In vivo interaction of AF-6 with activated Ras and ZO-1.
AF-6 contains two putative Ras-associating domains (RA domains) which are seen in several Ras effectors such as RalGDS and RIN1. We previously showed that an AF-6 fragment containing the amino-terminal (N-terminal) RA domain directly binds to activated Ras and ZO-1 in vitro. In this study, we showed that a single ... amino acid mutation in the N-terminal RA domain of AF-6 abolished the interaction of AF-6 with activated Ras and that the sites of this critical amino acid residue were similar to those for Raf-1 and RalGDS. The overexpression of the N-terminal RA domain of AF-6 inhibited the Ras-dependent c-fos promoter/enhancer stimulation in NIH3T3 cells. Endogenous AF-6 was coimmunoprecipitated with activated Ras from Rat1 cells expressing activated Ras. Moreover, we showed that AF-6 was coimmunoprecipitated with ZO-1 from Rat1 cells. Taken together, these results indicate that the Ras-interacting region on AF-6 is structurally similar to that on Raf-1 and on RalGDS and that AF-6 interacts with activated Ras and ZO-1 in vivo.
Mesh Terms:
3T3 Cells, Animals, Binding Sites, Cells, Cultured, Gene Expression Regulation, Genes, fos, Humans, Kinesin, Membrane Proteins, Mice, Mutagenesis, Site-Directed, Myosins, Phosphoproteins, Protein Binding, Rats, Recombinant Fusion Proteins, Transfection, ras Proteins
3T3 Cells, Animals, Binding Sites, Cells, Cultured, Gene Expression Regulation, Genes, fos, Humans, Kinesin, Membrane Proteins, Mice, Mutagenesis, Site-Directed, Myosins, Phosphoproteins, Protein Binding, Rats, Recombinant Fusion Proteins, Transfection, ras Proteins
Biochem. Biophys. Res. Commun.
Date: May. 27, 1999
PubMed ID: 10334923
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