Inhibition of poly(A) polymerase requires p34cdc2/cyclin B phosphorylation of multiple consensus and non-consensus sites

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.