Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The making and sealing of a tight junction (TJ) requires cell-cell contacts and Ca2+, and can be gauged through the development of transepithelial electrical resistance (TER) and the accumulation of ZO-1 peptide at the cell borders. We observe that pertussis toxin increases TER, while AIF3 and carbamil choline (carbachol) inhibit it, and 5-guanylylimidodiphosphate (GTPTs) blocks the development of a cell border pattern of ZO-1, suggesting that G-proteins are involved. Phospholipase C (PLC) and protein kinase C (PKC) probably participate in these processes since (i) activation of PLC by thyrotropin-1 releasing hormone increases TER, and its inhibition by neomycin blocks the development of this resistance; (ii) 1,2-dioctanoylglycerol, an activator of PKC, stimulates TER development, while polymyxin B and 1-(5-isoquinoline sulfonyl)-2-methyl-piperazine dihydrochloride (H7), which inhibit this enzyme, abolish TER. Addition of 3-isobutyl-1-methyl-xanthine, dB-cAMP or forskolin do not enhance the value of TER, but have just the opposite effect. Trifluoperazine and calmidazoline inhibit TER development, suggesting that calmodulin (CaM) also plays a role in junction formation. These results indicate that junction formation may be controlled by a network of reactions where G-proteins, phospholipase C, adenylate cyclase, protein kinase C and CaM are involved.
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PMID:Assembly and sealing of tight junctions: possible participation of G-proteins, phospholipase C, protein kinase C and calmodulin. 192 Mar 85

We have previously shown that protein phosphorylation plays an important role in the sorting and assembly of tight junctions. We have now examined in detail the role of protein kinases in intercellular junction biogenesis by using a combination of highly specific and broad-spectrum inhibitors that act by independent mechanisms. Our data indicate that protein kinase C (PKC) is required for the proper assembly of tight junctions. Low concentrations of the specific inhibitor of PKC, calphostin C, markedly inhibited development of transepithelial electrical resistance, a functional measure of tight-junction biogenesis. The effect of PKC inhibitors on the development of tight junctions, as measured by resistance, was paralleled by a delay in the sorting of the tight-junction protein, zona occludens 1 (ZO-1), to the tight junction. The assembly of desmosomes and the adherens junction were not detectably affected, as determined by immunocytochemical analysis. In addition, ZO-1 was phosphorylated subsequent to the initiation of cell-cell contact, and treatment with calphostin C prevented approximately 85% of the phosphorylation increase. Furthermore, in vitro measurements indicate that ZO-1 may be a direct target of PKC. Moreover, membrane-associated PKC activity more than doubled during junction assembly, and immunocytochemical analysis revealed a pool of PKC zeta that appeared to colocalize with ZO-1 at the tight junction. A preformed complex containing ZO-1, ZO-2, p130, as well as 330- and 65-kDa phosphoproteins was detected by coimmunoprecipitation in both the presence and absence of cell-cell contact. Identity of the 330- and 65-kDa phosphoproteins remains to be determined, but the 65-kDa protein may be occludin. The mass of this complex and the incorporation of ZO-1 into the Triton X-100-insoluble cytoskeleton were not PKC dependent.
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PMID:Regulated assembly of tight junctions by protein kinase C. 759 83

Extracellular Ca2+ triggers assembly and sealing of tight junctions (TJs) in MDCK cells. These events are modulated by G-proteins, phospholipase C, protein kinase C (PKC), and calmodulin. In the present work we observed that 1,2-dioctanoylglycerol (diC8) promotes the assembly of TJ in low extracellular Ca2+, as evidenced by translocation of the TJ-associated protein ZO-1 to the plasma membrane, formation of junctional fibrils observed in freeze-fracture replicas, decreased permeability of the intercellular space to [3H]mannitol, and reorganization of actin filaments to the cell periphery, visualized by fluorescence microscopy using rhodamine-phalloidin. In contrast, diC8 in low Ca2+ did not induce redistribution of the Ca-dependent adhesion protein E-cadherin (uvomorulin). Extracellular antibodies to E-cadherin block junction formation normally induced by adding Ca2+. diC8 counteracted this inhibition, suggesting that PKC may be in the signaling pathway activated by E-cadherin-mediated cell-cell adhesion. In addition, we found a novel phosphoprotein of 130 kD which coimmunoprecipitated with the ZO-1/ZO-2 complex. Although the assembly and sealing of TJs may involve the activation of PKC, the level of phosphorylation of ZO-1, ZO-2, and the 130-kD protein did not change after adding Ca2+ or a PKC agonist. The complex of these three proteins was present even in low extracellular Ca2+, suggesting that the addition of Ca2+ or diC8 triggers the translocation and assembly of preformed TJ subcomplexes.
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PMID:Assembly of the tight junction: the role of diacylglycerol. 840 13

