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.

l-Glutamine ameliorates acetaldehyde-induced increase in paracellular permeability in Caco-2 cell monolayer

2004 ◽  
Vol 287 (3) ◽  
pp. G510-G517 ◽  
Author(s):  
A. Seth ◽  
S. Basuroy ◽  
P. Sheth ◽  
R. K. Rao
Keyword(s):  
Barrier Function ◽  
Egf Receptor ◽  
Kinase Inhibitor ◽  
Cell Monolayer ◽  
Intercellular Junctions ◽  
Paracellular Permeability ◽  
Dependent Manner ◽  
Dose Dependent ◽  
E Cadherin

Role of l-glutamine in the protection of intestinal epithelium from acetaldehyde-induced disruption of barrier function was evaluated in Caco-2 cell monolayer. l-Glutamine reduced the acetaldehyde-induced decrease in transepithelilal electrical resistance and increase in permeability to inulin and lipopolysaccharide in a time- and dose-dependent manner; d-glutamine, l-aspargine, l-arginine, l-lysine, or l-alanine produced no significant protection. The glutaminase inhibitor 6-diazo-5-oxo-l-norleucine failed to affect the l-glutamine-mediated protection of barrier function. l-Glutamine reduced the acetaldehyde-induced redistribution of occludin, zonula occludens-1 (ZO-1), E-cadherin, and β-catenin from the intercellular junctions. Acetaldehyde dissociates occludin, ZO-1, E-cadherin, and β-catenin from the actin cytoskeleton, and this effect was reduced by l-glutamine. l-Glutamine induced a rapid increase in the tyrosine phosphorylation of EGF receptor, and the protective effect of l-glutamine was prevented by AG1478, the EGF-receptor tyrosine kinase inhibitor. These results indicate that l-glutamine prevents acetaldehyde-induced disruption of the tight junction and increase in the paracellular permeability in Caco-2 cell monolayer by an EGF receptor-dependent mechanism.

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.

Protein-tyrosine Kinase p72 syk Is Activated by Platelet Activating Factor in Platelets

1994 ◽  
Vol 72 (06) ◽  
pp. 937-941 ◽  
Author(s):  
Karim Rezaul ◽  
Shigeru Yanagi ◽  
Kiyonao Sada ◽  
Takanobu Taniguchi ◽  
Hirohei Yamamura
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Kinase Inhibitor ◽  
Critical Role ◽  
Platelet Activating Factor ◽  
Kinase Assay ◽  
Potential Candidate ◽  
Protein Tyrosine ◽  
Induced Aggregation

SummaryIt has been demonstrated that activation of platelets by platelet-activating factor (PAF) results in a dramatic increase in tyrosine phosphorylation of several cellular proteins. We report here that p72 syk is a potential candidate for the protein-tyrosine phosphorylation following PAF stimulation in porcine platelets. Immunoprecipitation kinase assay revealed that PAF stimulation resulted in a rapid activation of p72 syk which peaked at 10 s. The level of activation was found to be dose dependent and could be completely inhibited by the PAF receptor antagonist, CV3988. Phosphorylation at the tyrosine residues of p72 syk coincided with activation of yllsyk. Pretreatment of platelets with aspirin and apyrase did not affect PAF induced activation of p72 syk .Furthermore, genistein, a potent protein-tyrosine-kinase inhibitor, diminished PAF-induced p72 syk activation and Ca2+ mobilization as well as platelet aggregation. These results suggest that p72 syk may play a critical role in PAF-induced aggregation, possibly through regulation of Ca2+ mobilization.

Secretin regulates paracellular permeability in canine gastric monolayers by a Src kinase-dependent pathway

2002 ◽  
Vol 283 (4) ◽  
pp. G893-G899 ◽  
Author(s):  
Monica C. Chen ◽  
Travis E. Solomon ◽  
Eduardo Perez Salazar ◽  
Robert Kui ◽  
Enrique Rozengurt ◽  
...  
Keyword(s):  
Gastric Mucosa ◽  
Epithelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Tyrosine Phosphorylation ◽  
Molecular Mechanisms ◽  
Kinase Inhibitor ◽  
Paracellular Permeability ◽  
Maximal Response ◽  
Src Tyrosine Kinase

