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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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Purification of AF-6-interacting protein. (a) The bovine  brain cytosol was loaded onto glutathione-Sepharose 4B columns  coated with GST, GST-AF-6 (1130–1612 aa), or GST-CD44. The  bound proteins were coeluted with the respective GST fusion  proteins by adding glutathione. Aliquots of the eluates were resolved by SDS-PAGE, followed by silver staining. Lane 1, GST;  lane 2, GST-AF-6 (1130–1612 aa); lane 3, GST-CD44. The arrowhead denotes the position of p220. (b) Protein p220 was immunoblotted with the anti-Fam antibodies. Lane 1, the position of p220  by silver staining; lane 2, with preimmuneserum; lane 3, with the  anti-Fam antibody (N20); lane 4, with the anti-Fam antibody  (C114). The arrowhead denotes the position of p220. The results  shown are representative of three independent experiments.
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Figure 1: Purification of AF-6-interacting protein. (a) The bovine brain cytosol was loaded onto glutathione-Sepharose 4B columns coated with GST, GST-AF-6 (1130–1612 aa), or GST-CD44. The bound proteins were coeluted with the respective GST fusion proteins by adding glutathione. Aliquots of the eluates were resolved by SDS-PAGE, followed by silver staining. Lane 1, GST; lane 2, GST-AF-6 (1130–1612 aa); lane 3, GST-CD44. The arrowhead denotes the position of p220. (b) Protein p220 was immunoblotted with the anti-Fam antibodies. Lane 1, the position of p220 by silver staining; lane 2, with preimmuneserum; lane 3, with the anti-Fam antibody (N20); lane 4, with the anti-Fam antibody (C114). The arrowhead denotes the position of p220. The results shown are representative of three independent experiments.

Mentions: To detect molecules interacting with AF-6 (1130–1612 aa), which includes a proline-rich region, the bovine brain cytosolic fraction was loaded onto affinity columns coated with GST, GST-AF-6 (1130–1612 aa), or GST-CD44. The proteins bound to the affinity columns were coeluted with GST-AF-6 (1130–1612 aa) by adding glutathione. The glutathione-eluted fractions were subjected to SDS-PAGE followed by silver staining. A protein with a molecular mass of ∼220 kD (p220) was detected in the glutathione eluate from the GST-AF-6 (1130–1612 aa) affinity column, but not from the GST or GST-CD44 affinity columns (Fig. 1 a), indicating that p220 specifically interacts with AF-6 (1130–1612 aa) directly or indirectly. To identify the AF-6 (1130–1612 aa)-interacting molecule, p220 was subjected to amino acid sequencing as described in Materials and Methods. Two peptide sequences derived from p220 were determined. These were LSVPATFMLVSLD and NDYFEF. Both peptide sequences were identical to the deduced amino acid sequence of mouse Fam, which is one of the deubiquitinating enzymes (Wood et al., 1997).


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)

Purification of AF-6-interacting protein. (a) The bovine  brain cytosol was loaded onto glutathione-Sepharose 4B columns  coated with GST, GST-AF-6 (1130–1612 aa), or GST-CD44. The  bound proteins were coeluted with the respective GST fusion  proteins by adding glutathione. Aliquots of the eluates were resolved by SDS-PAGE, followed by silver staining. Lane 1, GST;  lane 2, GST-AF-6 (1130–1612 aa); lane 3, GST-CD44. The arrowhead denotes the position of p220. (b) Protein p220 was immunoblotted with the anti-Fam antibodies. Lane 1, the position of p220  by silver staining; lane 2, with preimmuneserum; lane 3, with the  anti-Fam antibody (N20); lane 4, with the anti-Fam antibody  (C114). The arrowhead denotes the position of p220. 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 1: Purification of AF-6-interacting protein. (a) The bovine brain cytosol was loaded onto glutathione-Sepharose 4B columns coated with GST, GST-AF-6 (1130–1612 aa), or GST-CD44. The bound proteins were coeluted with the respective GST fusion proteins by adding glutathione. Aliquots of the eluates were resolved by SDS-PAGE, followed by silver staining. Lane 1, GST; lane 2, GST-AF-6 (1130–1612 aa); lane 3, GST-CD44. The arrowhead denotes the position of p220. (b) Protein p220 was immunoblotted with the anti-Fam antibodies. Lane 1, the position of p220 by silver staining; lane 2, with preimmuneserum; lane 3, with the anti-Fam antibody (N20); lane 4, with the anti-Fam antibody (C114). The arrowhead denotes the position of p220. The results shown are representative of three independent experiments.
Mentions: To detect molecules interacting with AF-6 (1130–1612 aa), which includes a proline-rich region, the bovine brain cytosolic fraction was loaded onto affinity columns coated with GST, GST-AF-6 (1130–1612 aa), or GST-CD44. The proteins bound to the affinity columns were coeluted with GST-AF-6 (1130–1612 aa) by adding glutathione. The glutathione-eluted fractions were subjected to SDS-PAGE followed by silver staining. A protein with a molecular mass of ∼220 kD (p220) was detected in the glutathione eluate from the GST-AF-6 (1130–1612 aa) affinity column, but not from the GST or GST-CD44 affinity columns (Fig. 1 a), indicating that p220 specifically interacts with AF-6 (1130–1612 aa) directly or indirectly. To identify the AF-6 (1130–1612 aa)-interacting molecule, p220 was subjected to amino acid sequencing as described in Materials and Methods. Two peptide sequences derived from p220 were determined. These were LSVPATFMLVSLD and NDYFEF. Both peptide sequences were identical to the deduced amino acid sequence of mouse Fam, which is one of the deubiquitinating enzymes (Wood et al., 1997).

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