scholarly journals Acetaldehyde dissociates the PTP1B–E-cadherin–β-catenin complex in Caco-2 cell monolayers by a phosphorylation-dependent mechanism

2007 ◽  
Vol 402 (2) ◽  
pp. 291-300 ◽  
Author(s):  
Parimal Sheth ◽  
Ankur Seth ◽  
Katherine J. Atkinson ◽  
Tarun Gheyi ◽  
Gautam Kale ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Phosphatase ◽  
Direct Interaction ◽  
Intercellular Junctions ◽  
Glutathione S Transferase ◽  
Binding Studies ◽  
Cell Monolayers ◽  
Dependent Mechanism ◽  
Ptpase Activity ◽  
E Cadherin

Interactions between E-cadherin, β-catenin and PTP1B (protein tyrosine phosphatase 1B) are crucial for the organization of AJs (adherens junctions) and epithelial cell–cell adhesion. In the present study, the effect of acetaldehyde on the AJs and on the interactions between E-cadherin, β-catenin and PTP1B was determined in Caco-2 cell monolayers. Treatment of cell monolayers with acetaldehyde induced redistribution of E-cadherin and β-catenin from the intercellular junctions by a tyrosine phosphorylation-dependent mechanism. The PTPase activity associated with E-cadherin and β-catenin was significantly reduced and the interaction of PTP1B with E-cadherin and β-catenin was attenuated by acetaldehyde. Acetaldehyde treatment resulted in phosphorylation of β-catenin on tyrosine residues, and abolished the interaction of β-catenin with E-cadherin by a tyrosine kinase-dependent mechanism. Protein binding studies showed that the treatment of cells with acetaldehyde reduced the binding of β-catenin to the C-terminal region of E-cadherin. Pairwise binding studies using purified proteins indicated that the direct interaction between E-cadherin and β-catenin was reduced by tyrosine phosphorylation of β-catenin, but was unaffected by tyrosine phosphorylation of E-cadherin-C. Treatment of cells with acetaldehyde also reduced the binding of E-cadherin to GST (glutathione S-transferase)–PTP1B. The pairwise binding study showed that GST–E-cadherin-C binds to recombinant PTP1B, but this binding was significantly reduced by tyrosine phosphorylation of E-cadherin. Acetaldehyde increased the phosphorylation of β-catenin on Tyr-331, Tyr-333, Tyr-654 and Tyr-670. These results show that acetaldehyde induces disruption of interactions between E-cadherin, β-catenin and PTP1B by a phosphorylation-dependent mechanism.

scholarly journals ERK is involved in EGF-mediated protection of tight junctions, but not adherens junctions, in acetaldehyde-treated Caco-2 cell monolayers

2011 ◽  
Vol 301 (1) ◽  
pp. G50-G59 ◽  
Author(s):  
G. Samak ◽  
S. Aggarwal ◽  
R. K. Rao
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tight Junctions ◽  
P38 Mapk ◽  
Adherens Junctions ◽  
Intercellular Junctions ◽  
Dominant Negative ◽  
Cell Monolayers ◽  
Mitogen Activated Protein ◽  
Jnk Inhibitors ◽  
E Cadherin

