The Ras target AF-6 interacts with ZO-1 and serves as a peripheral component of tight junctions in epithelial cells

Abstract

The dynamic rearrangement of cell-cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2, and adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell-cell contacts. We previously identified the ALL-1 fusion partner from chromosome 6 (AF-6) as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes such as cell-cell contact sites. We investigated roles of Ras and AF-6 in the regulation of cell-cell contacts and found that AF-6 accumulated at the cell-cell contact sites of polarized MDCKII epithelial cells and had a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy revealed a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacted with ZO-1 in vitro. ZO-1 interacted with the Ras-binding domain of AF-6, and this interaction was inhibited by activated Ras. AF-6 accumulated with ZO-1 at the cell-cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells resulted in the perturbation of cell-cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell-cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell-cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras.

Figure 1

Immunoblot analysis of MDCKII, Rat1, and PC12 cell lysates with anti–AF-6 antibody. Lane 1, MDCKII cell lysate with preimmuneserum; lane 2, MDCKII cell lysate with anti–AF-6 antibody preincubated with recombinant AF-6; lane 3, MDCKII cell lysate with anti–AF-6 antibody; lane 4, Rat1 cell lysate with anti– AF-6 antibody; lane 5, PC12 cell lysate with anti–AF-6 antibody. The results shown are representative of three independent experiments. The arrowheads denote the position of AF-6.

Figure 2

Confocal microscope images of confluent MDCKII cells showing the distributions of AF-6, ZO-1, and α-catenin. Confluent MDCKII cells were doubly stained with a rabbit polyclonal antibody against AF-6 and a mouse monoclonal antibody against ZO-1 (a–c, h, and k) or a rat monoclonal antibody against α-catenin (d–f, j, and l), followed by FITC-conjugated anti–rabbit IgG and Texas red-conjugated anti–mouse IgG or Texas red-conjugated anti–rat IgG antibodies. 20 serial optical sections were obtained at 0.8-μm intervals, and three-dimensional images were generated. In images a, c, d, f, k, and l, AF-6 is shown in green, and ZO-1 (b, c, and k) or α-catenin (e, f, and l) is shown in red. The yellow area indicates the colocalization of AF-6 and ZO-1 (c and k) or α-catenin (f and l). Images a–f are unrotated en face view. In images k and l, images c and f are rotated latitudinally by 90°, respectively. Images g–j show five confocal sections for each staining. Images g and i show the distribution of AF-6, and images h and j show that of ZO-1 and of α-catenin, respectively. Subsequent sections from apical to basal were shown from left to right. The results shown are representative of three independent experiments. Bars, 10 μm.

Figure 2

Confocal microscope images of confluent MDCKII cells showing the distributions of AF-6, ZO-1, and α-catenin. Confluent MDCKII cells were doubly stained with a rabbit polyclonal antibody against AF-6 and a mouse monoclonal antibody against ZO-1 (a–c, h, and k) or a rat monoclonal antibody against α-catenin (d–f, j, and l), followed by FITC-conjugated anti–rabbit IgG and Texas red-conjugated anti–mouse IgG or Texas red-conjugated anti–rat IgG antibodies. 20 serial optical sections were obtained at 0.8-μm intervals, and three-dimensional images were generated. In images a, c, d, f, k, and l, AF-6 is shown in green, and ZO-1 (b, c, and k) or α-catenin (e, f, and l) is shown in red. The yellow area indicates the colocalization of AF-6 and ZO-1 (c and k) or α-catenin (f and l). Images a–f are unrotated en face view. In images k and l, images c and f are rotated latitudinally by 90°, respectively. Images g–j show five confocal sections for each staining. Images g and i show the distribution of AF-6, and images h and j show that of ZO-1 and of α-catenin, respectively. Subsequent sections from apical to basal were shown from left to right. The results shown are representative of three independent experiments. Bars, 10 μm.

