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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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Regulation of AF-6 by the ubiquitin–proteasome pathway. Rat1 and EL cells were treated with the vehicle (DMSO;  lanes 1 and 4), 25 μM ALLM (lanes 2 and 5), or 25 μM ALLN  (lanes 3 and 6) for 3 h. Cell lysates of Rat1 (lanes 1–3) and EL  (lanes 4–6) cells were immunoblotted with anti-AF-6 antibody  (#4; a), anti-β-catenin antibody (b), and anti-α-catenin antibody  (c). The arrowheads denote the position of the ubiquitinated AF-6  (a) and the ubiquitinated β-catenin (b), respectively. The results  shown are representative of three independent experiments.
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Figure 7: Regulation of AF-6 by the ubiquitin–proteasome pathway. Rat1 and EL cells were treated with the vehicle (DMSO; lanes 1 and 4), 25 μM ALLM (lanes 2 and 5), or 25 μM ALLN (lanes 3 and 6) for 3 h. Cell lysates of Rat1 (lanes 1–3) and EL (lanes 4–6) cells were immunoblotted with anti-AF-6 antibody (#4; a), anti-β-catenin antibody (b), and anti-α-catenin antibody (c). The arrowheads denote the position of the ubiquitinated AF-6 (a) and the ubiquitinated β-catenin (b), respectively. The results shown are representative of three independent experiments.

Mentions: As described above, AF-6 interacts with Fam, and the AF-6–interacting domain of Fam involves the deubiquitinating catalytic domain of Fam. This raises the possibility that AF-6 is the substrate of Fam, and that AF-6 is Ub-conjugated and subjected to the ubiquitin–proteasome pathway. First, to determine whether AF-6 is degraded by the proteasome-dependent proteolysis pathway, Rat1 fibroblasts and EL cells were treated with the proteasome inhibitor. EL cells are L cells stably expressing E-cadherin (Nagafuchi et al., 1994). It is well-known that the peptide aldehyde ALLN inhibits the proteasome-dependent proteolysis pathway, leading to an accumulation of proteins that are usually metabolized by the proteasome pathway (Coux et al., 1996). ALLM is the related peptide aldehyde and a calpain inhibitor, but it does not inhibit the proteasome pathway. Recently some groups reported that the turnover of β-catenin is regulated by the ubiquitin–proteasome pathway (Aberle et al., 1997; Orford et al., 1997). They showed that treating certain cell lines with ALLN resulted in accumulation of the higher molecular weight β-catenin. It has been reported that such a modification in α-catenin was not observed when cells were treated with ALLN. We obtained similar results as shown in Fig. 7, b and c, when Rat1 and EL cells were treated with ALLN. To determine whether AF-6 shows the similar modification, we immunoblotted the cell lysates with anti-AF-6 antibody. The higher molecular weight forms of AF-6 were detected by the treatment with ALLN, but little was detected by the treatment with ALLM under the same conditions (Fig. 7 a). These results suggest that AF-6 is degraded by the proteasome pathway.


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)

Regulation of AF-6 by the ubiquitin–proteasome pathway. Rat1 and EL cells were treated with the vehicle (DMSO;  lanes 1 and 4), 25 μM ALLM (lanes 2 and 5), or 25 μM ALLN  (lanes 3 and 6) for 3 h. Cell lysates of Rat1 (lanes 1–3) and EL  (lanes 4–6) cells were immunoblotted with anti-AF-6 antibody  (#4; a), anti-β-catenin antibody (b), and anti-α-catenin antibody  (c). The arrowheads denote the position of the ubiquitinated AF-6  (a) and the ubiquitinated β-catenin (b), respectively. The results  shown are representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 7: Regulation of AF-6 by the ubiquitin–proteasome pathway. Rat1 and EL cells were treated with the vehicle (DMSO; lanes 1 and 4), 25 μM ALLM (lanes 2 and 5), or 25 μM ALLN (lanes 3 and 6) for 3 h. Cell lysates of Rat1 (lanes 1–3) and EL (lanes 4–6) cells were immunoblotted with anti-AF-6 antibody (#4; a), anti-β-catenin antibody (b), and anti-α-catenin antibody (c). The arrowheads denote the position of the ubiquitinated AF-6 (a) and the ubiquitinated β-catenin (b), respectively. The results shown are representative of three independent experiments.
Mentions: As described above, AF-6 interacts with Fam, and the AF-6–interacting domain of Fam involves the deubiquitinating catalytic domain of Fam. This raises the possibility that AF-6 is the substrate of Fam, and that AF-6 is Ub-conjugated and subjected to the ubiquitin–proteasome pathway. First, to determine whether AF-6 is degraded by the proteasome-dependent proteolysis pathway, Rat1 fibroblasts and EL cells were treated with the proteasome inhibitor. EL cells are L cells stably expressing E-cadherin (Nagafuchi et al., 1994). It is well-known that the peptide aldehyde ALLN inhibits the proteasome-dependent proteolysis pathway, leading to an accumulation of proteins that are usually metabolized by the proteasome pathway (Coux et al., 1996). ALLM is the related peptide aldehyde and a calpain inhibitor, but it does not inhibit the proteasome pathway. Recently some groups reported that the turnover of β-catenin is regulated by the ubiquitin–proteasome pathway (Aberle et al., 1997; Orford et al., 1997). They showed that treating certain cell lines with ALLN resulted in accumulation of the higher molecular weight β-catenin. It has been reported that such a modification in α-catenin was not observed when cells were treated with ALLN. We obtained similar results as shown in Fig. 7, b and c, when Rat1 and EL cells were treated with ALLN. To determine whether AF-6 shows the similar modification, we immunoblotted the cell lysates with anti-AF-6 antibody. The higher molecular weight forms of AF-6 were detected by the treatment with ALLN, but little was detected by the treatment with ALLM under the same conditions (Fig. 7 a). These results suggest that AF-6 is degraded by the proteasome pathway.

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