scholarly journals Protein kinase Cζ phosphorylates occludin and promotes assembly of epithelial tight junctions

2011 ◽  
Vol 437 (2) ◽  
pp. 289-299 ◽  
Author(s):  
Suneet Jain ◽  
Takuya Suzuki ◽  
Ankur Seth ◽  
Geetha Samak ◽  
Radhakrishna Rao
Keyword(s):  
Protein Kinase ◽  
Tight Junction ◽  
Tight Junctions ◽  
Intercellular Junctions ◽  
Cell Monolayers ◽  
Terminal Domain ◽  
Tight Junction Regulation ◽  
Epithelial Tight Junctions ◽  
Zona Occludens

Protein kinases play an important role in the regulation of epithelial tight junctions. In the present study, we investigated the role of PKCζ (protein kinase Cζ) in tight junction regulation in Caco-2 and MDCK (Madin–Darby canine kidney) cell monolayers. Inhibition of PKCζ by a specific PKCζ pseudosubstrate peptide results in redistribution of occludin and ZO-1 (zona occludens 1) from the intercellular junctions and disruption of barrier function without affecting cell viability. Reduced expression of PKCζ by antisense oligonucleotide or shRNA (short hairpin RNA) also results in compromised tight junction integrity. Inhibition or knockdown of PKCζ delays calcium-induced assembly of tight junctions. Tight junction disruption by PKCζ pseudosubstrate is associated with the dephosphorylation of occludin and ZO-1 on serine and threonine residues. PKCζ directly binds to the C-terminal domain of occludin and phosphorylates it on threonine residues. Thr403, Thr404, Thr424 and Thr438 in the occludin C-terminal domain are the predominant sites of PKCζ-dependent phosphorylation. A T424A or T438A mutation in full-length occludin delays its assembly into the tight junctions. Inhibition of PKCζ also induces redistribution of occludin and ZO-1 from the tight junctions and dissociates these proteins from the detergent-insoluble fractions in mouse ileum. The present study demonstrates that PKCζ phosphorylates occludin on specific threonine residues and promotes assembly of epithelial tight junctions.

Tight junction regulation through vesicle trafficking: bringing cells together

2014 ◽  
Vol 42 (1) ◽  
pp. 195-200 ◽  
Author(s):  
Sarah J. Fletcher ◽  
Joshua Z. Rappoport
Keyword(s):  
Steady State ◽  
Tight Junction ◽  
Tight Junctions ◽  
Vesicle Trafficking ◽  
Present Review ◽  
Physiological Processes ◽  
Endosomal Recycling ◽  
Tight Junction Regulation ◽  
Epithelial Layers

Epithelial layers are integral for many physiological processes and are maintained by intercellular adhesive structures. During disease, these structures can disassemble, leading to breakdown of epithelia. TJs (tight junctions) are one type of intercellular adhesion. Loss of TJs has been linked to the pathogenesis of many diseases. The present review focuses on the role of vesicle trafficking in regulation of TJs, in particular trafficking of the TJ protein occludin. We examine how endocytosis and endosomal recycling modulate occludin localization under steady-state conditions and during stimulated TJ disassembly.

scholarly journals Contrasting effects of ERK on tight junction integrity in differentiated and under-differentiated Caco-2 cell monolayers

2010 ◽  
Vol 433 (1) ◽  
pp. 51-63 ◽  
Author(s):  
Sudhir Aggarwal ◽  
Takuya Suzuki ◽  
William L. Taylor ◽  
Aditi Bhargava ◽  
Radhakrishna K. Rao
Keyword(s):  
Protein Kinase ◽  
Tight Junction ◽  
Tight Junctions ◽  
Kinase Inhibitor ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Cell Monolayers ◽  
Differentiated Cells ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

ERK (extracellular-signal-regulated kinase) activation leads to disruption of tight junctions in some epithelial monolayers, whereas it prevents disruption of tight junctions in other epithelia. The factors responsible for such contrasting influences of ERK on tight junction integrity are unknown. The present study investigated the effect of the state of cell differentiation on ERK-mediated regulation of tight junctions in Caco-2 cell monolayers. EGF (epidermal growth factor) potentiated H2O2-induced tight junction disruption in under-differentiated cell monolayers, which was attenuated by the MEK [MAPK (mitogen-activated protein kinase)/ERK kinase] inhibitor U0126. In contrast, EGF prevented H2O2-induced disruption of tight junctions in differentiated cell monolayers, which was also attenuated by U0126. Knockdown of ERK1/2 enhanced tight junction integrity and accelerated assembly of tight junctions in under-differentiated cell monolayers, whereas it had the opposite effect in differentiated cell monolayers. Regulated expression of wild-type and constitutively active MEK1 disrupted tight junctions, and the expression of dominant-negative MEK1 enhanced tight junction integrity in under-differentiated cells, whereas contrasting responses were recorded in differentiated cells. EGF prevented both H2O2-induced association of PP2A (protein phosphatase 2A), and loss of association of PKCζ (protein kinase Cζ), with occludin by an ERK-dependent mechanism in differentiated cell monolayers, but not in under-differentiated cell monolayers. Active ERK was distributed in the intracellular compartment in under-differentiated cell monolayers, whereas it was localized mainly in the perijunctional region in differentiated cell monolayers. Thus ERK may exhibit its contrasting influences on tight junction integrity in under-differentiated and differentiated epithelial cells by virtue of differences in its subcellular distribution and ability to regulate the association of PKCζ and PP2A with tight junction proteins.

