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The Ras target AF-6 is a substrate of the fam deubiquitinating enzyme.

Taya S, Yamamoto T, Kano K, Kawano Y, Iwamatsu A, Tsuchiya T, Tanaka K, Kanai-Azuma M, Wood SA, Mattick JS, Kaibuchi K - J. Cell Biol. (1998)

Bottom Line: We found that Fam accumulated at the cell-cell contact sites of MDCKII cells, but not at free ends of plasma membranes.Fam was partially colocalized with AF-6 and interacted with AF-6 in vivo and in vitro.We also showed that AF-6 was ubiquitinated in intact cells, and that Fam prevented the ubiquitination of AF-6.

View Article: PubMed Central - PubMed

Affiliation: Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma 630-0101, Japan.

ABSTRACT
The Ras target AF-6 has been shown to serve as one of the peripheral components of cell-cell adhesions, and is thought to participate in cell-cell adhesion regulation downstream of Ras. We here purified an AF-6-interacting protein with a molecular mass of approximately 220 kD (p220) to investigate the function of AF-6 at cell-cell adhesions. The peptide sequences of p220 were identical to the amino acid sequences of mouse Fam. Fam is homologous to a deubiquitinating enzyme in Drosophila, the product of the fat facets gene. Recent genetic analyses indicate that the deubiquitinating activity of the fat facets product plays a critical role in controlling the cell fate. We found that Fam accumulated at the cell-cell contact sites of MDCKII cells, but not at free ends of plasma membranes. Fam was partially colocalized with AF-6 and interacted with AF-6 in vivo and in vitro. We also showed that AF-6 was ubiquitinated in intact cells, and that Fam prevented the ubiquitination of AF-6.

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Confocal microscope images of confluent MDCKII  cells showing the distributions of Fam, ZO-1 and β-catenin. (A)  Immunoblot analysis of MDCKII cell lysates with preimmune serum (lane 1) and with anti-Fam antibody (K2; lane 2). The arrowhead denotes the position of Fam. (B) Subconfluent MDCKII  cells stained with anti-Fam antibody (K2). Arrows indicate free  ends of plasma membrane. (C) Confluent MDCKII cells doubly  stained with a rabbit polyclonal antibody against Fam (K2; a and  d) and mouse monoclonal antibodies against ZO-1 (b) or β-catenin (e). Fam immunoreactivity is shown in green, and ZO-1 and  β-catenin immunoreactivities are shown in red. ZO-1 was concentrated at the apical sections (b), whereas β-catenin was found  at more basal sections (e). The yellow area indicates colocalization of Fam and ZO-1 at the apical sites (c), or Fam and β-catenin  at the basal sites (f). The results shown are representative of  three independent experiments. Bar, 10 μm.
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Figure 3: Confocal microscope images of confluent MDCKII cells showing the distributions of Fam, ZO-1 and β-catenin. (A) Immunoblot analysis of MDCKII cell lysates with preimmune serum (lane 1) and with anti-Fam antibody (K2; lane 2). The arrowhead denotes the position of Fam. (B) Subconfluent MDCKII cells stained with anti-Fam antibody (K2). Arrows indicate free ends of plasma membrane. (C) Confluent MDCKII cells doubly stained with a rabbit polyclonal antibody against Fam (K2; a and d) and mouse monoclonal antibodies against ZO-1 (b) or β-catenin (e). Fam immunoreactivity is shown in green, and ZO-1 and β-catenin immunoreactivities are shown in red. ZO-1 was concentrated at the apical sections (b), whereas β-catenin was found at more basal sections (e). The yellow area indicates colocalization of Fam and ZO-1 at the apical sites (c), or Fam and β-catenin at the basal sites (f). The results shown are representative of three independent experiments. Bar, 10 μm.

Mentions: To understand the functions of Fam, we examined its intracellular distribution in MDCKII epithelial cells that show characteristics of polarized epithelial cells and form the junctional complex, including the tight junction and adherens junction at cell–cell contact sites (Gonzalez-Mariscal et al., 1985). The immunoblot analysis of cell lysates from MDCKII cells showed that anti-Fam antibody recognized a single band corresponding to a molecular weight of ∼220 kD (Fig. 3 A) as in the case of bovine brain extract (Fig. 1 b). Antibody preincubation with the recombinant Fam abolished the immunoreactivity (data not shown). The immunoreactivity of Fam was specifically localized at sites where a cell contacted a neighboring cell, and not at free ends of plasma membranes (Fig. 3 B). The cytoplasm exhibited a relatively low level of immunoreactivity. To further examine whether Fam exists in the apical or basal site of the lateral membrane, cellular distribution of Fam was compared with that of ZO-1 and β-catenin (Fig. 3, C, b and e). ZO-1 was concentrated at the apical sections, whereas β-catenin was found at more basal sections as described previously (Nagafuchi and Takeichi, 1989; Itoh et al., 1993). In contrast, Fam immunoreactivity was colocalized at the immunofluorescence microscopic level with ZO-1 at the apical sites, and with β-catenin at the basal sites (Fig. 3, C, c and f). Since we have previously shown that AF-6 interacts with ZO-1 and is colocalized with ZO-1 at cell–cell contact sites including tight junctions (Yamamoto et al., 1997), part of the Fam that is localized at the same sites with ZO-1 may be colocalized with AF-6. We also found that accumulation of Fam is induced by the formation of cell–cell adhesions by using a Ca2+ switch assay in MDCKII cells (data not shown). These results suggest that Fam is partly colocalized with AF-6 at apical sites of the lateral membrane in confluent MDCKII cells.


