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. 2002 Apr;13(4):1227-37.
doi: 10.1091/mbc.01-08-0423.

Nonreceptor tyrosine kinase c-Yes interacts with occludin during tight junction formation in canine kidney epithelial cells

Yan-Hua Chen  1 , Qun Lu, Daniel A Goodenough, Beverly Jeansonne
Affiliations

Nonreceptor tyrosine kinase c-Yes interacts with occludin during tight junction formation in canine kidney epithelial cells

Yan-Hua Chen et al. Mol Biol Cell. 2002 Apr.

Abstract

Occludin is an integral membrane protein that is tyrosine phosphorylated when localized at tight junctions. When Ca(2+) was depleted from the culture medium, occludin tyrosine phosphorylation was diminished from Madin-Darby canine kidney epithelial cells in 2 min. This dephosphorylation was correlated with a significant reduction in transepithelial electrical resistance (TER), indicating a global loss of the tight junction barrier function. Reconstitution of Ca(2+) resulted in a robust tyrosine rephosphorylation of occludin that was temporally associated with an increase in TER. Moreover, we demonstrate in this study that occludin was colocalized with the nonreceptor tyrosine kinase c-Yes at cell junction areas and formed an immunoprecipitable complex with c-Yes in vivo. This complex dissociated when the cells were incubated in medium without Ca(2+) or treated with a c-Yes inhibitor, CGP77675. In the presence of CGP77675 after Ca(2+) repletion, occludin tyrosine phosphorylation was completely abolished and both tight junction formation and the increase of the TER were inhibited. Our study thus provides strong evidence that occludin tyrosine phosphorylation is tightly linked to tight junction formation in epithelial cells, and that the nonreceptor tyrosine kinase c-Yes is involved in the regulation of this process.

