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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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Ubiquitination of AF-6 in COS7 cells.AF-6 and HA-tagged ubiquitin (HA-Ub) were expressed in COS7 cells. COS7  cells were treated with or without 25 μM ALLN for 18 h. The immunoprecipitates were then collected with anti-AF-6 antibody  (#3) and subjected to an immunoblot analysis with anti-HA antibody. The arrowhead denotes the position of HA-Ub-conjugated  AF-6. The results shown are representative of three independent  experiments.
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Figure 8: Ubiquitination of AF-6 in COS7 cells.AF-6 and HA-tagged ubiquitin (HA-Ub) were expressed in COS7 cells. COS7 cells were treated with or without 25 μM ALLN for 18 h. The immunoprecipitates were then collected with anti-AF-6 antibody (#3) and subjected to an immunoblot analysis with anti-HA antibody. The arrowhead denotes the position of HA-Ub-conjugated AF-6. The results shown are representative of three independent experiments.

Mentions: Next, to determine whether AF-6 is ubiquitinated, we performed an assay to detect ubiquitinated proteins (Treier et al., 1994; Aberle et al., 1997). AF-6 and hemagglutinin-tagged ubiquitin (HA-Ub) were transiently expressed in COS7 cells. The immunoprecipitates were then collected with anti-AF-6 antibody and subjected to an immunoblot analysis with anti-HA antibody. As shown in Fig. 8, HA-Ub–conjugated AF-6 was detected in cells expressing HA-Ub, but not in control cells. We examined the effect of ALLN on AF-6 ubiquitination. HA-Ub–conjugated proteins were more strongly detected in the cells treated with ALLN than in the untreated cells (Fig. 8). The band below HA-Ub–conjugated AF-6 was as strong as HA-Ub–conjugated AF-6 in the lane of ALLN plus. The lower band was probably the degradation product of the full-length AF-6, because the lower band as well as the upper band were detected only when the AF-6 cDNA was transfected. It may be noted that the intensity of the lower band was more strongly enhanced by ALLN, though we can not give the precise reasons for this phenomenon. Taken together, these results indicate that AF-6 is ubiquitinated in vivo and suggest that the ubiquitinated 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)

Ubiquitination of AF-6 in COS7 cells.AF-6 and HA-tagged ubiquitin (HA-Ub) were expressed in COS7 cells. COS7  cells were treated with or without 25 μM ALLN for 18 h. The immunoprecipitates were then collected with anti-AF-6 antibody  (#3) and subjected to an immunoblot analysis with anti-HA antibody. The arrowhead denotes the position of HA-Ub-conjugated  AF-6. 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 8: Ubiquitination of AF-6 in COS7 cells.AF-6 and HA-tagged ubiquitin (HA-Ub) were expressed in COS7 cells. COS7 cells were treated with or without 25 μM ALLN for 18 h. The immunoprecipitates were then collected with anti-AF-6 antibody (#3) and subjected to an immunoblot analysis with anti-HA antibody. The arrowhead denotes the position of HA-Ub-conjugated AF-6. The results shown are representative of three independent experiments.
Mentions: Next, to determine whether AF-6 is ubiquitinated, we performed an assay to detect ubiquitinated proteins (Treier et al., 1994; Aberle et al., 1997). AF-6 and hemagglutinin-tagged ubiquitin (HA-Ub) were transiently expressed in COS7 cells. The immunoprecipitates were then collected with anti-AF-6 antibody and subjected to an immunoblot analysis with anti-HA antibody. As shown in Fig. 8, HA-Ub–conjugated AF-6 was detected in cells expressing HA-Ub, but not in control cells. We examined the effect of ALLN on AF-6 ubiquitination. HA-Ub–conjugated proteins were more strongly detected in the cells treated with ALLN than in the untreated cells (Fig. 8). The band below HA-Ub–conjugated AF-6 was as strong as HA-Ub–conjugated AF-6 in the lane of ALLN plus. The lower band was probably the degradation product of the full-length AF-6, because the lower band as well as the upper band were detected only when the AF-6 cDNA was transfected. It may be noted that the intensity of the lower band was more strongly enhanced by ALLN, though we can not give the precise reasons for this phenomenon. Taken together, these results indicate that AF-6 is ubiquitinated in vivo and suggest that the ubiquitinated 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