Figure 3

The ultrastructural localization of AF-6 and that of ZO-1 in confluent MDCKII cells. Immunoelectron micrographs of the junctional complex region in MDCKII cells stained with a rabbit polyclonal antibody against AF-6 (a), with a mouse monoclonal antibody against ZO-1 (b), or doubly stained with both anti–AF-6 polyclonal antibody (gold particles) and anti–ZO-1 monoclonal antibody (HRP reaction products; c and d). (a) The gold particles for AF-6 are accumulated on the cytoplasmic surface of the plasma membranes in the junctional complex region. (b) ZO-1 labeling is exclusively concentrated in the junctional complex region. (c and d) AF-6 immunoreactivity (gold particles) and ZO-1 immunoreactivity (HRP reaction products) are intermixed. The results shown are representative of three independent experiments. Bar, 500 nm.

Figure 4

Immunofluorescence localization of AF-6 and that of ZO-1 in Ca²⁺ switch experiments with MDCKII cells. Subconfluent MDCKII cells were grown in normal growth media (a–c) and transferred to low Ca²⁺ media (growth media containing 4 mM EGTA) for 6 h (d–f) and then transferred back to the normal Ca²⁺ medium for 2 h (g–i). The cells were doubly stained with a rabbit polyclonal antibody against AF-6 (b, e, and h) and a mouse monoclonal antibody against ZO-1 (c, f, and i), followed by FITC-conjugated anti–rabbit IgG and Texas red-conjugated anti– mouse IgG antibodies, and examined using laser scanning confocal microscopy. Images a, d, and g are phase contrast images. The results shown are representative of three independent experiments. Bar, 10 μm.

Figure 5

Immunofluorescence localization of AF-6 and that of ZO-1 in the frozen mouse intestinal epithelium. Cryosections of mouse intestine were doubly stained with a rabbit polyclonal antibody against AF-6 (a) and a mouse monoclonal antibody against ZO-1 (b), followed by FITC-conjugated anti–rabbit IgG and Texas red-conjugated anti–mouse IgG antibodies, and examined using laser scanning confocal microscopy. The results shown are representative of three independent experiments. Bar, 5 μm.

Figure 6

Colocalization of AF-6 with ZO-1 in Rat1 and PC12 cells. Subconfluent Rat1 (a–c) and PC12 (d–f) cells were doubly stained with a rabbit polyclonal antibody against AF-6 (a, c, d, and f) and a mouse monoclonal antibody against ZO-1 (b, c, e, and f), followed by FITC-conjugated anti–rabbit IgG and Texas red-conjugated anti–mouse IgG antibodies, and examined using laser scanning confocal microscopy. AF-6 is shown in green (a, c, d, and f) and ZO-1 (b, c, e, and f) is shown in red. In images c and f, the yellow area indicates the colocalization of AF-6 and ZO-1. The results shown are representative of three independent experiments. Bars: (a–c) 10 μm; (d–f) 5 μm.

Figure 7

Interaction of bovine AF-6 with ZO-1. Bovine brain membrane fraction was loaded onto affinity columns immobilized with GST (lane 1), GST-ZO-1 (lane 2), GST-occludin (lane 3), GST–E-cadherin (lane 4), GST–α-catenin (lane 5), GST–β-catenin (lane 6), and GST-CD44 (lane 7). The interacting proteins were eluted with GST proteins by the addition of glutathione. The eluates were subjected to SDS-PAGE, followed by immunoblot analysis with anti–AF-6 antibody. The arrowheads denote the position of bovine AF-6. The results shown are representative of three independent experiments.

Figure 8

Interaction of recombinant AF-6 with ZO-1. Crude lysates of Sf-9 cells infected with baculovirus carrying HA-AF-6 cDNA were loaded onto affinity columns immobilized with GST (lane 1), GST-ZO-1 (lane 2), GST-occludin (lane 3), and GST-CD44 (lane 4). The interacting proteins were eluted with GST fusion proteins by the addition of glutathione. The eluates were subjected to SDS-PAGE and followed by immunoblot analysis with anti–AF-6 antibody. The arrowheads denote the positions of HA-AF-6. The results shown are representative of three independent experiments.

