipitated full-length CFTR. Br-t also slowed rundown of CFTR channels, but, in marked contrast to genistein, it did inhibit all four protein phosphatases tested. Half-maximal inhibition of PP2A and PP2C was observed with 0.5 and 1.5 mM Br-t, respectively. Protein phosphatases were also sensitive to (+)- p-Br-t, a stereoisomer of Br-t that does not inhibit alkaline phosphatases. Br-t appeared to act exclusively through phosphatases since it did not affect CFTR channels in patches that had low apparent endogenous phosphatase activity (i.e., those lacking spontaneous rundown). We conclude that genistein and Br-t act through different mechanisms. Genistein stimulates CFTR without inhibiting phosphatases, whereas Br-t acts by inhibiting a membrane-associated protein phosphatase (probably PP2C) that presumably allows basal phosphorylation to accumulate.

Activation of NF-κB in airway epithelial cells is dependent on CFTR trafficking and Cl− channel function

2001 ◽  
Vol 281 (1) ◽  
pp. L71-L78 ◽  
Author(s):  
Adam J. Weber ◽  
Grace Soong ◽  
Ruth Bryan ◽  
Shahryar Saba ◽  
Alice Prince
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Airway Epithelial Cells ◽  
Cell Stress ◽  
Airway Epithelial ◽  
Ovary Cells ◽  
Channel Function ◽  
Cftr Mutations ◽  
Δf508 Cftr ◽  
Cf Transmembrane Conductance Regulator

Polymorphonuclear leukocyte-dominated airway inflammation is a major component of cystic fibrosis (CF) lung disease and may be associated with CF transmembrane conductance regulator (CFTR) dysfunction as well as infection. Mutant ΔF508 CFTR is mistrafficked, accumulates in the endoplasmic reticulum (ER), and may cause “cell stress” and activation of nuclear factor (NF)-κB. G551D mutants also lack Cl− channel function, but CFTR is trafficked normally. We compared the effects of CFTR mutations on the endogenous activation of an NF-κB reporter construct. In transfected Chinese hamster ovary cells, the mistrafficked ΔF508 allele caused a sevenfold activation of NF-κB compared with wild-type CFTR or the G551D mutant ( P < 0.001). NF-κB was also activated in 9/HTEo−/pCep-R cells and in 16HBE/p cftrantisense cell lines, which lack CFTR Cl− channel function but do not accumulate mutant protein in the ER. This endogenous activation of NF-κB was associated with elevated interleukin-8 expression. Impaired CFTR Cl− channel activity as well as cell stress due to accumulation of mistrafficked CFTR in the ER contributes to the endogenous activation of NF-κB in cells with the CFTR mutation.

Abstract 11947: Unraveling the Mechanism for the Stimulatory Role of Platelet GPIb-IX-V in Thrombin-Induced Platelet Activation

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Brian Estevez ◽  
Michael K Delaney ◽  
Aleksandra Stojanovic-Terpo ◽  
Xiaoping Du
Keyword(s):  
Platelet Activation ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Human Platelets ◽  
Platelet Glycoprotein ◽  
Inhibitory Peptide ◽  
Protease Activated Receptors ◽  
Signaling Mechanism ◽  
Ovary Cells

Numerous reports indicate that the platelet glycoprotein (GP) Ib-IX complex (GPIb-IX) binds directly to the potent platelet agonist thrombin and is important for promoting thrombin-induced platelet activation. However, how GPIb-IX contributes to thrombin-induced platelet activation is unclear. It has been suggested that thrombin binding to GPIb facilitates the cleavage, and thus activation, of the protease-activated receptors (PAR). Our data indicate that GPIb-IX promotes thrombin signaling through a GPIb-IX signaling mechanism. Pretreatment of human platelets with MPalphaC, an inhibitory peptide based on a critical 14-3-3 signaling protein binding site on the cytoplasmic domain of the GPIb alpha chain, inhibited thrombin-induced platelet activation. MPalphaC-treatment inhibited thrombin-induced activation of Rac1 and LIMK1, both of which are known to play essential roles in GPIb signaling. To more specifically determine the role of GPIb-IX, we reconstituted GPIb-IX-facilitated thrombin signaling in Chinese Hamster Ovary cells expressing PAR1. Thrombin induced signaling was significantly enhanced by GPIb-expression, and deletion of the cytoplasmic 14-3-3-binding domain of GPIb alpha abolished the stimulatory effect of GPIb on thrombin signaling. Furthermore, the role of GPIb-IX in promoting thrombin signaling requires Rac1, and GPIb-IX-dependent Rac1 activation and LIMK phosphorylation are abolished in delta 605 cells expressing a 14-3-3-binding defective mutant GPIb alpha. Taken together, these data suggest that the stimulatory role of GPIb in thrombin signaling requires a C-terminal 14-3-3-binding region which mediates activation of a Rac1/LIMK1 pathway that promotes thrombin signaling leading to platelet activation.

