Rho-dependent and -independent activation mechanisms of ezrin/radixin/moesin proteins: an essential role for polyphosphoinositides in vivo

2002 ◽  
Vol 115 (12) ◽  
pp. 2569-2580 ◽  
Author(s):  
Shigenobu Yonemura ◽  
Takeshi Matsui ◽  
Shoichiro Tsukita ◽  
Sachiko Tsukita
Keyword(s):  
Plasma Membranes ◽  
Kinase Inhibitor ◽  
Mdck Cells ◽  
Aminoglycoside Antibiotic ◽  
Activation Mechanism ◽  
Dependent Manner ◽  
A431 Cells ◽  
Erm Proteins ◽  
Protein Activation

Ezrin/radixin/moesin (ERM) proteins crosslink actin filaments to plasma membranes and are involved in the organization of the cortical cytoskeleton,especially in the formation of microvilli. ERM proteins are reported to be activated as crosslinkers in a Rho-dependent manner and are stabilized when phosphorylated at their C-terminal threonine residue to create C-terminal threonine-phosphorylated ERM proteins (CPERMs). Using a CPERM-specific mAb, we have shown, in vivo, that treatment with C3 transferase (a Rho inactivator) or staurosporine (a protein kinase inhibitor) leads to the dephosphorylation of CPERMs, the translocation of ERM proteins from plasma membranes to the cytoplasm and microvillar breakdown. We further elucidated that ERM protein activation does not require C-terminal phosphorylation in A431 cells stimulated with epidermal growth factor. In certain types of kidney-derived cells such as MDCK cells, however, ERM proteins appear to be activated in the absence of Rho activation and remain active without C-terminal phosphorylation. Interestingly, microinjection of an aminoglycoside antibiotic, neomycin, which binds to polyphosphoinositides, such as phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2],affected the activation of ERM proteins regardless of cell type. These findings not only indicate the existence of a Rho-independent activation mechanism of ERM proteins but also suggest that both Rho-dependent and-independent activation of ERM proteins require a local elevation of PtdIns(4,5)P2 concentration in vivo.

Specific coupling of a cation-sensing receptor to G protein alpha-subunits in MDCK cells

1997 ◽  
Vol 273 (1) ◽  
pp. F129-F135 ◽  
Author(s):  
J. M. Arthur ◽  
G. P. Collinsworth ◽  
T. W. Gettys ◽  
L. D. Quarles ◽  
J. R. Raymond
Keyword(s):  
G Protein ◽  
G Proteins ◽  
Plasma Membranes ◽  
Mdck Cells ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cellular Activation ◽  
Protein Activation ◽  
A Cell ◽  
Cation Sensing

Extracellular cations such as Ca2+ stimulate a G protein-coupled, cation-sensing receptor (CaR). We used microphysiometry to determine whether an extracellular cation-sensing mechanism exists in Madin-Darby canine kidney (MDCK) cells. The CaR agonists Ca2+ and Gd3+ caused cellular activation in a concentration-dependent manner. mRNA for the CaR was identified by reverse transcription and polymerase chain reaction (PCR) using nested CaR-specific primers, identification of an appropriately located restriction site, and sequencing of the subcloned fragment obtained by PCR. G protein activation was evaluated using the GTP photoaffinity label [alpha-32P]GTP azidoanalide (AA-GTP). After stimulation with Gd3+ and cross-linking, plasma membranes were solubilized and immunoprecipitated with antisera specific for Gq/11 alpha and Gi alpha family members. Gd3+ increased incorporation of AA-GTP into Gq/11 alpha precipitates by 146 +/- 48% and into G alpha i-2 and G alpha i-3 to a lesser extent but not into G alpha i-1. Direct effects of Gd3+ on the G proteins were ruled out using partially purified mammalian G proteins expressed in Escherichia coli or Sf9 cells. We conclude that MDCK cells possess a cell-surface CaR that activates Gq/11 alpha, G alpha i-2, and G alpha i-3 but not G alpha i-1.