Previous studies found that epidermal growth factor (EGF) decreased paracellular permeability in gastric mucosa, but the other physiological regulators and the molecular mechanisms mediating these responses remain undefined. We investigated the role of secretin and Src in regulating paracellular permeability because secretin regulates gastric chief cell function and Src mediates events involving the cytoskeletal-membrane interface, respectively. Confluent monolayers were formed from canine gastric epithelial cells in short-term culture on Transwell filter inserts. Resistance was monitored in the presence of secretin with or without specific kinase inhibitors. Tyrosine phosphorylation of Src at Tyr416 was measured with a site-specific phosphotyrosine antibody. Basolateral, but not apical, secretin at concentrations from 1 to 100 nM dose dependently increased resistance; this response was rapid and sustained over hours. PP2 (10 μM), a selective Src tyrosine kinase inhibitor, but not the inactive isomer PP3, abolished the increase in resistance by secretin but only modestly attenuated apical EGF effects. AG-1478 (100 nM), a specific EGF receptor tyrosine kinase inhibitor, attenuated the resistance increase to EGF but not secretin. Secretin, but not EGF, induced tyrosine phosphorylation of Src at Tyr416 in a dose-dependent fashion, with the maximal response observed at 1 min. PP2, but not PP3, dramatically inhibited this tyrosine phosphorylation. Secretin increases paracellular resistance in gastric mucosa through a Src-mediated pathway, while the effect of EGF is Src independent. Src appears to mediate the physiological effects of this Gs-coupled receptor in primary epithelial cells.

Activation of pp60(src) is critical for stretch-induced orienting response in fibroblasts

1999 ◽  
Vol 112 (9) ◽  
pp. 1365-1373 ◽  
Author(s):  
X. Sai ◽  
K. Naruse ◽  
M. Sokabe
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Time Course ◽  
Kinase Inhibitor ◽  
Orienting Response ◽  
Cyclic Stretch ◽  
Wild Type ◽  
Herbimycin A ◽  
Molecular Masses

When subjected to uni-axial cyclic stretch (120% in length, 1 Hz), fibroblasts (3Y1) aligned perpendicular to the stretch axis in a couple of hours. Concomitantly with this orienting response, protein tyrosine phosphorylation of cellular proteins (molecular masses of approximately 70 kDa and 120–130 kDa) increased and peaked at 30 minutes. Immuno-precipitation experiments revealed that paxillin, pp125(FAK), and pp130(CAS) were included in the 70 kDa, and 120–130 kDa bands, respectively. Treatment of the cells with herbimycin A, a tyrosine kinase inhibitor, suppressed the stretch induced tyrosine phosphorylation and the orienting response suggesting that certain tyrosine kinases are activated by stretch. We focused on pp60(src), the most abundant tyrosine kinase in fibroblasts. The kinase activity of pp60(src) increased and peaked at 20 minutes after the onset of cyclic stretch. Treatment of the cells with an anti-sense S-oligodeoxynucleotide (S-ODN) against pp60(src), but not the sense S-ODN, inhibited the stretch induced tyrosine phosphorylation and the orienting response. To further confirm the involvement of pp60(src), we performed the same sets of experiments using c-src-transformed 3Y1 (c-src-3Y1) fibroblasts. Cyclic stretch induced a similar orienting response in c-src-3Y1 to that in wild-type 3Y1, but with a significantly faster rate. The time course of the stretch-induced tyrosine phosphorylation was also much faster in c-src-3Y1 than in 3Y1 fibroblasts. These results strongly suggest that cyclic stretch induces the activation of pp60(src) and that pp60(src) is indispensable for the tyrosine phosphorylation of pp130(CAS), pp125(FAK) and paxillin followed by the orienting response in 3Y1 fibroblasts.

Stimulation of tyrosine phosphorylation of distinct proteins in response to antibody-mediated ligation and clustering of alpha 3 and alpha 6 integrins

1995 ◽  
Vol 108 (3) ◽  
pp. 1165-1174
Author(s):  
K. Jewell ◽  
C. Kapron-Bras ◽  
P. Jeevaratnam ◽  
S. Dedhar
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
Cell Attachment ◽  
Umbilical Vein ◽  
Integrin Receptors ◽  
Human Prostate Carcinoma ◽  
Beta 1 Integrin ◽  
Umbilical Vein Endothelial Cells ◽  
Stimulation Of