The Ras target AF-6 is a substrate of the fam deubiquitinating enzyme.

Taya S, Yamamoto T, Kano K, Kawano Y, Iwamatsu A, Tsuchiya T, Tanaka K, Kanai-Azuma M, Wood SA, Mattick JS, Kaibuchi K - J. Cell Biol. (1998)

Confocal microscope images of confluent MDCKII  cells showing the distributions of Fam, ZO-1 and β-catenin. (A)  Immunoblot analysis of MDCKII cell lysates with preimmune serum (lane 1) and with anti-Fam antibody (K2; lane 2). The arrowhead denotes the position of Fam. (B) Subconfluent MDCKII  cells stained with anti-Fam antibody (K2). Arrows indicate free  ends of plasma membrane. (C) Confluent MDCKII cells doubly  stained with a rabbit polyclonal antibody against Fam (K2; a and  d) and mouse monoclonal antibodies against ZO-1 (b) or β-catenin (e). Fam immunoreactivity is shown in green, and ZO-1 and  β-catenin immunoreactivities are shown in red. ZO-1 was concentrated at the apical sections (b), whereas β-catenin was found  at more basal sections (e). The yellow area indicates colocalization of Fam and ZO-1 at the apical sites (c), or Fam and β-catenin  at the basal sites (f). The results shown are representative of  three independent experiments. Bar, 10 μm.
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Figure 3: Confocal microscope images of confluent MDCKII cells showing the distributions of Fam, ZO-1 and β-catenin. (A) Immunoblot analysis of MDCKII cell lysates with preimmune serum (lane 1) and with anti-Fam antibody (K2; lane 2). The arrowhead denotes the position of Fam. (B) Subconfluent MDCKII cells stained with anti-Fam antibody (K2). Arrows indicate free ends of plasma membrane. (C) Confluent MDCKII cells doubly stained with a rabbit polyclonal antibody against Fam (K2; a and d) and mouse monoclonal antibodies against ZO-1 (b) or β-catenin (e). Fam immunoreactivity is shown in green, and ZO-1 and β-catenin immunoreactivities are shown in red. ZO-1 was concentrated at the apical sections (b), whereas β-catenin was found at more basal sections (e). The yellow area indicates colocalization of Fam and ZO-1 at the apical sites (c), or Fam and β-catenin at the basal sites (f). The results shown are representative of three independent experiments. Bar, 10 μm.
Mentions: To understand the functions of Fam, we examined its intracellular distribution in MDCKII epithelial cells that show characteristics of polarized epithelial cells and form the junctional complex, including the tight junction and adherens junction at cell–cell contact sites (Gonzalez-Mariscal et al., 1985). The immunoblot analysis of cell lysates from MDCKII cells showed that anti-Fam antibody recognized a single band corresponding to a molecular weight of ∼220 kD (Fig. 3 A) as in the case of bovine brain extract (Fig. 1 b). Antibody preincubation with the recombinant Fam abolished the immunoreactivity (data not shown). The immunoreactivity of Fam was specifically localized at sites where a cell contacted a neighboring cell, and not at free ends of plasma membranes (Fig. 3 B). The cytoplasm exhibited a relatively low level of immunoreactivity. To further examine whether Fam exists in the apical or basal site of the lateral membrane, cellular distribution of Fam was compared with that of ZO-1 and β-catenin (Fig. 3, C, b and e). ZO-1 was concentrated at the apical sections, whereas β-catenin was found at more basal sections as described previously (Nagafuchi and Takeichi, 1989; Itoh et al., 1993). In contrast, Fam immunoreactivity was colocalized at the immunofluorescence microscopic level with ZO-1 at the apical sites, and with β-catenin at the basal sites (Fig. 3, C, c and f). Since we have previously shown that AF-6 interacts with ZO-1 and is colocalized with ZO-1 at cell–cell contact sites including tight junctions (Yamamoto et al., 1997), part of the Fam that is localized at the same sites with ZO-1 may be colocalized with AF-6. We also found that accumulation of Fam is induced by the formation of cell–cell adhesions by using a Ca2+ switch assay in MDCKII cells (data not shown). These results suggest that Fam is partly colocalized with AF-6 at apical sites of the lateral membrane in confluent MDCKII cells.

Bottom Line: We found that Fam accumulated at the cell-cell contact sites of MDCKII cells, but not at free ends of plasma membranes.Fam was partially colocalized with AF-6 and interacted with AF-6 in vivo and in vitro.We also showed that AF-6 was ubiquitinated in intact cells, and that Fam prevented the ubiquitination of AF-6.

View Article: PubMed Central - PubMed

Affiliation: Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma 630-0101, Japan.

ABSTRACT
The Ras target AF-6 has been shown to serve as one of the peripheral components of cell-cell adhesions, and is thought to participate in cell-cell adhesion regulation downstream of Ras. We here purified an AF-6-interacting protein with a molecular mass of approximately 220 kD (p220) to investigate the function of AF-6 at cell-cell adhesions. The peptide sequences of p220 were identical to the amino acid sequences of mouse Fam. Fam is homologous to a deubiquitinating enzyme in Drosophila, the product of the fat facets gene. Recent genetic analyses indicate that the deubiquitinating activity of the fat facets product plays a critical role in controlling the cell fate. We found that Fam accumulated at the cell-cell contact sites of MDCKII cells, but not at free ends of plasma membranes. Fam was partially colocalized with AF-6 and interacted with AF-6 in vivo and in vitro. We also showed that AF-6 was ubiquitinated in intact cells, and that Fam prevented the ubiquitination of AF-6.

Show MeSH
Related in: MedlinePlus