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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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Hydrolysis of 125I-labeled Ub-PEST by Fam.The 125I-labeled Ub-PEST was incubated alone or with 1.6 μg of Fam purified by GST-AF-6 (1130–1612 aa) affinity column in the absence or presence of ubiquitin-aldehyde (Ub-CHO) for 1, 3, or  6 h at 37°C. After the incubation, the samples were subjected to  SDS-PAGE, followed by Coomassie Brilliant Blue staining (a),  and by autoradiography (b). The release of ubiquitin from Ub-PEST (a) or the production of the hydrolysis of 125I-labeled PEST  peptides from Ub-PEST (b). The results shown are representative of three independent experiments.
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Figure 2: Hydrolysis of 125I-labeled Ub-PEST by Fam.The 125I-labeled Ub-PEST was incubated alone or with 1.6 μg of Fam purified by GST-AF-6 (1130–1612 aa) affinity column in the absence or presence of ubiquitin-aldehyde (Ub-CHO) for 1, 3, or 6 h at 37°C. After the incubation, the samples were subjected to SDS-PAGE, followed by Coomassie Brilliant Blue staining (a), and by autoradiography (b). The release of ubiquitin from Ub-PEST (a) or the production of the hydrolysis of 125I-labeled PEST peptides from Ub-PEST (b). The results shown are representative of three independent experiments.

Mentions: First we examined whether Fam has deubiquitinating activity. We used the 200-mM NaCl eluate from the GST-AF-6 (1130–1612 aa) affinity column as a source of Fam. Fam was incubated with the 125I-labeled ubiquitin-conjugated PEST sequence (Ub-PEST) for various periods of time. As shown in Fig. 2, Fam could release ubiquitin from Ub-PEST (a), and could produce the hydrolyzed 125I-PEST peptides from Ub-PEST (b). In the Fam minus control of Fig. 2, the 200-mM NaCl buffer that did not contain any enzyme was used. These results indicate that Fam is able to generate free ubiquitin from Ub-PEST. It is well-known that ubiquitin-aldehyde (Ub-CHO) inhibits Ubps. Release of ubiquitin from Ub-PEST or production of the PEST peptides was almost abolished in the presence of Ub-CHO. The deubiquitinating activity was not detected in the 200-mM NaCl eluate from the GST affinity column (data not shown). These results demonstrated that Fam has deubiquitinating activity.


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)

Hydrolysis of 125I-labeled Ub-PEST by Fam.The 125I-labeled Ub-PEST was incubated alone or with 1.6 μg of Fam purified by GST-AF-6 (1130–1612 aa) affinity column in the absence or presence of ubiquitin-aldehyde (Ub-CHO) for 1, 3, or  6 h at 37°C. After the incubation, the samples were subjected to  SDS-PAGE, followed by Coomassie Brilliant Blue staining (a),  and by autoradiography (b). The release of ubiquitin from Ub-PEST (a) or the production of the hydrolysis of 125I-labeled PEST  peptides from Ub-PEST (b). The results shown are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: Hydrolysis of 125I-labeled Ub-PEST by Fam.The 125I-labeled Ub-PEST was incubated alone or with 1.6 μg of Fam purified by GST-AF-6 (1130–1612 aa) affinity column in the absence or presence of ubiquitin-aldehyde (Ub-CHO) for 1, 3, or 6 h at 37°C. After the incubation, the samples were subjected to SDS-PAGE, followed by Coomassie Brilliant Blue staining (a), and by autoradiography (b). The release of ubiquitin from Ub-PEST (a) or the production of the hydrolysis of 125I-labeled PEST peptides from Ub-PEST (b). The results shown are representative of three independent experiments.
Mentions: First we examined whether Fam has deubiquitinating activity. We used the 200-mM NaCl eluate from the GST-AF-6 (1130–1612 aa) affinity column as a source of Fam. Fam was incubated with the 125I-labeled ubiquitin-conjugated PEST sequence (Ub-PEST) for various periods of time. As shown in Fig. 2, Fam could release ubiquitin from Ub-PEST (a), and could produce the hydrolyzed 125I-PEST peptides from Ub-PEST (b). In the Fam minus control of Fig. 2, the 200-mM NaCl buffer that did not contain any enzyme was used. These results indicate that Fam is able to generate free ubiquitin from Ub-PEST. It is well-known that ubiquitin-aldehyde (Ub-CHO) inhibits Ubps. Release of ubiquitin from Ub-PEST or production of the PEST peptides was almost abolished in the presence of Ub-CHO. The deubiquitinating activity was not detected in the 200-mM NaCl eluate from the GST affinity column (data not shown). These results demonstrated that Fam has deubiquitinating activity.

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