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Figures

Figure 1
Figure 1
Changes in tyrosine phosphorylation of occludin and ZO-1 during Ca2+ depletion. Cells were grown in Ca2+-containing medium until they were confluent (0 min). Then cells were switched to the medium without Ca2+ for 2, 10, 30, and 60 min. After Ca2+ depletion at desired time points, cells were lysed in RIPA buffer, immunoprecipitated with anti-occludin (A) or anti-ZO-1 (B), and immunoblotted with anti-phosphotyrosine antibody.
Figure 2
Figure 2
TER measurements during Ca2+ depletion. TER was measured at 0, 2, 10, 30, and 60 min after cells were switched from Ca2+-containing medium to the medium without Ca2+. Each TER measurement was normalized to the TER value before switching to the Ca2+-free medium and was obtained after background subtraction (contributed by the filter and bath solution). The data represent the average ± SE from five experiments.
Figure 3
Figure 3
Immunolocalization of occludin, claudin-1, and ZO-1 after Ca2+ was removed from the medium. Cells were grown in normal culture medium until they were confluent, and then the cells were switched to the medium without Ca2+ for 2, 10, and 30 min. The results from immunofluorescence staining showed that 2 min after Ca2+ was depleted from the medium, cell-cell contacts were still intact and occludin, claudin-1, and ZO-1 were all localized at cell-cell contact area (A–C). However, after 10 min of Ca2+ depletion, the cell junctions were disrupted and the immunostaining of the tight junction proteins became discontinued (D–F). By 30 min of Ca2+ depletion, cells were detached from each other and the fluorescence staining patterns appeared either punctate (G and H) or dispersed in the cytoplasm (I). Bar, 20 μm. The inserts in G–I are enlarged images selected in the same fields (arrowheads indicate the same cells) to show the localization of the signals. Bar, 10 μm.
Figure 4
Figure 4
Tyrosine phosphorylation of occludin and ZO-1 during tight junction assembly after Ca2+ repletion. Cells were incubated in Ca2+-free medium for 1 h (designated as 0 h) and then switched to the Ca2+-containing medium for 0.5, 1, 2, 4, and 6 h in A and B, or for 2, 4, 8, and 24 h in C and D. After Ca2+ repletion at designated time points, cells were lysed in RIPA buffer, immunoprecipitated with anti-occludin (A and C) or anti-ZO-1 (B and D), and immunoblotted with anti-phosphotyrosine antibody. Tyrosine phosphorylation of occludin appeared clearly at 1-h time point after Ca2+ repletion (A) and reached maximum at 2- and 4-h time points (A and C). Tyrosine phosphorylation of ZO-1 did not change significantly over the time course of these experiments (B and D).
Figure 5
Figure 5
Time course of TER recovery during Ca2+ repletion. Cells were incubated in Ca2+-free medium for 1 h and then switched to the Ca2+-containing medium for 0, 0.5, 1, 2, 4, and 8 h at which time points the TER were measured. Each TER measurement was normalized to the initial TER value (before switching to the Ca2+-free medium) and was obtained after background subtraction (contributed by filter and bath solution). The data represent the average ± SE from five experiments.
Figure 6
Figure 6
Recruitments of occludin, claudin-1, and ZO-1 to the cell junction during Ca2+ repletion. After incubated in Ca2+-free medium for 1 h, cells were dissociated from each other, and occludin, claudin-1, and ZO-1 were largely present in the cytoplasm (A–C). After 0.5 h of Ca2+ repletion, occludin, claudin-1, and ZO-1 started to be recruited to some of the cell-cell contact area (D–F). At 1-h time point, tight junction proteins were well accumulated at cell junctions (G–I). The tight junction staining of occludin, claudin-1, and ZO-1 completely returned to normal by 2 h of Ca2+ repletion (J–L). Bar, 20 μm.
Figure 7
Figure 7
Colocalization and coimmunoprecipitation of occludin with nonreceptor tyrosine kinase c-Yes in canine kidney epithelial cells. Confluent cells were fixed in methanol and processed for double immunofluorescence by using the rabbit anti-occludin (A) polyclonal antibody and mouse anti-c-Yes (B) mAb antibody. Images of A and B were superimposed as C. Confocal fluorescence microscopy showed the z-axis colocalization of occludin (D) and c-Yes (E) and their superimposed image (F). Occludin and c-Yes formed a complex in vivo. Cells were lysed with RIPA buffer and immunoprecipitated with either anti-occludin and blotted with anti-c-Yes (G), or immunoprecipitated with anti-c-Yes and blotted with anti-occludin (H). As a negative control, cell lysates were also either immunoprecipitated with anti-occludin and blotted with anti-claudin-1 (I) or directly probed with anti-claudin-1 antibody (J). Bar, 20 μm.
Figure 8
Figure 8
Effects of CGP77675, a c-Yes inhibitor, on tyrosine phosphorylation of occludin and tight junction formation. (A) Treatment of MDCK II cells with CGP77675 led to occludin tyrosine dephosphorylation. Confluent cells were cultured in Ca2+-free medium overnight and then switched to Ca2+-containing medium without (C) or with the addition of 50 μM genistein (Geni) or 200 nM CGP77675 (CGP). After 2.5 h of treatment, cells were lysed in RIPA buffer, immunoprecipitated with rabbit anti-occludin polyclonal antibody, and blotted with anti-phosphotyrosine antibody. (B) CGP77675 treatment disrupted tight junction formation. Same as in A, cells were incubated in Ca2+-free medium overnight and switched to Ca2+-containing medium for 2.5 h without (C), or with the addition of 50 μM genistein (Geni) or 200 nM CGP77675 (CGP). Cells were fixed in methanol at −20°C and detected by indirect immunofluorescence staining of anti-occludin antibody or anti-ZO-1 antibody. Bar, 20 μm.
Figure 9
Figure 9
Recovery of TER after Ca2+ repletion was inhibited by CGP77675 treatment. Cells were incubated in Ca2+-free medium overnight and then switched to the Ca2+-containing medium for 0, 0.5, 1, 2, 3, and 4 h. At these time points TER measurements were taken. Each TER measurement was normalized to the initial TER value (before incubated in the Ca2+-free medium) and was obtained after background subtraction (contributed by filter and bath solution). The data represent the average ± SE from five experiments.
Figure 10
Figure 10
Occludin/c-Yes complex was absent in minus calcium condition and was disrupted by the CGP77675 treatment. (A) Cells were incubated in Ca2+-free medium for overnight and then switched to the Ca2+-containing medium for 2.5 h without (C), or with the addition of 200 nM CGP77675 (CGP). Lane -Ca2+ indicated the cells without switching back to Ca2+-containing medium. Cells were lysed in RIPA buffer, immunoprecipitated by mouse anti-c-Yes mAb, and blotted with rabbit anti-occludin antibody. (B) Same blot as in A, but was stripped by 100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl pH 7.6 for 30 min at 55°C, and then reprobed with anti-c-Yes antibody to show the equal amount of c-Yes immunoprecipitated in all three conditions.
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References

    1. Akiyama T, Ishida J, Nakagawa S, Ogawara H, Watanabe S, Itoh N, Shibuya M, Fukami Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem. 1987;262:5592–5595. - PubMed
    1. Ando-Akatsuka Y, Saitou M, Hirase T, Kishi M, Sakakibara A, Furuse M, Tsukita S. Interspecies diversity of the occludin sequence - cDNA cloning of human, mouse, dog, and rat-kangaroo homologues. J Cell Biol. 1996;133:43–47. - PMC - PubMed
    1. Arreaza G, Melkonian KA, LaFevre-Bernt M, Brown DA. Triton X-100-resistant membrane complexes from cultured kidney epithelial cells contain the Src family protein tyrosine kinase p62yes. J Biol Chem. 1994;269:19123–19127. - PubMed
    1. Atkinson KJ, Rao RK. Role of protein tyrosine phosphorylation in acetaldehyde-induced disruption of epithelial tight junctions. Am J Physiol. 2001;280:G1280–G1288. - PubMed
    1. Balda MS, González-Mariscal L, Contreras RG, Macias-Silva M, Torres-Marquez ME, Garcia-Sainz JA, Cereijido M. Assembly and sealing tight junctions: possible participation of G-proteins, phospholipase C, protein kinase C and calmodulin. J Membr Biol. 1991;122:193–202. - PubMed

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