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.
Yamamoto
T
T
Division of Signal Transduction, Nara Institute of Science and Technology, Ikoma, Nara, 630-0101, Japan.
Harada
N
N
Kawano
Y
Y
Taya
S
S
Kaibuchi
K
K
D016475:3T3 Cells; D000818:Animals; D001665:Binding Sites; D002478:Cells, Cultured; D005786:Gene Expression Regulation; D016762:Genes, fos / Q000235:genetics; D006801:Humans; D016547:Kinesins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D062826:Zonula Occludens-1 Protein; D018631:ras Proteins / Q000378:metabolism*
C095516:AFDN protein, human; C490657:Afdn protein, mouse; D008565:Membrane Proteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; C569042:TJP1 protein, human; C569043:Tjp1 protein, mouse; C569044:Tjp1 protein, rat; D062826:Zonula Occludens-1 Protein; D009218:Myosins; D016547:Kinesins; D018631:ras Proteins