scholarly journals Acetaldehyde disrupts tight junctions in Caco-2 cell monolayers by a protein phosphatase 2A-dependent mechanism

2012 ◽  
Vol 303 (12) ◽  
pp. G1356-G1364 ◽  
Author(s):  
Mitzi Dunagan ◽  
Kamaljit Chaudhry ◽  
Geetha Samak ◽  
R. K. Rao
Keyword(s):  
Tight Junctions ◽  
Kinase Inhibitor ◽  
Ex Vivo ◽  
Intercellular Junctions ◽  
Ethanol Administration ◽  
Intestinal Epithelial ◽  
Barrier Dysfunction ◽  
Inhibitory Peptide ◽  
Cell Monolayers ◽  
Epithelial Tight Junctions

Acetaldehyde is accumulated at high concentrations in the colonic lumen following ethanol administration. Previous studies demonstrated that acetaldehyde disrupts intestinal epithelial tight junctions and increases paracellular permeability. In the present study, we investigated the role of PP2A in the acetaldehyde-induced disruption of intestinal epithelial tight junctions. Caco-2 cell monolayers were exposed to 200–600 μM acetaldehyde for varying times, and the epithelial barrier function was evaluated by measuring transepithelial electrical resistance and inulin permeability. Acetaldehyde treatment resulted in a time-dependent increase in inulin permeability and redistribution of occludin and ZO-1 from the intercellular junctions. Treatment of cells with fostriecin (a PP2A-selective inhibitor) or knockdown of PP2A by siRNA blocked acetaldehyde-induced increase in inulin permeability and redistribution of occludin and ZO-1. The effects of fostriecin and acetaldehyde were confirmed in mouse intestine ex vivo. Acetaldehyde-induced tight junction disruption and barrier dysfunction were also attenuated by a PP2A-specific inhibitory peptide, TPDYFL. Coimmunoprecipitation studies showed that acetaldehyde increased the interaction of PP2A with occludin and induced dephosphorylation of occludin on threonine residues. Fostriecin and TPDYFL significantly reduced acetaldehyde-induced threonine dephosphorylation of occludin. Acetaldehyde failed to change the level of the methylated form of PP2A-C subunit. However, genistein (a tyrosine kinase inhibitor) blocked acetaldehyde-induced association of PP2A with occludin and threonine dephosphorylation of occludin. These results demonstrate that acetaldehyde-induced disruption of tight junctions is mediated by PP2A translocation to tight junctions and dephosphorylation of occludin on threonine residues.

The Role of the N-Terminal Domain in the Regulation of the “Constitutively Active” Conformation of Protein Kinase CK2α: Insight from a Molecular Dynamics Investigation

ChemMedChem ◽  
2011 ◽  
Vol 6 (7) ◽  
pp. 1207-1216 ◽  
Author(s):  
Andrea Cristiani ◽  
Giorgio Costa ◽  
Giorgio Cozza ◽  
Flavio Meggio ◽  
Leonardo Scapozza ◽  
...  
Keyword(s):  
Molecular Dynamics ◽  
Protein Kinase ◽  
Active Conformation ◽  
Terminal Domain ◽  
Constitutively Active

scholarly journals Rab13 regulates PKA signaling during tight junction assembly

2004 ◽  
Vol 165 (2) ◽  
pp. 175-180 ◽  
Author(s):  
Katja Köhler ◽  
Daniel Louvard ◽  
Ahmed Zahraoui
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Inhibitory Effect ◽  
Cell Junctions ◽  
Unknown Mechanism ◽  
Epithelial Tight Junctions ◽  
Actin Remodelling ◽  
Cell Cell

The GTPase Rab13 regulates the assembly of functional epithelial tight junctions (TJs) through a yet unknown mechanism. Here, we show that expression of the GTP-bound form of Rab13 inhibits PKA-dependent phosphorylation and TJ recruitment of the vasodilator-stimulated phosphoprotein, an actin remodelling protein. We demonstrate that Rab13GTP directly binds to PKA and inhibits its activity. Interestingly, activation of PKA abrogates the inhibitory effect of Rab13 on the recruitment of vasodilator-stimulated phosphoprotein, ZO-1, and claudin1 to cell–cell junctions. Rab13 is, therefore, the first GTPase that controls PKA activity and provides an unexpected link between PKA signaling and the dynamics of TJ assembly.