Immunofluorescence detection of ezrin/radixin/moesin (ERM) proteins with their carboxyl-terminal threonine phosphorylated in cultured cells and tissues

1999 ◽  
Vol 112 (8) ◽  
pp. 1149-1158 ◽  
Author(s):  
K. Hayashi ◽  
S. Yonemura ◽  
T. Matsui ◽  
S. Tsukita
Keyword(s):  
Plasma Membranes ◽  
Immunofluorescence Microscopy ◽  
Cultured Cells ◽  
Tissue Type ◽  
Cross Linking ◽  
Dependent Manner ◽  
Cortical Actin ◽  
Erm Proteins

Ezrin/radixin/moesin (ERM) proteins are thought to play an important role in organizing cortical actin-based cytoskeletons through cross-linkage of actin filaments with integral membrane proteins. Recent in vitro biochemical studies have revealed that ERM proteins phosphorylated on their COOH-terminal threonine residue (CPERMs) are active in their cross-linking activity, but this has not yet been evaluated in vivo. To immunofluorescently visualize CPERMs in cultured cells as well as tissues using a mAb specific for CPERMs, we developed a new fixation protocol using trichloroacetic acid (TCA) as a fixative. Immunoblotting analyses in combination with immunofluorescence microscopy showed that TCA effectively inactivated soluble phosphatases, which maintained the phosphorylation level of CPERMs during sample processing for immunofluorescence staining. Immunofluorescence microscopy with TCA-fixed samples revealed that CPERMs were exclusively associated with plasma membranes in a variety of cells and tissues, whereas total ERM proteins were distributed in both the cytoplasm and plasma membranes. Furthermore, the amounts of CPERMs were shown to be regulated in a cell and tissue type-dependent manner. These findings favored the notion that phosphorylation of the COOH-terminal threonine plays a key role in the regulation of the cross-linking activity of ERM proteins in vivo.

scholarly journals A Novel Transcriptional Repression Domain Mediates p21WAF1/CIP1 Induction of p300 Transactivation

2000 ◽  
Vol 20 (8) ◽  
pp. 2676-2686 ◽  
Author(s):  
Andrew W. Snowden ◽  
Lisa A. Anderson ◽  
Gill A. Webster ◽  
Neil D. Perkins
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Transcriptional Repression ◽  
Kinase Inhibitor ◽  
Core Promoter ◽  
Dependent Manner ◽  
Creb Binding Protein ◽  
Cycle Dependent ◽  
Repression Domain

ABSTRACT The transcriptional coactivators p300 and CREB binding protein (CBP) are important regulators of the cell cycle, differentiation, and tumorigenesis. Both p300 and CBP are targeted by viral oncoproteins, are mutated in certain forms of cancer, are phosphorylated in a cell cycle-dependent manner, interact with transcription factors such as p53 and E2F, and can be found complexed with cyclinE-Cdk2 in vivo. Moreover, p300-deficient cells show defects in proliferation. Here we demonstrate that transcriptional activation by both p300 and CBP is stimulated by coexpression of the cyclin-dependent kinase inhibitor p21WAF/CIP1. Significantly this stimulation is independent of both the inherent histone acetyltransferase (HAT) activity of p300 and CBP and of the previously reported carboxyl-terminal binding site for cyclinE-Cdk2. Rather, we describe a previously uncharacterized transcriptional repression domain (CRD1) within p300. p300 transactivation is stimulated through derepression of CRD1 by p21. Significantly p21 regulation of CRD1 is dependent on the nature of the core promoter. We suggest that CRD1 provides a novel mechanism through which p300 and CBP can switch activities between the promoters of genes that stimulate growth and those that enhance cell cycle arrest.

scholarly journals Coelonin, an Anti-Inflammation Active Component of Bletilla striata and Its Potential Mechanism

2019 ◽  
Vol 20 (18) ◽  
pp. 4422 ◽  
Author(s):  
Fusheng Jiang ◽  
Meiya Li ◽  
Hongye Wang ◽  
Bin Ding ◽  
Chunchun Zhang ◽  
...  
Keyword(s):  
Molecular Mechanism ◽  
Kinase Inhibitor ◽  
Negative Regulator ◽  
Antibody Array ◽  
Dependent Manner ◽  
Active Ingredients ◽  
Bletilla Striata ◽  
Tnf Α ◽  
Anti Inflammatory