Reduction of endogenous GRP78 levels improves secretion of a heterologous protein in CHO cells

1988 ◽  
Vol 8 (10) ◽  
pp. 4063-4070
Author(s):  
A J Dorner ◽  
M G Krane ◽  
R J Kaufman
Keyword(s):  
Endoplasmic Reticulum ◽  
Chinese Hamster Ovary ◽  
Heterologous Protein ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Glycosylation Site ◽  
Ovary Cells ◽  
Tissue Plasminogen ◽  
Stable Association

GRP78 is localized in the endoplasmic reticulum and associates with improperly folded or underglycosylated proteins. The role of GRP78 in secretion was studied in Chinese hamster ovary cells expressing a tissue plasminogen activator (tPA) variant which lacks potential N-linked glycosylation site sequences because of mutagenesis. The expression of variant tPA resulted in elevated levels of GRP78 and its stable association with tPA. The introduction of antisense GRP78 genes resulted in a two- to threefold reduction in GRP78 levels compared with those of the original cells. Cells with reduced levels of GRP78 secreted two- to threefold-higher levels of tPA activity. tPA expressed in these cells displayed reduced association with GRP78, and a greater proportion was processed to the mature form and secreted. These results demonstrate that reduction of GRP78 level can improve the secretion of an associated protein.

Role of the α-subunit326GRV sequence in the surface expression of fibrinogen and vitronectin receptors

1998 ◽  
Vol 275 (5) ◽  
pp. C1239-C1246 ◽  
Author(s):  
Milagros Ferrer ◽  
Matilde S. Ayuso ◽  
Nora Butta ◽  
Roberto Parrilla ◽  
Consuelo González-Manchón
Keyword(s):  
Functional Role ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Surface Expression ◽  
Platelet Surface ◽  
Α Subunit ◽  
Vitronectin Receptor ◽  
Ovary Cells ◽  
High Affinity

The platelet GPIIb-GPIIIa heterodimer (integrin αIIbβ3) binds fibrinogen with high affinity in response to activation by agonists, leading to platelet aggregation and formation of a hemostatic plug. The326GRV motif in GPIIb is highly conserved in the α-subunit of other integrins, suggesting that it might play an important functional role. Moreover, Arg327→His substitution in GPIIb has been associated with defective platelet surface expression of GPIIb-IIIa and thrombasthenic phenotype. This work aimed at elucidating whether the absence of Arg327or its substitution by His was responsible for the impaired surface expression of GPIIb-IIIa complexes. Transfection of cDNA encoding [Ala327]GPIIb, [Gln327]GPIIb, or [Phe327]GPIIb into Chinese hamster ovary cells inherently expressing GPIIIa permitted surface exposure of GPIIb-IIIa complexes, whereas [Glu327]GPIIb did not. These observations indicate that it is not the loss of [Arg327]GPIIb but the presence of His327or a negatively charged residue like Glu at position 327 of GPIIb that prevents the surface exposure of GPIIb-IIIa heterodimers. In contrast, changing Gln344, the homologue to Arg327in the α-subunit of the vitronectin receptor, to His did not prevent the surface expression of αv-GPIIIa complexes. Thus the conformational constraint imposed by His327seems to be rather specific for the heterodimerization and/or processing of GPIIb-IIIa complexes.

The role of O6-alkylguanine in cell killing and mutagenesis in Chinese hamster ovary cells

1991 ◽  
Vol 12 (1) ◽  
pp. 83-89 ◽  
Author(s):  
William C. Dunn ◽  
Keizo Tano ◽  
Gregory J. Horesovsky ◽  
R.Julian Preston ◽  
Sankar Mitra
Keyword(s):  
Chinese Hamster Ovary ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Cell Killing ◽  
Ovary Cells

The Role of Ion Electrophoresis in Electroporation-Mediated Molecular Delivery

2009 ◽  
Author(s):  
Jianbo Li ◽  
Hao Lin
Keyword(s):  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Stem Cell Differentiation ◽  
Equation System ◽  
Cellular Membrane ◽  
Dominant Mode ◽  
Ovary Cells ◽  
Fluorescence Measurements ◽  
Molecular Delivery

Electroporation is a widely applied technique to deliver active molecules into the cellular compartment, to perform tasks such as gene therapy and directed stem cell differentiation, among many others. In this technique, an electric field transiently permeabilizes the cellular membrane to facilitate molecular exchange. While the permeabilization process is relatively well understood, the transport mechanisms for molecular delivery are still under debate. In this work, the role of ion electrophoresis in electroporation-mediated molecular delivery is investigated using numerical simulation. The Nernst-Planck equations for ionic transport in the extracellular and intracellular spaces are solved, respectively, and are coupled through a permeabilization model on the membrane. For the latter, an asymptotic Smoluchowski equation system is adopted, following the work of Krassowska and co-authors. The simulation is used to investigate the delivery of calcium ions into Chinese hamster ovary cells. The results indicate that ion electrophoresis is the dominant mode of transport in the delivery of small charged molecules. Furthermore, the achievable intracellular concentration is strongly influenced by the conductivity difference between the cytoplasm and the buffer, a phenomenon known as “field-amplified sample stacking”. The results agree qualitatively with the fluorescence measurements by Gabriel and Teissie´ (1999), and suggest a new possibility to simultaneously improve cell viability and efficiency in electroporation-mediated molecular delivery.

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