scholarly journals Numb controls E-cadherin endocytosis through p120 catenin with aPKC

2011 ◽  
Vol 22 (17) ◽  
pp. 3103-3119 ◽  
Author(s):  
Kazuhide Sato ◽  
Takashi Watanabe ◽  
Shujie Wang ◽  
Mai Kakeno ◽  
Kenji Matsuzawa ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Apical Membrane ◽  
Dominant Negative ◽  
Binding Ability ◽  
P120 Catenin ◽  
Adhesion Sites ◽  
Kinase C ◽  
Apicobasal Polarity ◽  
Intercellular Adhesions ◽  
E Cadherin

Cadherin trafficking controls tissue morphogenesis and cell polarity. The endocytic adaptor Numb participates in apicobasal polarity by acting on intercellular adhesions in epithelial cells. However, it remains largely unknown how Numb controls cadherin-based adhesion. Here, we found that Numb directly interacted with p120 catenin (p120), which is known to interact with E-cadherin and prevent its internalization. Numb accumulated at intercellular adhesion sites and the apical membrane in epithelial cells. Depletion of Numb impaired E-cadherin internalization, whereas depletion of p120 accelerated internalization. Expression of the Numb-binding fragment of p120 inhibited E-cadherin internalization in a dominant-negative fashion, indicating that Numb interacts with the E-cadherin/p120 complex and promotes E-cadherin endocytosis. Impairment of Numb induced mislocalization of E-cadherin from the lateral membrane to the apical membrane. Atypical protein kinase C (aPKC), a member of the PAR complex, phosphorylated Numb and inhibited its association with p120 and α-adaptin. Depletion or inhibition of aPKC accelerated E-cadherin internalization. Wild-type Numb restored E-cadherin internalization in the Numb-depleted cells, whereas a phosphomimetic mutant or a mutant with defective α-adaptin-binding ability did not restore the internalization. Thus, we propose that aPKC phosphorylates Numb to prevent its binding to p120 and α-adaptin, thereby attenuating E-cadherin endocytosis to maintain apicobasal polarity.

Effect of HCO3 − on TPA- and IBMX-induced anion conductances in Necturus gallbladder epithelial cells

2000 ◽  
Vol 279 (5) ◽  
pp. C1385-C1392 ◽  
Author(s):  
Pamela Lyall ◽  
William McD. Armstrong ◽  
Vijay Lyall
Keyword(s):  
Membrane Potential ◽  
Protein Kinase ◽  
Epithelial Cells ◽  
Channel Blocker ◽  
Apical Membrane ◽  
Necturus Gallbladder ◽  
Voltage Ratio ◽  
Kinase C ◽  
Free Media ◽  
Kinase A

Effects of HCO3 − on protein kinase C (PKC)- and protein kinase A (PKA)-induced anion conductances were investigated in Necturus gallbladder epithelial cells. In HCO3 −-free media, activation of PKC via 12- O-tetradecanoylphorbol 13-acetate (TPA) depolarized apical membrane potential ( V a) and decreased fractional apical voltage ratio (FR). These effects were blocked by mucosal 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB), a Cl− channel blocker. In HCO3 −media, TPA induced significantly greater changes in V a and FR. These effects were blocked only when NPPB was present in both mucosal and basolateral compartments. The data suggest that TPA activates NPPB-sensitive apical Cl− conductance ( g Cl a) in the absence of HCO3 −; in its presence, TPA stimulated both NPPB-sensitive g Cl a and basolateral Cl− conductance ( g Cl b). Activation of PKA via 3-isobutyl-1-methylxanthine (IBMX) also decreased V a and FR; however, these changes were not affected by external HCO3 −. We conclude that HCO3 − modulates the effects of PKC on g Cl b. In HCO3 − medium, TPA and IBMX also induced an initial transient hyperpolarization and increase in intracellular pH. Because these changes were independent of mucosal Na+ and Cl−, it is suggested that TPA and IBMX induce a transient increase in apical HCO3 −conductance.