The interaction of cells with components of the extracellular matrix through their integrin receptors results in the stimulation of tyrosine phosphorylation of several proteins, suggesting that these receptors play a key role in signal transduction. Here we report that antibody-mediated ligation and clustering of alpha 3 beta 1 and alpha 6 beta 1/alpha 6 beta 4 integrins resulted in the stimulation of tyrosine phosphorylation of proteins that are specific for each heterodimer. Thus, ligation and clustering of the alpha 3 beta 1 integrin on human prostate carcinoma cells (PC-3) and human umbilical vein endothelial cells (HUVEC) with anti-alpha 3 antibodies resulted in the stimulation of tyrosine phosphorylation of a 55 kDa protein. In contrast, ligation and clustering of the alpha 6 beta 1 integrin on these cells with anti-alpha 6 antibody resulted in the dramatic stimulation of tyrosine phosphorylation of a 90 kDa protein in addition to a 52 kDa protein, and ligation and clustering of alpha 5 beta 1 on HUVEC did not result in the apparent stimulation of tyrosine phosphorylation of any proteins. Clustering with anti-beta 1 antibodies triggered the tyrosine phosphorylation of all of these proteins, whereas ligation and clustering of PC-3 cells with an anti-beta 4 antibody resulted in the tyrosine phosphorylation of a distinct 62 kDa protein. Since the PC-3 cells express both alpha 6 beta 1 and alpha 6 beta 4, these data suggest that these two receptors can transduce distinct signals. All of the phosphorylations could be inhibited by treating the cells with Genistein, a tyrosine kinase inhibitor. Antibody-mediated ligation and clustering of integrins on the two types of cells did not result in the stimulation of tyrosine phosphorylation of pp125 focal adhesion kinase, although this was observed upon cell attachment and spreading on fibronectin, laminin and anti-alpha 3 monoclonal antibody. Collectively, these data demonstrate that cross-linking of different integrin heterodimers can stimulate tyrosine kinase activities, leading to the phosphorylation of distinct proteins, which are also different from those observed when cells are allowed to spread on a matrix.

Oxidative stress-induced cell death of human oral neutrophils

2003 ◽  
Vol 284 (4) ◽  
pp. C1048-C1053 ◽  
Author(s):  
Eisuke F. Sato ◽  
Masahiro Higashino ◽  
Kazuo Ikeda ◽  
Ryotaro Wake ◽  
Mitsuyoshi Matsuo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Caspase 3 ◽  
Nuclear Dna ◽  
Kinase Inhibitor ◽  
Tissue Injury ◽  
Morphological Changes ◽  
Limited Information ◽  
And Function

Polymorphonuclear leukocytes (PMN) play crucial roles in protecting hosts against invading microbes and in the pathogenesis of inflammatory tissue injury. Although PMN migrate into mucosal layers of digestive and respiratory tracts, only limited information is available of their fate and function in situ. We previously reported that, unlike circulating PMN (CPMN), PMN in the oral cavity spontaneously generate superoxide radical and nitric oxide (NO) in the absence of any stimuli. When cultured for 12 h under physiological conditions, oral PMN (OPMN) showed morphological changes that are characteristic of those of apoptosis. Upon agarose gel electrophoresis, nuclear DNA samples isolated from OPMN revealed ladder-like profiles characteristic of nucleosomal fragmentation.l-cysteine, reduced glutathione (GSH), and herbimycin A, a protein tyrosine kinase inhibitor, suppressed the activation of caspase-3 and apoptosis of OPMN. Neither thiourea, superoxide dismutase (SOD), nor catalase inhibited the activation of caspase-3 and apoptosis. Moreover, N-acetyl-Asp-Glu-Val-Asp-aldehyde (Ac-DEVD-CHO), inhibitor for caspase-3, inhibited the fragmentation of DNA. These results suggested that oxidative stress and/or tyrosine-kinase-dependent pathway(s) activated caspase-3 in OPMN, thereby inducing their apoptosis.

