The MAPK (mitogen-activated protein kinase) pathway is a major intracellular signalling pathway involved in EGF (epithelial growth factor) receptor-mediated cell growth and differentiation. A novel function of MAPK activity in the mechanism of EGF-mediated protection of TJs (tight junctions) from H2O2 was examined in Caco-2 cell monolayers. EGF-mediated prevention of H2O2-induced increase in paracellular permeability was associated with the prevention of H2O2-induced Tyr-phosphorylation, Thr-dephosphorylation and cellular redistribution of occludin and ZO-1 (zonula occludin-1). EGF also prevented H2O2-induced disruption of the actin cytoskeleton and the dissociation of occludin and ZO-1 from the actin-rich detergent-insoluble fractions. MEK (MAPK/ERK kinase, where ERK stands for extracellular signal related kinase) inhibitors, PD98059 and U0126, completely blocked these protective effects of EGF on TJs. EGF rapidly increased the levels of phosphorylated MEK (p-MEK) in detergent-soluble fractions and phosphorylated ERK (p-ERK) in detergent-insoluble fractions. p-ERK was colocalized and co-immunoprecipitated with occludin. GST (glutathione S-transferase) pull-down assay showed that the C-terminal tail of occludin binds to p-ERK in Caco-2 cell extracts. Pair-wise binding studies using recombinant proteins demonstrated that ERK1 directly interacts with the C-terminal tail of occludin. Therefore the present study shows that ERK interacts with the C-terminal region of occludin and mediates the prevention of H2O2-induced disruption of TJs by EGF.
Occludin is hyperphosphorylated on Ser and Thr residues in intact epithelial tight junction (TJ); however, the role of this phosphorylation in the assembly of TJ is unclear. The influence of protein phosphatases PP2A and PP1 on the assembly of TJ and phosphorylation of occludin was evaluated in Caco-2 cells. Protein phosphatase inhibitors and reduced expression of PP2A-C␣ and PP1␣ accelerated the calcium-induced increase in transepithelial electrical resistance and barrier to inulin permeability and also enhanced the junctional organization of occludin and ZO-1 during TJ assembly. Phosphorylation of occludin on Thr residues, but not on Ser residues, was dramatically reduced during the disassembly of TJ and was gradually increased during the reassembly. PP2A and PP1 co-immunoprecipitate with occludin, and this association was reduced during the assembly of TJ. Glutathione S-transferase (GST) pulldown assay using recombinant GST-occludin demonstrated that cellular PP2A and PP1 bind to the C-terminal tail of occludin, and these interactions were also reduced during the assembly of TJ. A pairwise binding assay using GST-occludin and purified PP2A and PP1 demonstrates that PP2A and PP1 directly interacts with the C-terminal tail of occludin. In vitro incubation of phospho-occludin with PP2A or PP1 indicated that PP2A dephosphorylates occludin on phospho-Thr residues, whereas PP1 dephosphorylates it on phospho-Ser. This study shows that PP2A and PP1 directly interact with occludin and negatively regulate the assembly of TJ by modulating the phosphorylation status of occludin. An important function of the epithelial tight junction (TJ)2 is to form a barrier to the diffusion of pathogens, toxins, and allergens from the external environment into the tissues. The disruption of TJ plays a crucial role in the pathogenesis of a number of diseases related to the gastrointestinal tract, lung, and kidney (1-3). The TJ is organized by specific interactions between a wide spectrum of proteins. Three types of transmembrane proteins, occludin (4), claudins (5), and junctional adhesion molecule (6) interact with other intracellular plaque proteins such as ZO-1, ZO-2, ZO-3, cingulin, and 7H6, which in turn anchor the transmembrane proteins to the actin cytoskeleton (7-9).A significant body of evidence indicates that the activities of various intracellular signaling molecules regulate the integrity of TJ. The signaling pathways involving protein kinases and GTPase switch proteins regulate the TJ permeability in different epithelial monolayer (10 -22). Tyrosine kinases such as c-Yes, c-Src, and focal adhesion kinase are localized in the vicinity of TJ (4). A number of previous studies have shown that oxidative stress induces disruption of TJ and an increase in paracellular permeability by a tyrosine kinase-dependent mechanism (15-17, 19, 20). Oxidative stress induces Tyr phosphorylation of a wide spectrum of proteins, including occludin, ZO-1, E-cadherin, and -catenin (19). Phosphorylation of occludin on Tyr residues results in the lo...