Ethanol extract of Bletilla striata has remarkable anti-inflammatory and anti-pulmonary fibrosis activities in the rat silicosis model. However, its active substances and molecular mechanism are still unclear. To uncover the active ingredients and potential molecular mechanism of the Bletilla striata extract, the lipopolysaccharide (LPS)-induced macrophage inflammation model and phospho antibody array were used. Coelonin, a dihydrophenanthrene compound was isolated and identified. It significantly inhibited LPS-induced interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) expression at 2.5 μg/mL. The microarray data indicate that the phosphorylation levels of 32 proteins in the coelonin pre-treated group were significantly down-regulated. In particular, the phosphorylation levels of the key inflammatory regulators factor nuclear factor-kappa B (NF-κB) were significantly reduced, and the negative regulator phosphatase and tensin homologue on chromosome ten (PTEN) was reduced. Moreover, the phosphorylation level of cyclin dependent kinase inhibitor 1B (p27Kip1), another downstream molecule regulated by PTEN was also reduced significantly. Western blot and confocal microscopy results confirmed that coelonin inhibited LPS-induced PTEN phosphorylation in a dose-dependent manner, then inhibited NF-κB activation and p27Kip1 degradation by regulating the phosphatidylinositol-3-kinases/ v-akt murine thymoma viral oncogene homolog (PI3K/AKT) pathway negatively. However, PTEN inhibitor co-treatment analysis indicated that the inhibition of IL-1β, IL-6 and TNF-α expression by coelonin was independent of PTEN, whereas the inhibition of p27Kip1 degradation resulted in cell-cycle arrest in the G1 phase, which was dependent on PTEN. The anti-inflammatory activity of coelonin in vivo, which is one of the main active ingredients of Bletilla striata, deserves further study.

scholarly journals RacG Regulates Morphology, Phagocytosis, and Chemotaxis

2006 ◽  
Vol 5 (10) ◽  
pp. 1648-1663 ◽  
Author(s):  
Baggavalli P. Somesh ◽  
Georgia Vlahou ◽  
Miho Iijima ◽  
Robert H. Insall ◽  
Peter Devreotes ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Actin Polymerization ◽  
Kinase Inhibitor ◽  
Dominant Negative ◽  
Dependent Manner ◽  
Free System ◽  
Mild Phenotype ◽  
Particle Uptake ◽  
Phosphoinositide 3 Kinase

ABSTRACTRacG is an unusual member of the complex family of Rho GTPases inDictyostelium. We have generated a knockout (KO) strain, as well as strains that overexpress wild-type (WT), constitutively active (V12), or dominant negative (N17) RacG. The protein is targeted to the plasma membrane, apparently in a nucleotide-dependent manner, and induces the formation of abundant actin-driven filopods. RacG is enriched at the rim of the progressing phagocytic cup, and overexpression of RacG-WT or RacG-V12 induced an increased rate of particle uptake. The positive effect of RacG on phagocytosis was abolished in the presence of 50 μM LY294002, a phosphoinositide 3-kinase inhibitor, indicating that generation of phosphatidylinositol 3,4,5-trisphosphate is required for activation of RacG. RacG-KO cells showed a moderate chemotaxis defect that was stronger in the RacG-V12 and RacG-N17 mutants, in part because of interference with signaling through Rac1. The in vivo effects of RacG-V12 could not be reproduced by a mutant lacking the Rho insert region, indicating that this region is essential for interaction with downstream components. Processes like growth, pinocytosis, exocytosis, cytokinesis, and development were unaffected in Rac-KO cells and in the overexpressor mutants. In a cell-free system, RacG induced actin polymerization upon GTPγS stimulation, and this response could be blocked by an Arp3 antibody. While the mild phenotype of RacG-KO cells indicates some overlap with one or moreDictyosteliumRho GTPases, like Rac1 and RacB, the significant changes found in overexpressors show that RacG plays important roles. We hypothesize that RacG interacts with a subset of effectors, in particular those concerned with shape, motility, and phagocytosis.

Generation and analyses of R8L barttin knockin mouse

2011 ◽  
Vol 301 (2) ◽  
pp. F297-F307 ◽  
Author(s):  
Naohiro Nomura ◽  
Masato Tajima ◽  
Noriko Sugawara ◽  
Tetsuji Morimoto ◽  
Yoshiaki Kondo ◽  
...  
Keyword(s):  
Chloride Channels ◽  
Plasma Membranes ◽  
Chloride Transport ◽  
Mdck Cells ◽  
Intracellular Localization ◽  
Bartter Syndrome ◽  
K Channel ◽  
Type Iv ◽  
Knockin Mouse