scholarly journals Changes in Tight Junctional Resistance of the Cervical Epithelium Are Associated with Modulation of Content and Phosphorylation of Occludin 65-Kilodalton and 50-Kilodalton Forms

Endocrinology ◽  
2006 ◽  
Vol 147 (2) ◽  
pp. 977-989 ◽  
Author(s):  
Ling Zhu ◽  
Xin Li ◽  
Robin Zeng ◽  
George I. Gorodeski
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tight Junction ◽  
Tyrosine Phosphorylation ◽  
Tight Junctions ◽  
Early Stage ◽  
Cervical Epithelium ◽  
Kinase C ◽  
Low Levels ◽  
Junctional Resistance

Treatment of human cervical epithelial CaSki cells with ATP or with the diacylglyceride sn-1,2-dioctanoyl diglyceride (diC8) induced a staurosporine-sensitive transient increase, followed by a late decrease, in tight-junctional resistance (RTJ). CaSki cells express two immunoreactive forms of occludin, 65 and 50 kDa. Treatments with ATP and diC8 decreased the density of the 65-kDa form and increased the density of the 50-kDa form. ATP also decreased threonine phosphorylation of the 65-kDa form and increased threonine phosphorylation of the 50-kDa form and tyrosine phosphorylation of the 65- and 50-kDa forms. Staurosporine decreased acutely threonine and tyrosine phosphorylation of the two isoforms and in cells pretreated with staurosporine ATP increased acutely the density of the 65-kDa form and threonine phosphorylation of the 65-kDa form. Treatment with N-acetyl-leucinyl-leucinyl-norleucinal increased the densities of the 65- and 50-kDa forms. Pretreatment with N-acetyl-leucinyl-leucinyl-norleucinal attenuated the late decreases in RTJ induced by ATP and diC8 and the decrease in the 65-kDa and increase in the 50-kDa forms induced by ATP. Correlation analyses showed that high levels of RTJ correlated with the 65-kDa form, whereas low levels of RTJ correlated negatively with the 65-kDa form and positively with the 50-kDa form. The results suggest that in CaSki cells 1) occludin determines gating of the tight junctions, 2) changes in occludin phosphorylation status and composition regulate the RTJ, 3) protein kinase-C-mediated, threonine dephosphorylation of the 65-kDa occludin form increases the resistance of assembled tight junctions, 4) the early stage of tight junction disassembly involves calpain-mediated breakdown of occludin 65-kDa form to the 50-kDa form, and 5) increased levels of the 50-kDa form interfere with occludin gating of the tight junctions.

Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation

1997 ◽  
Vol 273 (4) ◽  
pp. C1378-C1385 ◽  
Author(s):  
Jerrold R. Turner ◽  
Brian K. Rill ◽  
Susan L. Carlson ◽  
Denise Carnes ◽  
Rachel Kerner ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Kinase Inhibitors ◽  
Tight Junction Regulation ◽  
Tight Junction Permeability ◽  
Mlc Phosphorylation

Tight junctions serve as the rate-limiting barrier to passive movement of hydrophilic solutes across intestinal epithelia. After activation of Na+-glucose cotransport, the permeability of intestinal tight junctions is increased. Because previous analyses of this physiological tight junction regulation have been restricted to intact mucosae, dissection of the mechanisms underlying this process has been limited. To characterize this process, we have developed a reductionist model consisting of Caco-2 intestinal epithelial cells transfected with the intestinal Na+-glucose cotransporter, SGLT1. Monolayers of SGLT1 transfectants demonstrate physiological Na+-glucose cotransport. Activation of SGLT1 results in a 22 ± 5% fall in transepithelial resistance (TER) ( P< 0.001). Similarly, inactivation of SGLT1 by addition of phloridzin increases TER by 24 ± 2% ( P < 0.001). The increased tight junction permeability is size selective, with increased flux of small nutrient-sized molecules, e.g., mannitol, but not of larger molecules, e.g., inulin. SGLT1-dependent increases in tight junction permeability are inhibited by myosin light-chain kinase inhibitors (20 μM ML-7 or 40 μM ML-9), suggesting that myosin regulatory light-chain (MLC) phosphorylation is involved in tight junction regulation. Analysis of MLC phosphorylation showed a 2.08-fold increase after activation of SGLT1 ( P< 0.01), which was inhibited by ML-9 ( P < 0.01). Thus monolayers incubated with glucose and myosin light-chain kinase inhibitors are comparable to monolayers incubated with phloridzin. ML-9 also inhibits SGLT1-mediated tight junction regulation in small intestinal mucosa ( P < 0.01). These data demonstrate that epithelial cells are the mediators of physiological tight junction regulation subsequent to SGLT1 activation. The intimate relationship between tight junction regulation and MLC phosphorylation suggests that a critical step in regulation of epithelial tight junction permeability may be myosin ATPase-mediated contraction of the perijunctional actomyosin ring and subsequent physical tension on the tight junction.

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