Recent evidence suggests that the formation and permeability of tight junctions are actively regulated by second-messenger-generating systems involving G proteins and protein kinase C (PKC). A possible specific target for these regulatory proteins is the tight junction protein ZO-1. An extensive immunocytochemical study was performed in cultured epithelial monolayers of MDCK and Caco-2 cells to identify which isoforms of G proteins and PKC are present at or near zonula occludens complex. Antibodies against alpha-subunits of each one of the four major subfamilies were used for the localization of the G proteins. For the PKC localization, antibodies against eight different isoforms were used. In confluent monolayers, G alpha 12 and PKC zeta, were the only isoforms of these proteins at the cell borders. In subconfluent monolayers, G alpha 12 and PKC zeta were found at the plasma membrane only along the areas of lateral cell-cell contact. These isoforms formed a pattern of distribution very similar to the ZO-1 protein. The present findings indicate that G alpha 12 and PKC zeta may be part of the zonula occludens complex and may locally regulate formation and permeability of tight junctions.
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PMID:Identification of isoforms of G proteins and PKC that colocalize with tight junctions. 880 52

The tight junction (TJ) of polarized epithelial cells is critical for maintaining an impermeant barrier and epithelial cell polarity. The signaling events important for TJ assembly require regulated calcium stores and protein kinase C (PKC), but the earliest signaling events in the cascade have not been well defined. We now show that Galphai2 in Madin Darby canine kidney (MDCK) cells localizes to a region overlapping with the TJ. To further analyze the localization of Galpha subunits in epithelial cells, rat Galphao, Q205Lalphao (Galphao "activated" by point mutation) and plasmid without insert (PC) were transfected into MDCK cells and localized by immunofluorescence and confocal microscopy. Similar to endogenous Galphai2, Galphao-MDCK cells localize Galphao, (84% similar to Galphai2) in the subapical region overlapping with ZO-1 (zona occludens-I), a key component of the TJ. PC-MDCK cells have no detectable Galphao. In Galphao-MDCK cells, a physical association of Galphao with components of the TJ was detectable by immunoprecipitation of ZO-1. Immunoprecipitates of ZO-1 from Galphao-MDCK cells consistently coprecipitated Galphao. Constitutively active Q205LGalphao localized to the subapical lateral membrane similar to wild-type Galphao. To determine if constitutively activated Galpha subunits can affect TJ biogenesis, the formation of tight junctions in PC, Galphao, and Q205Lalphao-MDCK cells was followed by measurement of transepithelial resistance (TER) during the Ca2+ switch, a model widely used to study mechanisms of junctional assembly. Baseline and post Ca2+ switch TER values did not differ among the cell lines. However, constitutively activated Q205Lalphao-MDCK cells developed TER significantly faster than PC and Galphao cells in the early phase (0-4 h) (54 +/- 4 versus 23 +/- 3 (PC); 12 +/- 1 (Galphao) Omega.m2/h) and late phase (4-h peak) (117 +/- 10 versus 45 +/- 5 (PC); 66 +/- 7 (Galphao) Omega.m2/h) after Ca2+ switch. Peak TER values were significantly higher in Q205Lalphao-MDCK cells (1168 +/- 107 versus 437 +/- 37 (PC); 548 +/- 54 (Galphao) Omega.cm2). These results indicate that Galphao and Q205Lalphao expressed in MDCK cells are localized near the junctional complex, associate with at least one TJ protein, and that activated Galphao accelerates TJ biogenesis without significantly affecting the maintenance of the TJ. Together, these results suggest an important role for heterotrimeric G proteins in TJ assembly.
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PMID:Involvement of a heterotrimeric G protein alpha subunit in tight junction biogenesis. 882 2

Classically, the contiguous epithelial cells show intercellular tight junctions (TJ) positioned as a continuous belt of cell-cell contacts at the boundary of apical and basolateral plasma membranes. Among the epithelia, the "typical" cerebral ependyma is very peculiar due to the absence of TJ, in contrast to the ependyma of the circumventricular organs (e.g. choroid plexus and the subcommissural organ) which shows well differentiated TJ. Since the "typical" ependyma is covered in the rat with a plexus of intraventricular nervous fibres not present at the surface of the circumventricular organs, we hypothesized local repression of TJ by molecules (serotonin, GABA, etc.) released by the supra-ependymal varicosities. Neither the denervation of the "typical" ependyma nor the ex vivo activation of protein kinase C (which increases the transepithelial resistance as it has been reported in other epithelia) produced junctional fibrils as shown by freeze-fracture. As the protein ZO-1 was not detectable in the "typical" ependyma by immunocytochemistry, there is probably repression of the genes responsible for TJ biosynthesis by unidentified endogenous factors.
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PMID:The supra-ependymal innervation is not responsible for the repression of tight junctions in the rat cerebral ependyma. 904 45