The role of mitogen-activated protein kinases (MAPK) in the mechanism of EGF-mediated prevention of acetaldehyde-induced tight junction disruption was evaluated in Caco-2 cell monolayers. Pretreatment of cell monolayers with EGF attenuated acetaldehyde-induced decrease in resistance and increase in inulin permeability and redistribution of occludin, zona occludens-1 (ZO-1), E-cadherin, and β-catenin from the intercellular junctions. EGF rapidly increased the levels of phospho-ERK1/2, phospho-p38 MAPK, and phospho-JNK1. Pretreatment of cell monolayers with U-0126 (inhibitor of ERK activation), but not SB-202190 and SP-600125 (p38 MAPK and JNK inhibitors), significantly attenuated EGF-mediated prevention of acetaldehyde-induced changes in resistance, inulin permeability, and redistribution of occludin and ZO-1. U-0126, but not SB-202190 and SP-600125, also attenuated EGF-mediated prevention of acetaldehyde effect on the midregion F-actin ring. However, EGF-mediated preservation of junctional distribution of E-cadherin and β-catenin was unaffected by all three inhibitors. Expression of wild-type or constitutively active MEK1 attenuated acetaldehyde-induced redistribution of occludin and ZO-1, whereas dominant-negative MEK1 prevented EGF-mediated preservation of occludin and ZO-1 in acetaldehyde-treated cells. MEK1 expression did not alter E-cadherin distribution in acetaldehyde-treated cells in the presence or absence of EGF. Furthermore, EGF attenuated acetaldehyde-induced tyrosine-phosphorylation of occludin, ZO-1, claudin-3, and E-cadherin. U-0126, but not SB-202190 and SP-600125, prevented EGF effect on tyrosine-phosphorylation of occludin and ZO-1, but not claudin-3, E-cadherin, or β-catenin. These results indicate that EGF-mediated protection of tight junctions from acetaldehyde requires the activity of ERK1/2, but not p38 MAPK or JNK1/2, and that EGF-mediated protection of adherens junctions is independent of MAPK activities.

scholarly journals Tyrosine phosphorylation and dissociation of occludin–ZO-1 and E-cadherin–β-catenin complexes from the cytoskeleton by oxidative stress

2002 ◽  
Vol 368 (2) ◽  
pp. 471-481 ◽  
Author(s):  
Radhakrishna K. RAO ◽  
Shyamali BASUROY ◽  
Vijay U. RAO ◽  
Karl J. KARNAKY ◽  
Akshay GUPTA
Keyword(s):  
Oxidative Stress ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Protein Complexes ◽  
Cell Monolayer ◽  
Intercellular Junctions ◽  
Junctional Complexes ◽  
Dependent Mechanism ◽  
E Cadherin

The oxidative-stress-induced alteration in paracellular junctional complexes was analysed in Caco-2 cell monolayer. Oxidative stress induced a rapid increase in tyrosine phosphorylation of occludin, zonula occludens (ZO)-1, E-cadherin and β-catenin. An oxidative-stress-induced decrease in transepithelial electrical resistance was associated with a redistribution of occludin—ZO-1 and E-cadherin—β-catenin complexes from the intercellular junctions. Genistein, a tyrosine kinase inhibitor, prevented the oxidative-stress-induced decrease in resistance and redistribution of protein complexes. Occludin, ZO-1, E-cadherin and β-catenin in the Triton-insoluble cytoskeletal fraction were reduced by oxidative stress, which was prevented by genistein. Oxidative stress also reduced the co-immunoprecipitation of ZO-1 with occludin, which was prevented by genistein. Co-immunoprecipitation of β-catenin with E-cadherin was unaffected by oxidative stress or genistein. ZO-1, E-cadherin and β-catenin in the plasma membrane or membrane-cytoskeleton were either slightly reduced or unaffected by oxidative stress or genistein. These results show that oxidative stress induces tyrosine phosphorylation and cellular redistribution of occludin—ZO-1 and E-cadherin—β-catenin complexes by a tyrosine-kinase-dependent mechanism.

scholarly journals Dynamic Interaction of PTPμ with Multiple Cadherins In Vivo

1998 ◽  
Vol 141 (1) ◽  
pp. 287-296 ◽  
Author(s):  
Susann M. Brady-Kalnay ◽  
Tracy Mourton ◽  
Joseph P. Nixon ◽  
Gregory E. Pietz ◽  
Michael Kinch ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Phosphatase ◽  
Cross Reactivity ◽  
Receptor Protein ◽  
Temperature Sensitive ◽  
Mutant Form ◽  
Cell Biol ◽  
E Cadherin