Leukotriene D(4) affects localisation of vinculin in intestinal epithelial cells via distinct tyrosine kinase and protein kinase C controlled events

2001 ◽  
Vol 114 (10) ◽  
pp. 1925-1934 ◽  
Author(s):  
R. Massoumi ◽  
A. Sjolander
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Epithelial Cells ◽  
Tyrosine Kinase ◽  
Intestinal Epithelial Cells ◽  
Focal Adhesions ◽  
Epithelial Cell Line ◽  
Intestinal Epithelial ◽  
Kinase C ◽  
E Cadherin

Local inflammatory reactions affect the integrity of intestinal epithelial cells, such as E-cadherin-mediated cell-cell interactions. To elucidate this event, we investigated the effects of an inflammatory mediator, leukotriene D(4)(LTD(4)), on the phosphorylation status and properties of vinculin, a multi-binding protein known to interact with both the E-cadherin-catenin complex and the cytoskeleton. Treatment of an intestinal epithelial cell line with LTD(4)induced rapid tyrosine phosphorylation of vinculin, which was blocked by the Src family tyrosine kinase inhibitor PP1. Simultaneously, LTD(4) caused an increased association between vinculin and actin, and that association was decreased by PP1. LTD(4) also induced dissociation of vinculin from (α)-catenin without affecting the catenin complex itself. This dissociation was not blocked by PP1 but was mimicked by the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Also, the PKC inhibitor GF109203X abolished both the LTD(4)- and the TPA-induced dissociation of vinculin from (α)-catenin. Furthermore, LTD(4) caused a colocalisation of vinculin with PKC-(α) in focal adhesions. This accumulation of vinculin was blocked by transfection with a dominant negative inhibitor of PKC (PKC regulatory domain) and also by preincubation with either GF109203X or PP1. Thus, various LTD(4)-induced phosphorylations of vinculin affect the release of this protein from catenin complexes and its association with actin, two events that are necessary for accumulation of vinculin in focal adhesions. Functionally this LTD(4)-induced redistribution of vinculin was accompanied by a PKC-dependent upregulation of active (β)1 integrins on the cell surface and an enhanced (β)1 integrin-dependent adhesion of the cells to collagen IV.

scholarly journals Mistargeting of a truncated Na-K-2Cl cotransporter in epithelial cells

2018 ◽  
Vol 315 (2) ◽  
pp. C258-C276 ◽  
Author(s):  
Rainelli Koumangoye ◽  
Salma Omer ◽  
Eric Delpire
Keyword(s):  
Epithelial Cells ◽  
De Novo ◽  
Apical Membrane ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Dominant Negative ◽  
De Novo Mutation ◽  
Wild Type ◽  
Saliva Secretion

We recently reported the case of a young patient with multisystem failure carrying a de novo mutation in SLC12A2, the gene encoding the Na-K-2Cl cotransporter-1 (NKCC1). Heterologous expression studies in nonepithelial cells failed to demonstrate dominant-negative effects. In this study, we examined expression of the mutant cotransporter in epithelial cells. Using Madin-Darby canine kidney (MDCK) cells grown on glass coverslips, permeabilized support, and Matrigel, we show that the fluorescently tagged mutant cotransporter is expressed in cytoplasm and at the apical membrane and affects epithelium integrity. Expression of the mutant transporter at the apical membrane also results in the mislocalization of some of the wild-type transporter to the apical membrane. This mistargeting is specific to NKCC1 as the Na+-K+-ATPase remains localized on the basolateral membrane. To assess transporter localization in vivo, we created a mouse model using CRISPR/cas9 that reproduces the 11 bp deletion in exon 22 of Slc12a2. Although the mice do not display an overt phenotype, we show that the colon and salivary gland expresses wild-type NKCC1 abundantly at the apical pole, confirming the data obtained in cultured epithelial cells. Enough cotransporter must remain, however, on the basolateral membrane to participate in saliva secretion, as no significant decrease in saliva production was observed in the mutant mice.