PKC is expressed predominantly in the epithelial tissues; however, its role in the regulation of epithelial tight junctions (TJs) is unknown. We present evidence that PKC phosphorylates occludin on threonine residues (T403 and T404) and plays a crucial role in the assembly and/or maintenance of TJs in Caco-2 and MDCK cell monolayers. Inhibition of PKC by specific pseudo substrate inhibitor or knockdown of PKC by specific shRNA disrupts the junctional distribution of occludin and ZO-1 and compromises the epithelial barrier function. Expression of dominant negative, PKC K394R disrupts the TJ and barrier function, whereas wild-type PKC and constitutively active PKC A161E enhance the TJ integrity. Inhibition and knockdown of PKC or expression of PKC K394R induce dephosphorylation of occludin on threonine residues, whereas active PKC elevates occludin phosphorylation. PKC directly interacts with the C-terminal domain of occludin and phosphorylates it on highly conserved T403 and T404. T403/404A mutations result in the loss of occludin's ability to localize at the TJs, whereas T403/404D mutations attenuates the PKC inhibitormediated redistribution of occludin from the intercellular junctions. These results reveal an important mechanism of epithelial TJ regulation by PKC .differentiation ͉ epithelium ͉ protein kinase T he epithelial tight junctions (TJs) on one hand determine the cell polarity by forming a fence between the apical and basolateral membranes (1), and on the other hand, it prevents the diffusion of toxins, allergens, and pathogens from the lumen into the tissue (2). Additionally, TJs play essential roles in the regulation of cell-cell adhesion and the epithelium-tomesenchymal transition (3). Dysfunctional TJs are associated with the pathogenesis of inflammatory diseases (2) and tumor metastasis (3). Therefore, understanding the molecular structure of TJs and the regulatory mechanisms that control the integrity of TJs is essential to advance our knowledge in epithelial homeostasis in health and disease.The assembly of TJs involves at least 3 types of transmembrane proteins, occludin, claudins, and junctional adhesion molecule (4). The intracellular domains of occludin and claudins interact with the plaque proteins such as ZO-1, ZO-2, and ZO-3, which form the platforms for recruitment of scaffold proteins such as cingulin, Par-3, Par-6, etc.; this TJ protein complex is anchored into the perijunctional actomyosin ring. Although occludin knockout mice showed the formation of intact TJs in different epithelia (5), several studies indicated that occludin does play an important role in the regulation of TJ integrity (6, 7).Protein kinases (8-10) and protein phosphatases (11) are either localized at the TJs or interact directly with the TJ proteins. Whereas atypical PKCs (PKC and PKC / ) directly interact with the TJs (10), PKC and PKCI may indirectly regulate the integrity of TJs (12). PKC , a novel PKC isoform, is predominantly expressed in epithelial tissues (13). The function of PKC in the epithelial tissues...
Occludin is phosphorylated on tyrosine residues during the oxidative stress-induced disruption of tight junction, and in vitro phosphorylation of occludin by c-Src attenuates its binding to ZO-1. In the present study mass spectrometric analyses of C-terminal domain of occludin identified Tyr-379 and Tyr-383 in chicken occludin as the phosphorylation sites, which are located in a highly conserved sequence of occludin, YETDYTT; Tyr-398 and Tyr-402 are the corresponding residues in human occludin. Deletion of YETDYTT motif abolished the c-Src-mediated phosphorylation of occludin and the regulation of ZO-1 binding. Y398A and Y402A mutations in human occludin also abolished the c-Src-mediated phosphorylation and regulation of ZO-1 binding. Y398D/Y402D mutation resulted in a dramatic reduction in ZO-1 binding even in the absence of c-Src. Similar to wild type occludin, its Y398A/Y402A mutant was localized at the plasma membrane and cell-cell contact sites in Rat-1 cells. However, Y398D/Y402D mutants of occludin failed to localize at the cell-cell contacts. Calcium-induced reassembly of Y398D/Y402D mutant occludin in Madin-Darby canine kidney cells was significantly delayed compared with that of wild type occludin or its T398A/T402A mutant. Furthermore, expression of Y398D/Y402D mutant of occludin sensitized MDCK cells for hydrogen peroxide-induced barrier disruption. This study reveals a unique motif in the occludin sequence that is involved in the regulation of ZO-1 binding by reversible phosphorylation of specific Tyr residues.
We have developed a novel, high-throughput scintillation proximity assay to measure the membraneassociated steps (stages 2 and 3) of peptidoglycan synthesis in Escherichia coli. At least five enzymes are involved in these two stages, all of which are thought to be essential for the survival of the cell. The individual enzymes are difficult to assay since the substrates are lipidic and difficult to isolate in large quantities and analysis is done by paper chromatography. We have assayed all five enzymes in a single mixture by monitoring synthesis of cross-linked peptidoglycan, which is the final product of the pathway. E. coli membranes are incubated with the two sugar precursors, UDP-N-acetyl muramylpentapeptide and UDP-[ 3 H]-N-acetylglucosamine. The radiolabel is incorporated into peptidoglycan, which is captured using wheat germ agglutinincoated scintillation proximity assay beads. The assay monitors the activity of the translocase (MraY), the transferase (MurG), the lipid pyrophosphorylase, and the transglycosylase and transpeptidase activities of the penicillin-binding proteins. Vancomyin, tunicamycin, nisin, moenomycin, bacitracin, and penicillin inhibit the assay, and these inhibitors have been used to validate the assay. The search for new antimicrobial agents that act via the late stages of peptidoglycan biosynthesis can now be performed in high throughput in a microtiter plate.
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