There is a growing body of evidence to implicate reversible tyrosine phosphorylation as an important mechanism in the control of the adhesive function of cadherins. We previously demonstrated that the receptor protein tyrosine phosphatase PTPμ associates with the cadherin–catenin complex in various tissues and cells and, therefore, may be a component of such a regulatory mechanism (Brady-Kalnay, S.M., D.L. Rimm, and N.K. Tonks. 1995. J. Cell Biol. 130:977– 986). In this study, we present further characterization of this interaction using a variety of systems. We observed that PTPμ interacted with N-cadherin, E-cadherin, and cadherin-4 (also called R-cadherin) in extracts of rat lung. We observed a direct interaction between PTPμ and E-cadherin after coexpression in Sf9 cells. In WC5 cells, which express a temperature-sensitive mutant form of v-Src, the complex between PTPμ and E-cadherin was dynamic, and conditions that resulted in tyrosine phosphorylation of E-cadherin were associated with dissociation of PTPμ from the complex. Furthermore, we have demonstrated that the COOH-terminal 38 residues of the cytoplasmic segment of E-cadherin was required for association with PTPμ in WC5 cells. Zondag et al. (Zondag, G., W. Moolenaar, and M. Gebbink. 1996. J. Cell Biol. 134: 1513–1517) have asserted that the association we observed between PTPμ and the cadherin–catenin complex in immunoprecipitates of the phosphatase arises from nonspecific cross-reactivity between BK2, our antibody to PTPμ, and cadherins. In this study we have confirmed our initial observation and demonstrated the presence of cadherin in immunoprecipitates of PTPμ obtained with three antibodies that recognize distinct epitopes in the phosphatase. In addition, we have demonstrated directly that the anti-PTPμ antibody BK2 that we used initially did not cross-react with cadherin. Our data reinforce the observation of an interaction between PTPμ and E-cadherin in vitro and in vivo, further emphasizing the potential importance of reversible tyrosine phosphorylation in regulating cadherin function.

Glutathione oxidation and PTPase inhibition by hydrogen peroxide in Caco-2 cell monolayer

2000 ◽  
Vol 279 (2) ◽  
pp. G332-G340 ◽  
Author(s):  
R. K. Rao ◽  
L. Li ◽  
R. D. Baker ◽  
S. S. Baker ◽  
A. Gupta
Keyword(s):  
Xanthine Oxidase ◽  
Ferrous Sulfate ◽  
Tyrosine Phosphatase ◽  
Cell Monolayer ◽  
Paracellular Permeability ◽  
Cell Monolayers ◽  
Protein Thiols ◽  
Glutathione Oxidation ◽  
Ptpase Activity

The role of H2O2and protein thiol oxidation in oxidative stress-induced epithelial paracellular permeability was investigated in Caco-2 cell monolayers. Treatment with a H2O2 generating system (xanthine oxidase + xanthine) or H2O2 (20 μM) increased the paracellular permeability. Xanthine oxidase-induced permeability was potentiated by superoxide dismutase and prevented by catalase. H2O2-induced permeability was prevented by ferrous sulfate and potentiated by deferoxamine and 1,10-phenanthroline. GSH, N-acetyl-l-cysteine, dithiothreitol, mercaptosuccinate, and diethylmaleate inhibited H2O2-induced permeability, but it was potentiated by 1,3-bis(2-chloroethyl)-1-nitrosourea. H2O2 reduced cellular GSH and protein thiols and increased GSSG. H2O2-mediated reduction of GSH-to-GSSG ratio was prevented by ferrous sulfate, GSH, N-acetyl-l-cysteine, diethylmaleate, and mercaptosuccinate and potentiated by 1,10-phenanthroline and 1,3-bis(2-chloroethyl)-1-nitrosourea. Incubation of soluble fraction of cells with GSSG reduced protein tyrosine phosphatase (PTPase) activity, which was prevented by coincubation with GSH. PTPase activity was also lower in H2O2-treated cells. This study indicates that H2O2, but not O2 −· or ·OH, increases paracellular permeability of Caco-2 cell monolayer by a mechanism that involves oxidation of GSH and inhibition of PTPases.

