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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 MBP-Fam (1476–1918 aa) and GST-AF-6  (1130–1612 aa) in vitro.The result of the kinetic study of the  binding of MBP-Fam (1476–1918 aa) with GST-AF-6 (1130–1612  aa) are shown. Various amounts of MBP-Fam (1476–1918 aa)  were mixed with GST (lanes 1, 3, 5, 7, 9, and 11) or GST-AF-6  (1130–1612 aa) (lanes 2, 4, 6, 8, 10, and 12)-coated glutathione-Sepharose 4B beads. The interacting proteins were coeluted with  GST fusion proteins by adding glutathione. (a) The eluted MBP-Fam (1476–1918 aa) was detected by Coomassie Brilliant Blue  staining. (b) The bound MBP-Fam (1476–1918 aa) was visualized  and estimated with a densitograph. The values shown are means ±  SEM of triplicate experiments.
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Figure 6: Interaction of MBP-Fam (1476–1918 aa) and GST-AF-6 (1130–1612 aa) in vitro.The result of the kinetic study of the binding of MBP-Fam (1476–1918 aa) with GST-AF-6 (1130–1612 aa) are shown. Various amounts of MBP-Fam (1476–1918 aa) were mixed with GST (lanes 1, 3, 5, 7, 9, and 11) or GST-AF-6 (1130–1612 aa) (lanes 2, 4, 6, 8, 10, and 12)-coated glutathione-Sepharose 4B beads. The interacting proteins were coeluted with GST fusion proteins by adding glutathione. (a) The eluted MBP-Fam (1476–1918 aa) was detected by Coomassie Brilliant Blue staining. (b) The bound MBP-Fam (1476–1918 aa) was visualized and estimated with a densitograph. The values shown are means ± SEM of triplicate experiments.

Mentions: To test the specificity of the binding, we carried out a kinetic study of the binding of Fam to AF-6. We first examined whether GST-AF-6 (1130–1612 aa) could bind to MBP-Fam (1476–1918 aa) involving the deubiquitinating catalytic domain. Affinity beads coated with GST or GST-AF-6 (1130–1612 aa) were mixed with MBP-Fam (1476– 1918 aa). The bound MBP-Fam (1476–1918 aa) was then coeluted with the GST fusion proteins by adding glutathione, and the eluted MBP-Fam (1476–1918 aa) was detected by Coomassie Brilliant Blue staining. MBP-Fam (1476–1918 aa) was detected in the eluates from the GST-AF-6 (1130–1612 aa) affinity beads, but only slightly in those from the GST affinity beads (data not shown). In addition, as shown in Fig. 6, MBP-Fam (1476–1918 aa) bound to GST-AF-6 (1130–1612 aa) in a dose-dependent manner, and this binding was saturable when the amounts of MBP-Fam (1476–1918 aa) were increased (Fig. 6, a and b). The apparent Kd value for binding MBP-Fam (1476– 1918 aa) to GST-AF-6 (1130–1612 aa) was estimated to be ∼810 nM under the conditions used. These results indicate that mainly 1476–1918 aa of Fam is responsible for binding Fam to AF-6.


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 MBP-Fam (1476–1918 aa) and GST-AF-6  (1130–1612 aa) in vitro.The result of the kinetic study of the  binding of MBP-Fam (1476–1918 aa) with GST-AF-6 (1130–1612  aa) are shown. Various amounts of MBP-Fam (1476–1918 aa)  were mixed with GST (lanes 1, 3, 5, 7, 9, and 11) or GST-AF-6  (1130–1612 aa) (lanes 2, 4, 6, 8, 10, and 12)-coated glutathione-Sepharose 4B beads. The interacting proteins were coeluted with  GST fusion proteins by adding glutathione. (a) The eluted MBP-Fam (1476–1918 aa) was detected by Coomassie Brilliant Blue  staining. (b) The bound MBP-Fam (1476–1918 aa) was visualized  and estimated with a densitograph. The values shown are means ±  SEM of triplicate experiments.
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Related In: Results  -  Collection

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Figure 6: Interaction of MBP-Fam (1476–1918 aa) and GST-AF-6 (1130–1612 aa) in vitro.The result of the kinetic study of the binding of MBP-Fam (1476–1918 aa) with GST-AF-6 (1130–1612 aa) are shown. Various amounts of MBP-Fam (1476–1918 aa) were mixed with GST (lanes 1, 3, 5, 7, 9, and 11) or GST-AF-6 (1130–1612 aa) (lanes 2, 4, 6, 8, 10, and 12)-coated glutathione-Sepharose 4B beads. The interacting proteins were coeluted with GST fusion proteins by adding glutathione. (a) The eluted MBP-Fam (1476–1918 aa) was detected by Coomassie Brilliant Blue staining. (b) The bound MBP-Fam (1476–1918 aa) was visualized and estimated with a densitograph. The values shown are means ± SEM of triplicate experiments.
Mentions: To test the specificity of the binding, we carried out a kinetic study of the binding of Fam to AF-6. We first examined whether GST-AF-6 (1130–1612 aa) could bind to MBP-Fam (1476–1918 aa) involving the deubiquitinating catalytic domain. Affinity beads coated with GST or GST-AF-6 (1130–1612 aa) were mixed with MBP-Fam (1476– 1918 aa). The bound MBP-Fam (1476–1918 aa) was then coeluted with the GST fusion proteins by adding glutathione, and the eluted MBP-Fam (1476–1918 aa) was detected by Coomassie Brilliant Blue staining. MBP-Fam (1476–1918 aa) was detected in the eluates from the GST-AF-6 (1130–1612 aa) affinity beads, but only slightly in those from the GST affinity beads (data not shown). In addition, as shown in Fig. 6, MBP-Fam (1476–1918 aa) bound to GST-AF-6 (1130–1612 aa) in a dose-dependent manner, and this binding was saturable when the amounts of MBP-Fam (1476–1918 aa) were increased (Fig. 6, a and b). The apparent Kd value for binding MBP-Fam (1476– 1918 aa) to GST-AF-6 (1130–1612 aa) was estimated to be ∼810 nM under the conditions used. These results indicate that mainly 1476–1918 aa of Fam is responsible for binding Fam to AF-6.

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