Barttin, a gene product of BSND, is one of four genes responsible for Bartter syndrome. Coexpression of barttin with ClC-K chloride channels dramatically induces the expression of ClC-K current via insertion of ClC-K-barttin complexes into plasma membranes. We previously showed that stably expressed R8L barttin, a disease-causing missense mutant, is retained in the endoplasmic reticulum (ER) of Madin-Darby canine kidney (MDCK) cells, with the barttin β-subunit remaining bound to ClC-K α-subunits (Hayama A, Rai T, Sasaki S, Uchida S. Histochem Cell Biol 119: 485–493, 2003). However, transient expression of R8L barttin in MDCK cells was reported to impair ClC-K channel function without affecting its subcellular localization. To investigate the pathogenesis in vivo, we generated a knockin mouse model of Bartter syndrome that carries the R8L mutation. These mice display disease-like phenotypes (hypokalemia, metabolic alkalosis, and decreased NaCl reabsorption in distal tubules) under a low-salt diet. Immunofluorescence and immunoelectron microscopy revealed that the plasma membrane localization of both R8L barttin and the ClC-K channel was impaired in these mice, and transepithelial chloride transport in the thin ascending limb of Henle's loop (tAL) as well as thiazide-sensitive chloride clearance were significantly reduced. This reduction in transepithelial chloride transport in tAL, which is totally dependent on ClC-K1/barttin, correlated well with the reduction in the amount of R8L barttin localized to plasma membranes. These results suggest that the major cause of Bartter syndrome type IV caused by R8L barttin mutation is its aberrant intracellular localization.

scholarly journals Dasatinib increases endothelial permeability leading to pleural effusion

2018 ◽  
Vol 51 (1) ◽  
pp. 1701096 ◽  
Author(s):  
Carole Phan ◽  
Etienne-Marie Jutant ◽  
Ly Tu ◽  
Raphaël Thuillet ◽  
Andrei Seferian ◽  
...  
Keyword(s):  
Pleural Effusion ◽  
Kinase Inhibitor ◽  
Umbilical Vein ◽  
Endothelial Permeability ◽  
Cell Junctions ◽  
Dependent Manner ◽  
Vascular Endothelial ◽  
Umbilical Vein Endothelial Cells

Pleural effusion is a frequent side-effect of dasatinib, a second-generation tyrosine kinase inhibitor used in the treatment of chronic myelogenous leukaemia. However, the underlying mechanisms remain unknown. We hypothesised that dasatinib alters endothelial integrity, resulting in increased pulmonary vascular endothelial permeability and pleural effusion.To test this, we established the first animal model of dasatinib-related pleural effusion, by treating rats with a daily regimen of high doses of dasatinib (10 mg·kg−1·day−1 for 8 weeks).Pleural ultrasonography revealed that rats chronically treated with dasatinib developed pleural effusion after 5 weeks. Consistent with these in vivo observations, dasatinib led to a rapid and reversible increase in paracellular permeability of human pulmonary endothelial cell monolayers as reflected by increased macromolecule passage, loss of vascular endothelial cadherin and zonula occludens-1 from cell–cell junctions, and the development of actin stress fibres. These results were replicated using human umbilical vein endothelial cells and confirmed by decreased endothelial resistance. Interestingly, we demonstrated that this increased endothelial permeability is a reactive oxygen species (ROS)-dependent mechanism in vitro and in vivo using a cotreatment with an antioxidant agent, N-acetylcysteine.This study shows that dasatinib alters pulmonary endothelial permeability in a ROS-dependent manner in vitro and in vivo leading to pleural effusion.

Myo-inositol does not modulate PI turnover in MDCK cells under hyperosmolar conditions

1990 ◽  
Vol 258 (5) ◽  
pp. F1282-F1287
Author(s):  
J. A. Shayman ◽  
D. Wu
Keyword(s):  
Plasma Membranes ◽  
Mdck Cells ◽  
Inositol Trisphosphate ◽  
Enzyme Reaction ◽  
Gas Liquid Chromatography ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Competitive Binding Assay ◽  
Pi Turnover ◽  
Phosphatidylinositol Turnover

The relationship between free cellular myo-inositol concentration and phosphatidylinositol turnover was evaluated in Madin-Darby canine kidney (MDCK) cells under isosmolar and hyperosmolar conditions. MDCK cells exposed to high extracellular sodium chloride were documented to increase their free myo-inositol content as measured by gas-liquid chromatography in both a time- and concentration-dependent manner. Measurement of phosphatidylinositol, phosphatidylinositol 4-monophosphate, and phosphatidylinositol 4,5-bisphosphate mass failed to reveal changes under conditions where the myo-inositol concentration was more than threefold higher compared with control conditions. CDP diacylglycerol:myo-inositol 3-phosphatidyltransferase activity, measured in plasma membranes from MDCK cells grown under control and hyperosmolar conditions, was kinetically similar with comparable observed Michaelis constant (Km) and maximal rate of enzyme reaction. Moreover, the apparent Km was significantly below the estimated intracellular myo-inositol concentration consistent with exposure of the transferase to saturating concentrations of myo-inositol under both conditions. Finally, bradykinin-stimulated myo-inositol trisphosphate mass was measured by use of a competitive binding assay. Both basal and hormone-stimulated myo-inositol 1,4,5-trisphosphate levels were not significantly different under control and hyperosmolar conditions. These data indicate that bulk free myo-inositol content is unlikely to regulate phosphatidylinositol turnover and myo-inositol trisphosphate formation under hyperosmolar conditions.

