Regular Article
In VivoInteraction of AF-6 with Activated Ras and ZO-1

https://doi.org/10.1006/bbrc.1999.0731Get rights and content

Abstract

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-1in 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-fospromoter/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-1in vivo.

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    Abbreviations used: RA domain, Ras-associating domain; N-terminal, amino-terminal; C-terminal, carboxyl-terminal; Raf-RBD, Ras-binding domain in Raf; RalGDS-RID, Ras-interacting domain in RalGDS; GST, glutathione-S-transferase; MBP, maltose-binding protein; DTT, dithiothreitol; IPTG, isopropyl-β-D-thiogalactoside; GTPγS, guanosine 5′-(3-O-thio)-triphosphate; DSP, dithiobis(succinimidylpropionate); RGL-RID, RGL (RalGDS like) Ras-interacting domain

    1

    To whom correspondence should be addressed at Division of Signal Transduction, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. Fax: +81-743-72-5449. E-mail:[email protected].

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