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The Ras target AF-6 is a substrate of the fam deubiquitinating enzyme
1Division 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.
Insights
Researchers identified Fam, a deubiquitinating enzyme, interacting with AF-6 at cell-cell adhesion sites. Fam prevents AF-6 ubiquitination, suggesting a role in regulating cell adhesion and cell fate.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- AF-6 is a Ras target involved in cell-cell adhesion regulation.
- Understanding AF-6's function at cell adhesions requires identifying interacting proteins.
- Deubiquitinating enzymes play critical roles in cellular processes, including cell fate determination.
Purpose of the Study:
- To identify and characterize AF-6-interacting proteins at cell-cell adhesions.
- To investigate the functional relationship between AF-6 and its interacting partners in cell adhesion.
Main Methods:
- Purification of AF-6-interacting proteins.
- Peptide sequencing and identification of the interacting protein as mouse Fam.
- Immunofluorescence microscopy to determine Fam localization in MDCKII cells.
- In vivo and in vitro interaction assays between Fam and AF-6.
- Analysis of AF-6 ubiquitination status in the presence and absence of Fam.
Main Results:
- An approximately 220 kD protein interacting with AF-6 was purified and identified as mouse Fam.
- Fam, homologous to a deubiquitinating enzyme, localized to cell-cell contact sites.
- Fam partially colocalized and interacted with AF-6 in vivo and in vitro.
- Fam inhibited the ubiquitination of AF-6 in intact cells.
Conclusions:
- Fam is a novel AF-6-interacting protein localized to cell-cell adhesions.
- Fam's deubiquitinating activity may regulate AF-6 function at cell-cell junctions.
- This interaction suggests a mechanism for controlling cell adhesion and potentially cell fate via AF-6 ubiquitination status.