Polarized epithelial cells separate two extremely different cellular milieus. The tight junction (TJ) is the most apical component of the junctional complex and serves as the permeability barrier between these environments. The tight junctional complex appears to be a dynamic and regulated structure. Some of its protein components have been identified and include the transmembrane protein occludin. Nontransmembrane proteins on the cytosolic leaflet including ZO-1, ZO-2, cingulin, 7H6, and several unidentified phosphoproteins are also believed to be part of the TJ. Interactions of some of these proteins with the actin cytoskeleton are a major determinant of TJ structure and may also play a role in the regulation of TJ assembly. Recent progress using the "calcium switch" and the "ATP depletion-repletion" model of TJ formation offers new insight regarding how these structures form. TJ biogenesis appears to be regulated, in part, by classic signal transduction pathways involving heterotrimeric G proteins, release of intracellular Ca2+, and activation of protein kinase C. Although many of the details of the signaling pathways have yet to be defined, these observations may provide insight into how TJs form during tubular development. Furthermore, it may be possible to suggest potential therapeutic targets for intervention in a variety of diseases (e.g., ischemia, toxic injury to the kidney and other epithelial tissue) where TJ integrity has been compromised and reassembly is required.
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PMID:Molecular structure and assembly of the tight junction. 945 17

Enterohemorrhagic Escherichia coli (EHEC) infection is associated with watery diarrhea and can lead to complications, including hemorrhagic colitis and the hemolytic-uremic syndrome. The mechanisms by which these organisms produce diarrheal disease remain to be elucidated. Changes in T84 epithelial cell electrophysiology were examined following EHEC infection. T84 cell monolayers infected with EHEC O157:H7 displayed a time-dependent decrease in transepithelial resistance. Increases in the transepithelial flux of both [3H]mannitol and 51Cr-EDTA accompanied the EHEC-induced decreases in T84 resistance. Altered barrier function induced by EHEC occurred at the level of the tight junction since immunofluorescent staining of the tight-junction-associated protein ZO-1 was disrupted when examined by confocal microscopy. Decreased resistance induced by EHEC involved a protein kinase C (PKC)-dependent pathway as the highly specific PKC inhibitor, CGP41251, abrogated the EHEC-induced drop in resistance. PKC activity was also increased in T84 cells infected with EHEC. Calmodulin and myosin light chain kinase played a role in EHEC-induced resistance changes as inhibition of these effector molecules partially reversed the effects of EHEC on barrier function. These studies demonstrate that intracellular signal transduction pathways activated following EHEC infection link the increases in T84 epithelial permeability induced by this pathogen.
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PMID:Signal transduction pathways involved in enterohemorrhagic Escherichia coli-induced alterations in T84 epithelial permeability. 952 98

The tight junction creates a regulated barrier in the paracellular pathway and, together with the actin-rich adherens junction, forms a functional unit called the apical junction complex. A growing number of tight junction-associated proteins have been identified, but functions are defined for only a few. The intercellular barrier is formed by rows of the transmembrane protein occludin, which is bound on the cytoplasmic surface to ZO-1 and ZO-2. These proteins are members of the membrane-associated guanylate kinase (MAGUK) protein family and are likely to have both structural and signaling roles. Junctional plaque proteins without known functions include cingulin, p130, and 7H6; single reports describe ZA-1TJ and symplekin. Many cellular signaling pathways affect assembly and sealing of junctions. Transducing proteins, which localize within the junction, include both heterotrimeric and rho-related GTP-binding proteins, PKC-zeta and nonreceptor tyrosine kinases. Control of perijunctional actin may be the unifying mechanism for regulating paracellular permeability.
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PMID:Molecular architecture of tight junctions. 955 57

The molecular composition and functional properties of cell-cell junctions of choroid plexus epithelial cells and the ependyma of the lateral ventricular wall were investigated in the rat brain. Expression studies of cadherin and alpha- and beta-catenins, as well as expression of occludin and ZO-1, indicated that cell adherens and tight junctions were present in both choroid plexus epithelial cells and in ependymal cells. We then tested the hypothesis that phorbolester in vivo can induce changes in the expression level of adherens and tight junction molecules at the blood-cerebrospinal fluid (CSF) barrier as well as in the ependyma. In addition, the functional properties of the ependymal junctions were tested by injection of dextran 3000 into the striatum after phorbolester application. Twenty-four hours after phorbolester-injection into the lateral ventricle of the rat brain, the expression patterns of tight and adherens junction molecules were markedly changed in the epithelial cells of the choroid plexus. The adherens junction proteins cadherin and beta-catenin were reduced in both the ependymal cells of the lateral ventricle and choroid plexus epithelial cells. In addition, the occludin-immunoreactivity of the choroid plexus epithelial cells was strongly reduced. However, the ZO-1 immunoreactivity was not affected by the phorbol ester-treatment and the alpha-catenin immunoreactivity was not changed. Furthermore, phorbol ester injection induced a reduction of the volume of intrastriatal injected biotinylated dextran (m.w. 3000), which is consistent with a modulatory influence of protein kinase C activation on the clearance capacity of the brain.
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PMID:Phorbol ester induced changes in tight and adherens junctions in the choroid plexus epithelium and in the ependyma. 1078 22


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