Role for tyrosine kinases in carbachol-regulated Ca entry into colonic epithelial cells

1995 ◽  
Vol 268 (1) ◽  
pp. C154-C161 ◽  
Author(s):  
G. Bischof ◽  
B. Illek ◽  
W. W. Reenstra ◽  
T. E. Machen
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Divalent Cations ◽  
Tyrosine Phosphatase ◽  
Inhibitory Effect ◽  
Colonic Epithelial Cells

We studied a possible role of tyrosine kinases in the regulation of Ca entry into colonic epithelial cells HT-29/B6 using digital image processing of fura 2 fluorescence. Both carbachol and thapsigargin increased Ca entry to a similar extent and Ca influx was reduced by the tyrosine kinase inhibitor genistein (50 microM). Further experiments were performed in solutions containing 95 mM K to depolarize the membrane potential, and the effects of different inhibitors on influx of Ca, Mn, and Ba were compared. Genistein, but not the inactive analogue daidzein nor the protein kinase C inhibitor 1-(5-isoquinolinylsulfonyl)-2- methylpiperazine, decreased entry of all three divalent cations by 47-59%. In high-K solutions, carbachol or thapsigargin both caused intracellular Ca to increase to a plateau of 223 +/- 19 nM. This plateau was reduced by the tyrosine kinase inhibitors genistein (to 95 +/- 8 nM), lavendustin A (to 155 +/- 17 nM), and methyl-2,5-dihydroxycinnamate (to 39 +/- 3 nM). Orthovanadate, a protein tyrosine phosphatase inhibitor, prevented the inhibitory effect of genistein. Ca pumping was unaffected by genistein. Carbachol increased tyrosine phosphorylation (immunoblots with anti-phosphotyrosine antibodies) of 110-, 75-, and 70-kDa proteins, and this phosphorylation was inhibited by genistein. We conclude that carbachol and thapsigargin increase Ca entry, and tyrosine phosphorylation of some key proteins may be important for regulating this pathway.

Platelet/Polymorphonuclear Leukocyte Interaction: P-Selectin Triggers Protein-Tyrosine Phosphorylation–Dependent CD11b/CD18 Adhesion: Role of PSGL-1 as a Signaling Molecule

Blood ◽  
1999 ◽  
Vol 93 (3) ◽  
pp. 876-885 ◽  
Author(s):  
Virgilio Evangelista ◽  
Stefano Manarini ◽  
Rita Sideri ◽  
Serenella Rotondo ◽  
Nicola Martelli ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinase Inhibitors ◽  
Polymorphonuclear Leukocyte ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Chinese Hamster ◽  
Mixed Cell ◽  
Β2 Integrin ◽  
Activated Platelets

Abstract Polymorphonuclear leukocyte (PMN) adhesion to activated platelets is important for the recruitment of PMN at sites of vascular damage and thrombus formation. We have recently shown that binding of activated platelets to PMN in mixed cell suspensions under shear involves P-selectin and the activated β2-integrin CD11b/CD18. Integrin activation required signaling mechanisms that were sensitive to tyrosine kinase inhibitors.1 Here we show that mixing activated, paraformaldehyde (PFA)-fixed platelets with PMNs under shear conditions leads to rapid and fully reversible tyrosine phosphorylation of a prominent protein of 110 kD (P∼110). Phosphorylation was both Ca2+ and Mg2+ dependent and was blocked by antibodies against P-selectin or CD11b/CD18, suggesting that both adhesion molecules need to engage with their respective ligands to trigger phosphorylation of P∼110. The inhibition of P∼110 phosphorylation by tyrosine kinase inhibitors correlates with the inhibition of platelet/PMN aggregation. Similar effects were observed when platelets were substituted by P-selectin–transfected Chinese hamster ovary (CHO-P) cells or when PMN were stimulated with P-selectin–IgG fusion protein. CHO-P/PMN mixed-cell aggregation and P-selectin–IgG–triggered PMN/PMN aggregation as well as P∼110 phosphorylation were all blocked by antibodies against P-selectin or CD18. In each case PMN adhesion was sensitive to the tyrosine kinase inhibitor genistein. The antibody PL-1 against P-selectin glycoprotein ligand-1 (PSGL-1) blocked platelet/PMN aggregation, indicating that PSGL-1 was the major tethering ligand for P-selectin in this experimental system. Moreover, engagement of PSGL-1 with a nonadhesion blocking antibody triggered β2-integrin–dependent genistein-sensitive aggregation as well as tyrosine phosphorylation in PMN. This study shows that binding of P-selectin to PSGL-1 triggers tyrosine kinase–dependent mechanisms that lead to CD11b/CD18 activation in PMN. The availability of the β2-integrin to engage with its ligands on the neighboring cells is necessary for the tyrosine phosphorylation of P∼110.

Sign in / Sign up

Export Citation Format

Share Document