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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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Coimmunoprecipitation of Fam with AF-6. MDCKII  cells were lysed and solubilized with buffer C (see Materials and  Methods). This sample was incubated with preimmuneserum  (lane 1), with anti-Fam antibody (K2; lane 2), or with anti-AF-6  antibody (#3; lane 3). The immunocomplexes were then precipitated with protein A-Sepharose 4B, and subjected to immunoblot  analysis using anti-AF-6 antibody (#4; a) or anti-Fam antibody  (N20; b). The arrowheads denote the positions of AF-6 (a) and  Fam (b), respectively. The results shown are representative of  three independent experiments.
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Figure 4: Coimmunoprecipitation of Fam with AF-6. MDCKII cells were lysed and solubilized with buffer C (see Materials and Methods). This sample was incubated with preimmuneserum (lane 1), with anti-Fam antibody (K2; lane 2), or with anti-AF-6 antibody (#3; lane 3). The immunocomplexes were then precipitated with protein A-Sepharose 4B, and subjected to immunoblot analysis using anti-AF-6 antibody (#4; a) or anti-Fam antibody (N20; b). The arrowheads denote the positions of AF-6 (a) and Fam (b), respectively. The results shown are representative of three independent experiments.

Mentions: Because Fam was partly colocalized with AF-6 at apical sites of the lateral membrane, we examined whether Fam interacts with AF-6 in vivo. When Fam was immunoprecipitated with anti-Fam antibody from confluent MDCKII cells, AF-6 was coimmunoprecipitated with Fam (Fig. 4 a). AF-6 was not coimmunoprecipitated with control preimmuneserum. AF-6 appeared to associate with Fam with a stoichiometry of about one AF-6 per ten Fam under these conditions. Similarly, Fam was also coimmunoprecipitated with AF-6 (Fig. 4 b) when AF-6 was immunoprecipitated with anti-AF-6 antibody from confluent MDCKII cells. These results indicate that Fam interacts with AF-6 in vivo.


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)

Coimmunoprecipitation of Fam with AF-6. MDCKII  cells were lysed and solubilized with buffer C (see Materials and  Methods). This sample was incubated with preimmuneserum  (lane 1), with anti-Fam antibody (K2; lane 2), or with anti-AF-6  antibody (#3; lane 3). The immunocomplexes were then precipitated with protein A-Sepharose 4B, and subjected to immunoblot  analysis using anti-AF-6 antibody (#4; a) or anti-Fam antibody  (N20; b). The arrowheads denote the positions of AF-6 (a) and  Fam (b), respectively. The results shown are representative of  three independent experiments.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2132865&req=5

Figure 4: Coimmunoprecipitation of Fam with AF-6. MDCKII cells were lysed and solubilized with buffer C (see Materials and Methods). This sample was incubated with preimmuneserum (lane 1), with anti-Fam antibody (K2; lane 2), or with anti-AF-6 antibody (#3; lane 3). The immunocomplexes were then precipitated with protein A-Sepharose 4B, and subjected to immunoblot analysis using anti-AF-6 antibody (#4; a) or anti-Fam antibody (N20; b). The arrowheads denote the positions of AF-6 (a) and Fam (b), respectively. The results shown are representative of three independent experiments.
Mentions: Because Fam was partly colocalized with AF-6 at apical sites of the lateral membrane, we examined whether Fam interacts with AF-6 in vivo. When Fam was immunoprecipitated with anti-Fam antibody from confluent MDCKII cells, AF-6 was coimmunoprecipitated with Fam (Fig. 4 a). AF-6 was not coimmunoprecipitated with control preimmuneserum. AF-6 appeared to associate with Fam with a stoichiometry of about one AF-6 per ten Fam under these conditions. Similarly, Fam was also coimmunoprecipitated with AF-6 (Fig. 4 b) when AF-6 was immunoprecipitated with anti-AF-6 antibody from confluent MDCKII cells. These results indicate that Fam interacts with AF-6 in vivo.

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