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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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Interaction of in vitro–translated Fam (1210–2100 aa)  with GST-AF-6 (1130–1612 aa). (a) Domain diagram of Fam and  the recombinant fragments used for the in vitro binding assay.  D.U., catalytic site of deubiquitinating enzyme including cysteine  and histidine domains. Bold lines show the recombinant fragments used for the in vitro binding assay. (b) In vitro–translated  Fam (1–669 aa; lanes 1 and 2), Fam (670–1213 aa; lanes 3 and 4),  Fam (1210–2100 aa; lanes 5 and 6), and Fam (2097–2554 aa; lanes  7 and 8) were mixed with GST (lanes 1, 3, 5, and 7) or GST-AF-6  (1130–1612 aa; lanes 2, 4, 6, and 8)-coated glutathione-Sepharose  4B beads. The interacting proteins were coeluted with GST fusion proteins by adding glutathione. The eluates were subjected  to SDS-PAGE and vacuum-dried. The in vitro–translated Fam  fragments were visualized with an image analyzer. The arrowhead denotes the position of the in vitro-translated Fam (1210– 2100 aa). The results shown are representative of three independent experiments.
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Figure 5: Interaction of in vitro–translated Fam (1210–2100 aa) with GST-AF-6 (1130–1612 aa). (a) Domain diagram of Fam and the recombinant fragments used for the in vitro binding assay. D.U., catalytic site of deubiquitinating enzyme including cysteine and histidine domains. Bold lines show the recombinant fragments used for the in vitro binding assay. (b) In vitro–translated Fam (1–669 aa; lanes 1 and 2), Fam (670–1213 aa; lanes 3 and 4), Fam (1210–2100 aa; lanes 5 and 6), and Fam (2097–2554 aa; lanes 7 and 8) were mixed with GST (lanes 1, 3, 5, and 7) or GST-AF-6 (1130–1612 aa; lanes 2, 4, 6, and 8)-coated glutathione-Sepharose 4B beads. The interacting proteins were coeluted with GST fusion proteins by adding glutathione. The eluates were subjected to SDS-PAGE and vacuum-dried. The in vitro–translated Fam fragments were visualized with an image analyzer. The arrowhead denotes the position of the in vitro-translated Fam (1210– 2100 aa). The results shown are representative of three independent experiments.

Mentions: We examined which domain of Fam interacts with AF-6 using in vitro–translated Fam such as Fam (1–669 aa), Fam (670–1213 aa), Fam (1210–2100 aa), and Fam (2097–2554 aa; Fig. 5 a). Affinity beads coated with GST or GST-AF-6 (1130–1612 aa) were mixed with the in vitro–translated Fam (1–669 aa), Fam (670–1213 aa), Fam (1210–2100 aa), and Fam (2097–2554 aa), and the interacting proteins were then coeluted with GST fusion proteins by adding glutathione. As shown in Fig. 5 b, Fam (1210–2100 aa) bound to GST-AF-6 (1130–1612 aa), but Fam (1–669 aa), Fam (670–1213 aa), and Fam (2097–2554 aa) did not. Fam (1210–2100 aa) did not bind to control GST.


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)

Interaction of in vitro–translated Fam (1210–2100 aa)  with GST-AF-6 (1130–1612 aa). (a) Domain diagram of Fam and  the recombinant fragments used for the in vitro binding assay.  D.U., catalytic site of deubiquitinating enzyme including cysteine  and histidine domains. Bold lines show the recombinant fragments used for the in vitro binding assay. (b) In vitro–translated  Fam (1–669 aa; lanes 1 and 2), Fam (670–1213 aa; lanes 3 and 4),  Fam (1210–2100 aa; lanes 5 and 6), and Fam (2097–2554 aa; lanes  7 and 8) were mixed with GST (lanes 1, 3, 5, and 7) or GST-AF-6  (1130–1612 aa; lanes 2, 4, 6, and 8)-coated glutathione-Sepharose  4B beads. The interacting proteins were coeluted with GST fusion proteins by adding glutathione. The eluates were subjected  to SDS-PAGE and vacuum-dried. The in vitro–translated Fam  fragments were visualized with an image analyzer. The arrowhead denotes the position of the in vitro-translated Fam (1210– 2100 aa). The results shown are representative of three independent experiments.
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Related In: Results  -  Collection

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Figure 5: Interaction of in vitro–translated Fam (1210–2100 aa) with GST-AF-6 (1130–1612 aa). (a) Domain diagram of Fam and the recombinant fragments used for the in vitro binding assay. D.U., catalytic site of deubiquitinating enzyme including cysteine and histidine domains. Bold lines show the recombinant fragments used for the in vitro binding assay. (b) In vitro–translated Fam (1–669 aa; lanes 1 and 2), Fam (670–1213 aa; lanes 3 and 4), Fam (1210–2100 aa; lanes 5 and 6), and Fam (2097–2554 aa; lanes 7 and 8) were mixed with GST (lanes 1, 3, 5, and 7) or GST-AF-6 (1130–1612 aa; lanes 2, 4, 6, and 8)-coated glutathione-Sepharose 4B beads. The interacting proteins were coeluted with GST fusion proteins by adding glutathione. The eluates were subjected to SDS-PAGE and vacuum-dried. The in vitro–translated Fam fragments were visualized with an image analyzer. The arrowhead denotes the position of the in vitro-translated Fam (1210– 2100 aa). The results shown are representative of three independent experiments.
Mentions: We examined which domain of Fam interacts with AF-6 using in vitro–translated Fam such as Fam (1–669 aa), Fam (670–1213 aa), Fam (1210–2100 aa), and Fam (2097–2554 aa; Fig. 5 a). Affinity beads coated with GST or GST-AF-6 (1130–1612 aa) were mixed with the in vitro–translated Fam (1–669 aa), Fam (670–1213 aa), Fam (1210–2100 aa), and Fam (2097–2554 aa), and the interacting proteins were then coeluted with GST fusion proteins by adding glutathione. As shown in Fig. 5 b, Fam (1210–2100 aa) bound to GST-AF-6 (1130–1612 aa), but Fam (1–669 aa), Fam (670–1213 aa), and Fam (2097–2554 aa) did not. Fam (1210–2100 aa) did not bind to control GST.

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