scholarly journals The pan-class I phosphatidyl-inositol-3 kinase inhibitor NVP-BKM120 demonstrates anti-leukemic activity in acute myeloid leukemia

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Matteo Allegretti ◽  
Maria Rosaria Ricciardi ◽  
Roberto Licchetta ◽  
Simone Mirabilii ◽  
Stefania Orecchioni ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Phosphatidyl Inositol ◽  
Class I ◽  
Clinical Activity ◽  
Dependent Manner ◽  
Acute Myeloid

Abstract Aberrant activation of the PI3K/Akt/mTOR pathway is a common feature of acute myeloid leukemia (AML) patients contributing to chemoresistance, disease progression and unfavourable outcome. Therefore, inhibition of this pathway may represent a potential therapeutic approach in AML. The aim of this study was to evaluate the pre-clinical activity of NVP-BKM120 (BKM120), a selective pan-class I PI3K inhibitor, on AML cell lines and primary samples. Our results demonstrate that BKM120 abrogates the activity of the PI3K/Akt/mTOR signaling, promoting cell growth arrest and significant apoptosis in a dose- and time-dependent manner in AML cells but not in the normal counterpart. BKM120-induced cytotoxicity is associated with a profound modulation of metabolic behaviour in both cell lines and primary samples. In addition, BKM120 synergizes with the glycolitic inhibitor dichloroacetate enhancing apoptosis induction at lower doses. Finally, in vivo administration of BKM120 to a xenotransplant mouse model of AML significantly inhibited leukemia progression and improved the overall survival of treated mice. Taken together, our findings indicate that BKM120, alone or in combination with other drugs, has a significant anti-leukemic activity supporting its clinical development as a novel therapeutic agent in AML.

scholarly journals Rho kinase inhibitor HA-1077 prevents Rho-mediated myosin phosphatase inhibition in smooth muscle cells

2000 ◽  
Vol 278 (1) ◽  
pp. C57-C65 ◽  
Author(s):  
Hiromitsu Nagumo ◽  
Yasuharu Sasaki ◽  
Yoshitaka Ono ◽  
Hiroyuki Okamoto ◽  
Minoru Seto ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Phosphatase Activity ◽  
Kinase Inhibitor ◽  
Rho Kinase ◽  
Muscle Cells ◽  
Dependent Manner ◽  
Myosin Phosphatase ◽  
Rho Kinase Inhibitor

In smooth muscle, a Rho-regulated system of myosin phosphatase exists; however, it has yet to be established whether Rho kinase, one of the downstream effectors of Rho, mediates the regulation of myosin phosphatase activity in vivo. In the present study, we demonstrate in permeabilized vascular smooth muscle cells (SMCs) that the vasodilator 1-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077), which we show to be a potent inhibitor of Rho kinase, dose dependently inhibits Rho-mediated enhancement of Ca2+-induced 20-kDa myosin light chain (MLC20) phosphorylation due to abrogating Rho-mediated inhibition of MLC20dephosphorylation. By an immune complex phosphatase assay, we found that guanosine 5′- O-(3-thiotriphosphate) (GTPγS) stimulation of permeabilized SMCs caused a decrease in myosin phosphatase activity with an increase in the extent of phosphorylation of the 130-kDa myosin-binding regulatory subunit (MBS) of myosin phosphatase in a Rho-dependent manner. HA-1077 abolished both of the Rho-mediated events. Moreover, we observed that the pleckstrin homology/cystein-rich domain protein of Rho kinase, a dominant negative inhibitor of Rho kinase, inhibited GTPγS-induced phosphorylation of MBS. These results provide direct in vivo evidence that Rho kinase mediates inhibition of myosin phosphatase activity with resultant enhancement of MLC20phosphorylation in smooth muscle and reveal the usefulness of HA-1077 as a Rho kinase inhibitor.

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