The Transient Increase of Tight Junction Permeability Induced by Bryostatin 1 Correlates with Rapid Downregulation of Protein Kinase C-α

2000 ◽  
Vol 261 (1) ◽  
pp. 239-249 ◽  
Author(s):  
H. Clarke ◽  
N. Ginanni ◽  
K.V. Laughlin ◽  
J.B. Smith ◽  
G.R. Pettit ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tight Junction ◽  
Transient Increase ◽  
Bryostatin 1 ◽  
Kinase C ◽  
Tight Junction Permeability

scholarly journals Dephosphorylation of the catenins p120 and p100 in endothelial cells in response to inflammatory stimuli

1999 ◽  
Vol 338 (2) ◽  
pp. 471-478 ◽  
Author(s):  
Marianne J. RATCLIFFE ◽  
Caroline SMALES ◽  
James M. STADDON
Keyword(s):  
Endothelial Cells ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Tight Junction ◽  
Adherens Junction ◽  
Receptor Activation ◽  
Protein Kinase C Inhibitors ◽  
Kinase C ◽  
Inflammatory Stimuli ◽  
Tight Junction Permeability

Inflammatory mediators such as histamine and thrombin increase the tight-junction permeability of endothelial cells. Tight-junction permeability may be independently controlled, but is dependent on the adherens junction, where adhesion is achieved through homotypic interaction of cadherins, which in turn are associated with cytoplasmic proteins, the catenins. p120, also termed p120cas/p120ctn, and its splice variant, p100, are catenins. p120, originally discovered as a substrate of the tyrosine kinase Src, is also a target for a protein kinase C-stimulated pathway in epithelial cells, causing its serine/threonine dephosphorylation. The present study shows that pharmacological activation of protein kinase C stimulated a similar pathway in endothelial cells. Activation of receptors for agents such as histamine (H1), thrombin and lysophosphatidic acid in the endothelial cells also caused serine/threonine dephosphorylation of p120 and p100, suggesting physiological relevance. However, protein kinase C inhibitors, although blocking the effect of pharmacological activation of protein kinase C, did not block the effects due to receptor activation. Calcium mobilization and the myosin-light-chain-kinase pathway do not participate in p120/p100 signalling. In conclusion, endothelial cells possess protein kinase C-dependent and -independent pathways regulating p120/p100 serine/threonine phosphorylation. These data describe a new connection between inflammatory agents, receptor-stimulated signalling and pathways potentially influencing intercellular adhesion in endothelial cells.

scholarly journals Protein kinase C activation leads to dephosphorylation of occludin and tight junction permeability increase in LLC-PK1 epithelial cell sheets

2000 ◽  
Vol 113 (18) ◽  
pp. 3187-3196 ◽  
Author(s):  
H. Clarke ◽  
A.P. Soler ◽  
J.M. Mullin
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Tight Junction ◽  
Phorbol Ester ◽  
Electrical Resistance ◽  
Rapid Change ◽  
Transepithelial Electrical Resistance ◽  
Kinase C ◽  
Tight Junction Permeability ◽  
Triton X

Activation of protein kinase C by exposure of LLC-PK1 renal epithelial cells to 10(−7) M TPA, a tumor promoting phorbol ester, results in a rapid and sustained increase in paracellular permeability as evidenced by a decrease in transepithelial electrical resistance. Occludin, the first identified transmembrane protein to be localized to the tight junction of both epithelial and endothelial cells is thought play an important role in tight junction barriers. Although transepithelial electrical resistance fell to less than 20% of initial values within 1 hour of TPA exposure, transmission electron microscopy showed no change in the gross morphology of the tight junction of cells treated with 10(−7) M TPA for up to 2 hours. Immunofluorescence microscopy revealed a more rapid change in the membrane distribution of ZO-1 compared to occludin in the TPA-treated cells. Immunoblot analysis indicated that occludin levels in total cell lysates as well as cytosolic, membrane (Triton-X soluble) and cytoskeletal (Triton-X insoluble) fractions remained unchanged for at least 2 hours in cells treated with 10(−7) M TPA compared to their corresponding control cells. As the phosphorylation state of occludin is thought to be important in both tight junction assembly and regulation, the effect of phorbol ester treatment on the phosphorylation of occludin was investigated. Surprisingly, activation of protein kinase C with 10(−7) M TPA resulted in a time-dependent decrease in threonine phosphorylation of occludin which correlated closely with the rapid decrease in transepithelial electrical resistance. This dephosphorylation of occludin, occurring after activation of a serine/threonine kinase by TPA, suggested that protein kinase C was not acting directly on this tight junction target protein. If occludin dephosphorylation is involved in increasing tight junction permeability, then protein kinase C is apparently further upstream in the signaling pathway regulating epithelial barrier function, with a downstream serine/threonine phosphatase acting upon occludin.

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.

Dephosphorylation of activated protein kinase C contributes to downregulation by bryostatin

1996 ◽  
Vol 271 (1) ◽  
pp. C304-C311 ◽  
Author(s):  
H. W. Lee ◽  
L. Smith ◽  
G. R. Pettit ◽  
J. Bingham Smith
Keyword(s):  
Alkaline Phosphatase ◽  
Protein Synthesis ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Bryostatin 1 ◽  
Rapid Loss ◽  
Renal Epithelial Cells ◽  
Kinase C ◽  
Phosphatase Treatment ◽  
Pkc Alpha

We show that bryostatin 1 (Bryo) rapidly produces an inactive, incompetent 76-kDa form of protein kinase C-alpha (PKC-alpha) in the LLC-MK2 line of renal epithelial cells. Bryo, like phorbol 12-myristate 13-acetate (PMA), acutely activated PKC, as indicated by autophosphorylation and translocation of PKC-alpha, the predominant PMA-sensitive isoform expressed by the cells. Bryo concomitantly increased the 32P labeling of 80-kDa PKC-alpha by autophosphorylation and produced a 76-kDa form of PKC-alpha that lacked detectable 32P. The 76-kDa form was in the particulate rather than the cytosolic fraction, which suggests that it was produced from activated kinase. Alkaline phosphatase treatment of immunoprecipitated PKC-alpha converted the 80-kDa form to 76 kDa, but it had no effect on the mobility of the 76-kDa form, suggesting that it was not phosphorylated. Pulse-chase labeling of PKC-alpha with [35S]Met/Cys indicated that there is a precursor-product relationship between the 80- and 76-kDa forms, respectively. Inhibition of protein synthesis had no effect on the production of 76-kDa PKC-alpha by Bryo. PMA also produced 76-kDa PKC-alpha but was less potent and efficacious than Bryo. Bryo produced a more rapid loss of 80-kDa PKC-alpha protein and total Ca(2+)- and phospholipid-dependent PKC activity than PMA. The 76-kDa form is inactive and incompetent because it lacked detectable 32P under conditions that strongly autophosphorylated the 80-kDa form. We suggest that dephosphorylation predisposes PKC to proteolysis, and greater production of the 76-kDa form explains the more efficient downregulation of the kinase by Bryo vs. PMA.

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