scholarly journals Haemin enhancement of glucose transport in human lymphocytes: stimulation of protein tyrosine phosphatase and activation of p56lck tyrosine kinase

1993 ◽  
Vol 291 (1) ◽  
pp. 281-287 ◽  
Author(s):  
H M Lander ◽  
D M Levine ◽  
A Novogrodsky
Keyword(s):  
Tyrosine Kinase ◽  
Glucose Uptake ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Human Lymphocytes ◽  
Protein Tyrosine Phosphorylation ◽  
Protein Tyrosine ◽  
Ptpase Activity ◽  
Stimulation Of

Following our previous observation that haemin is mitogenic for human lymphocytes, we investigated the ability of haemin to enhance glucose uptake in these cells. We found that preincubation of human peripheral-blood mononuclear cells (PBMC) with haemin for 60 min increased up to 5-fold the rate of 2-deoxy-D-[1-3H]glucose uptake by the cells. Actinomycin D and cycloheximide did not inhibit the effect, and cytochalasin B completely blocked it. Among the metalloporphyrins tested (Fe-, Ni-, Co-, Zn- and Sn-protoporphyrin), only haemin (Fe-protoporphyrin) induced a marked increase in glucose uptake. Thiourea, a scavenger of oxygen free radicals, and 3-amino-1,2,4-triazole inhibited haemin-induced glucose uptake. Oxidants such as H2O2 and phenylarsine oxide were previously reported to stimulate protein tyrosine phosphorylation and to enhance glucose uptake. We found that incubation of PBMC with haemin resulted in an increase in protein tyrosine phosphatase (PTPase) activity, probably that identified as CD45. Similarly to haemin, we found that phytohaemagglutinin also enhanced PTPase activity. Haemin also activated the tyrosine kinase p56lck, which is negatively controlled by phosphorylation of Tyr-505 at the C-terminus, and increased protein tyrosine phosphorylation in these cells. Tyrphostins, specific inhibitors of tyrosine kinases, at low concentrations markedly enhanced glucose uptake and synergized with haemin in enhancing glucose uptake. At high doses, tyrphostins inhibited the effect of haemin. Taken together, we postulate that haemin enhancement of glucose uptake in human lymphocytes results from its stimulation of PTPase, followed by activation of tyrosine kinase p56lck, leading to an increase in protein tyrosine phosphorylation.

scholarly journals Hydrogen peroxide activates focal adhesion kinase and c-Src by a phosphatidylinositol 3 kinase-dependent mechanism and promotes cell migration in Caco-2 cell monolayers

2010 ◽  
Vol 299 (1) ◽  
pp. G186-G195 ◽  
Author(s):  
Shyamali Basuroy ◽  
Mitzi Dunagan ◽  
Parimal Sheth ◽  
Ankur Seth ◽  
R. K. Rao
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Cell Migration ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Kinase Activity ◽  
Src Kinase ◽  
Pi3 Kinase ◽  
Cell Monolayers ◽  
Dependent Mechanism