Figure 9

Interaction of in vitro–translated AF-6 (36–494 amino acids) with ZO-1. (a) Domain diagram of AF-6 and the recombinant fragments used for the in vitro binding assay. RB, Ras-binding domain; Myosin V, Myosin V–like domain; PDZ, PDZ domain. Bold lines show the recombinant fragments used for the in vitro binding assay. (b–e) In vitro–translated AF-6 (36–494 amino acids; b), AF-6 (495–909 amino acids; c), AF-6 (914–1,129 amino acids; d), and AF-6 (1,130–1,612 amino acids; e) were mixed with GST (lane 1), GST-ZO-1 (lane 2), GST-occludin (lane 3), and GST-CD44 (lane 4) immobilized to glutathione Sepharose 4B beads. The interacting proteins were eluted with GST fusion proteins by the addition of glutathione. The eluates were subjected to SDS-PAGE and vacuum dried. The in vitro–translated AF-6 fragments were visualized with an image analyzer. The arrowheads denote the position of in vitro–translated AF-6 fragments. The results shown are representative of three independent experiments.

Figure 10

Dissociation of the Ras-interacting domain of AF-6 from ZO-1 by activated Ras. (a) Interaction of MBP-AF-6 (36– 206 amino acids) with ZO-1. Crude lysates of E. coli expressing MBP-AF-6 (36–206 amino acids) were loaded onto affinity columns immobilized with GST (lane 1), GST-ZO-1 (lane 2), GDP/ GST–Ha-Ras (lane 3), GTPγS/GST–Ha-Ras (lane 4), and GST-CD44 (lane 5). The interacting proteins were eluted with GST fusion proteins by the addition of glutathione. The eluates were subjected to SDS-PAGE and followed by immunoblot analysis with anti-MBP antibody. The arrowheads denote the positions of MBP-AF-6 (36–206 amino acids). (b) The kinetic study on the binding of MBP-AF-6 (36–206 amino acids) to GST-ZO-1. E. coli lysates containing various concentrations of MBP-AF-6 (36–206 amino acids) were loaded onto the GST-ZO-1 and GST-CD44 affinity columns (0.1 nmol). The proteins bound to GST-ZO-1 columns were eluted with GST-ZO-1 by the addition of glutathione. The eluates were subjected to SDS-PAGE and followed by immunoblot analysis with anti-MBP antibody. The immunodetected MBP-AF-6 were visualized and estimated with a densitograph. The values shown are means ± SEM of triplicate experiments. ▪, with GST-ZO-1; ▴, with GST-CD44. (c) Dissociation of MBP-AF-6 (36–206 amino acids) from ZO-1 by activated Ras. Crude lysates of E. coli expressing MBP-AF-6 (36–206 amino acids) were loaded onto affinity columns immobilized with GST-ZO-1. The proteins bound to the GST-ZO-1 columns were eluted by the addition of buffer containing GDP alone (lane 1), GTPγS alone (lane 2), GDP/Ras (lane 3), GTPγS/Ras (lane 4), and GTPγS/Rac (lane 5). The eluates were subjected to SDS-PAGE and followed by immunoblot analysis with anti-MBP antibody. The arrowhead denotes the position of MBP-AF-6 (36–206 amino acids). The results shown are representative of three independent experiments.

Figure 11

Localization of AF-6 in Ras-transformed Rat1 cells. (A) Immunoblot analysis of Rat1 RasVal A1 cells. The expression of Ras^V12 was induced in Rat1 RasVal A1 cells (lanes 3) by the addition of IPTG, and the cell lysates were subjected to SDS-PAGE, followed by immunoblot analysis with anti-Ras antibody (Rask4). Lane 1, Wild-type Rat1 cells; lane 2, Rat1 RasVal A1 cells in the absence of IPTG; lane 3, Rat1 RasVal A1 cells treated with IPTG for 24 h. (B) Localization of AF-6 in Ras-transformed Rat1 cells. Wild-type Rat1 cells (a and b), Rat1 RasVal A1 cells in the absence of IPTG (c and d), and Rat1 RasVal A1 cells treated with 5 mM IPTG for 24 h (e and f) were doubly stained with a rabbit polyclonal antibody against AF-6 (a, c, and e) and a mouse monoclonal antibody against ZO-1 (b, d, and f), followed by FITC-conjugated anti–rabbit IgG and Texas red-conjugated anti– mouse IgG antibodies. The results shown are representative of three independent experiments. Bar, 10 μm.