scholarly journals Intestinal HT-29 cells with dysfunction of E-cadherin show increased pp60src activity and tyrosine phosphorylation of p120-catenin

1996 ◽  
Vol 317 (1) ◽  
pp. 279-284 ◽  
Author(s):  
Anouchka SKOUDY ◽  
Maria del Mont LLOSAS ◽  
Antonio GARCÍA de HERREROS
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Kinase Inhibitor ◽  
The Other ◽  
P120 Catenin ◽  
Cell Contacts ◽  
Herbimycin A ◽  
Kinase C ◽  
Ht 29 ◽  
E Cadherin

1. HT-29 M6 cells are a subpopulation of HT-29 cells that, contrarily to the parental cells, establish tight cell contacts and differentiate. Cell-to-cell contacts in HT-29 M6 cells are also regulated by protein kinase C; addition of the phorbol ester phorbol 12-myristate 13-acetate (PMA) decreases the homotypic contacts of these cells. We show here that HT-29 cells or HT-29 M6 cells treated with PMA contain lower levels of functional E-cadherin, determined by analysing the association of this protein with the cytoskeleton. No significant differences in the localization of α-, β-, or p120-catenins were detected under the three different conditions. 2. Dysfunction of E-cadherin can be reversed by incubation of HT-29 cells with the tyrosine kinase inhibitor herbimycin A. On the other hand an augmentation of c-src activity in HT-29 cells or HT-29 M6 cells treated with PMA was observed with respect to control HT-29 M6 cells. The phosphorylation status of catenins was also investigated; in HT-29 or in HT-29 M6 cells treated with PMA, dysfunction of E-cadherin was accompanied by an increased phosphorylation of p120-catenin and by an elevated association of this protein to E-cadherin. These results suggest a role for pp60src and the pp60src substrate p120-catenin in the control of E-cadherin function in HT-29 cells.

scholarly journals Kv1.1 and Kv1.3 channels contribute to the delayed-rectifying K+ conductance in rat choroid plexus epithelial cells

2004 ◽  
Vol 286 (3) ◽  
pp. C611-C620 ◽  
Author(s):  
Tracey Speake ◽  
Jonathan D. Kibble ◽  
Peter D. Brown
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Epithelial Cells ◽  
Choroid Plexus ◽  
Apical Membrane ◽  
Paired Data ◽  
Kinase C ◽  
Kv1.3 Channels ◽  
Activate Protein Kinase ◽  
Choroid Plexus Epithelial

The choroid plexuses secrete, and maintain the composition of, the cerebrospinal fluid. K+ channels play an important role in these processes. In this study the molecular identity and properties of the delayed-rectifying K+ (Kv) conductance in rat choroid plexus epithelial cells were investigated. Whole cell K+ currents were significantly reduced by 10 nM dendrotoxin-K and 1 nM margatoxin, which are specific inhibitors of Kv1.1 and Kv1.3 channels, respectively. A combination of dendrotoxin-K and margatoxin caused a depolarization of the membrane potential in current-clamp experiments. Western blot analysis indicated the presence of Kv1.1 and Kv1.3 proteins in the choroid plexus. Furthermore, the Kv1.3 and Kv1.1 proteins appear to be expressed in the apical membrane of the epithelial cells in immunocytochemical studies. The Kv conductance was inhibited by 1 μM serotonin (5-HT), with maximum inhibition to 48% of control occurring in 8 min ( P < 0.05 by Student's t-test for paired data). Channel inhibition by 5-HT was prevented by the 5-HT2C antagonist mesulergine (300 nM). It was also attenuated in the presence of calphostin C (a protein kinase C inhibitor). The conductance was partially inhibited by 1,2-dioctanoyl- sn-glycerol and phorbol 12-myristate 13-acetate, both of which activate protein kinase C. These data suggest that 5-HT acts at 5-HT2C receptors to activate protein kinase C, which inhibits the Kv channels. In conclusion, Kv1.1 and Kv1.3 channels make a significant contribution to K+ efflux at the apical membrane of the choroid plexus.

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