Recent studies showed that c-Src and phosphatidylinositol 3 (PI3) kinase mediate the oxidative stress-induced disruption of tight junctions in Caco-2 cell monolayers. The present study evaluated the roles of PI3 kinase and Src kinase in the oxidative stress-induced activation of focal adhesion kinase (FAK) and acceleration of cell migration. Oxidative stress, induced by xanthine and xanthine oxidase system, rapidly increased phosphorylation of FAK on Y397, Y925, and Y577 in the detergent-insoluble and soluble fractions and increased its tyrosine kinase activity. The PI3 kinase inhibitors, wortmannin and LY294002, and the Src kinase inhibitor, 4-amino-5[chlorophyll]-7-[t-butyl]pyrazolo[3–4-d]pyrimidine, attenuated tyrosine phosphorylation of FAK. Oxidative stress induced phosphorylation of c-Src on Y418 by a PI3 kinase-dependent mechanism, whereas oxidative stress-induced activation of PI3 kinase was independent of Src kinase activity. Hydrogen peroxide accelerated Caco-2 cell migration in a concentration-dependent manner. Promotion of cell migration by hydrogen peroxide was attenuated by LY294002 and PP2. Reduced expression of FAK by siRNA attenuated hydrogen peroxide-induced acceleration of cell migration. The expression of constitutively active c-SrcY527F enhanced cell migration, whereas the expression of dominant negative c-SrcK296R/Y528F attenuated hydrogen peroxide-induced stimulation of cell migration. Oxidative stress-induced activation of c-Src and FAK was associated with a rapid increase in the tyrosine phosphorylation and the levels of paxillin and p130CAS in actin-rich, detergent-insoluble fractions. This study shows that oxidative stress activates FAK and accelerates cell migration in an intestinal epithelium by a PI3 kinase- and Src kinase-dependent mechanism.

Tropomyosin interacts with phosphorylated HSP27 in agonist-induced contraction of smooth muscle

2004 ◽  
Vol 286 (6) ◽  
pp. C1290-C1301 ◽  
Author(s):  
Sita Somara ◽  
Khalil N. Bitar
Keyword(s):  
Smooth Muscle ◽  
Smooth Muscle Cells ◽  
Direct Interaction ◽  
Muscle Cells ◽  
Glutathione S Transferase ◽  
Binding Studies ◽  
Mutant Proteins ◽  
Protein Protein Interaction ◽  
Hsp27 Phosphorylation

Displacement of the contractile protein tropomyosin from actin filament exposes the myosin-binding sites on actin, resulting in actin-myosin interaction and muscle contraction. The objective of the present study was to better understand the interaction of tropomyosin with heat shock protein (HSP)27 in contraction of smooth muscle cells of the colon. We investigated the possibility of a direct protein-protein interaction of tropomyosin with HSP27 and the role of phosphorylated HSP27 in this interaction. Immunoprecipitation studies on rabbit smooth muscle cells indicate that upon acetylcholine-induced contraction tropomyosin shows increased association with HSP27 phosphorylated at Ser82 and Ser78. Transfection of smooth muscle cells with HSP27 phosphorylation mutants indicated that the association of tropomyosin with HSP27 could be affected by HSP27 phosphorylation. In vitro binding studies with glutathione S-transferase (GST)-tagged HSP27 mutant proteins show that tropomyosin has greater direct interaction to phosphomimic HSP27 mutant compared with wild-type and nonphosphomimic HSP27. Our data suggest that, in response to a contractile agonist, HSP27 undergoes a rapid phosphorylation that may strengthen its interaction with tropomyosin.

scholarly journals The Effect of Transient Global Ischemia on the Interaction of Src and Fyn with the N-Methyl-d-Aspartate Receptor and Postsynaptic Densities: Possible Involvement of Src Homology 2 Domains

1999 ◽  
Vol 19 (8) ◽  
pp. 880-888 ◽  
Author(s):  
Norio Takagi ◽  
Herman H. Cheung ◽  
Nankie Bissoon ◽  
Lucy Teves ◽  
M. Christopher Wallace ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Phosphatase ◽  
Sh2 Domain ◽  
Signaling Proteins ◽  
Glutathione S Transferase ◽  
Nmda Receptor Subunits ◽  
Sh2 Domains ◽  
Src Homology 2 ◽  
Src Homology

Transient ischemia increases tyrosine phosphorylation of N-methyl-d-aspartate (NMDA) receptor subunits NR2A and NR2B in the rat hippocampus. The authors investigated the effects of this increase on the ability of the receptor subunits to bind to the Src homology 2 (SH2) domains of Src and Fyn expressed as glutathione-S-transferase–SH2 fusion proteins. The NR2A and NR2B bound to each of the SH2 domains and binding was increased approximately twofold after ischemia and reperfusion. Binding was prevented by prior incubation of hippocampal homogenates with a protein tyrosine phosphatase or by a competing peptide for the Src SH2 domain. Ischemia induced a marked increase in the tyrosine phosphorylation of several proteins in the postsynaptic density (PSD), including NR2A and NR2B, but had no effect on the amounts of individual NMDA receptor subunits in the PSD. The level of Src and Fyn in PSDs, but not in other subcellular fractions, was increased after ischemia. The ischemia-induced increase in the interaction of NR2A and NR2B with the SH2 domains of Src and Fyn suggests a possible mechanism for the recruitment of signaling proteins to the PSD and may contribute to altered signal transduction in the postischemic hippocampus.

scholarly journals Binding Studies Reveal Phospholipid Specificity and Its Role in the Calcium-Dependent Mechanism of Action of Daptomycin

2021 ◽  
Author(s):  
Ioli Kotsogianni ◽  
Thomas M. Wood ◽  
Francesca M. Alexander ◽  
Stephen A. Cochrane ◽  
Nathaniel I. Martin
Keyword(s):  
Mechanism Of Action ◽  
Binding Studies ◽  
Calcium Dependent ◽  
Dependent Mechanism

scholarly journals A Novel Pharmacological Approach to Enhance the Integrity and Accelerate Restitution of the Intestinal Epithelial Barrier

2020 ◽  
Vol 26 (9) ◽  
pp. 1340-1352
Author(s):  
Xuelei Cao ◽  
Lei Sun ◽  
Susana Lechuga ◽  
Nayden G Naydenov ◽  
Alex Feygin ◽  
...  
Keyword(s):  
Wound Healing ◽  
Epithelial Cell ◽  
Epithelial Barrier ◽  
Intestinal Epithelial ◽  
Intestinal Epithelial Barrier ◽  
Cell Monolayers ◽  
Barrier Disruption ◽  
Epithelial Cell Monolayers ◽  
Immunofluorescence Labeling ◽  
E Cadherin

Abstract Background Disruption of the gut barrier is an essential mechanism of inflammatory bowel diseases (IBDs) contributing to the development of mucosal inflammation. A hallmark of barrier disruption is the disassembly of epithelial adherens junctions (AJs) driven by decreased expression of a major AJ protein, E-cadherin. A group of isoxazole compounds, such as E-cadherin-upregulator (ECU) and ML327, were previously shown to stimulate E-cadherin expression in poorly differentiated human cancer cells. This study was designed to examine whether these isoxazole compounds can enhance and protect model intestinal epithelial barriers in vitro. Methods The study was conducted using T84, SK-CO15, and HT-29 human colonic epithelial cell monolayers. Disruption of the epithelial barrier was induced by pro-inflammatory cytokines, tumor necrosis factor-α, and interferon-γ. Barrier integrity and epithelial junction assembly was examined using different permeability assays, immunofluorescence labeling, and confocal microscopy. Epithelial restitution was analyzed using a scratch wound healing assay. Results E-cadherin-upregulator and ML327 treatment of intestinal epithelial cell monolayers resulted in several barrier-protective effects, including reduced steady-state epithelial permeability, inhibition of cytokine-induced barrier disruption and junction disassembly, and acceleration of epithelial wound healing. Surprisingly, these effects were not due to upregulation of E-cadherin expression but were mediated by multiple mechanisms including inhibition of junction protein endocytosis, attenuation of cytokine-induced apoptosis, and activation of promigratory Src and AKT signaling. Conclusions Our data highlight ECU and ML327 as promising compounds for developing new therapeutic strategies to protect the integrity and accelerate the restitution of the intestinal epithelial barrier in IBD and other inflammatory disorders.

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