Identification of a Link between the SAMP Repeats of Adenomatous Polyposis Coli Tumor Suppressor and the Src Homology 3 Domain of DDEF*
- ‡KAN Research Institute, Inc., Kobe MI R&D Center, Chuo-ku, Kobe, 650-0047, Japan and §Laboratory of Structural Proteomics, Institute for Protein Research, Osaka University, Suita, Osaka 565-0871, Japan
- 2 To whom correspondence should be addressed: KAN Research Institute, Inc. 3F, Kobe MI R&D Center, 6-7-3 Minatojima-minamimachi, Chuo-ku, Kobe 650-0047, Japan. Fax: 81-78-306-5920; E-mail: y-kiyosue{at}kan.eisai.co.jp.
Abstract
The adenomatous polyposis coli (APC) tumor suppressor protein is a multifunctional protein with a well characterized role in the Wnt signal transduction pathway and in cytoskeletal regulation. The SAMP repeats region of APC, an Axin-binding site, is known to be important for tumor suppression and for the developmental function of APC. We performed a yeast two-hybrid screening using the first SAMP motif-containing region of Xenopus APC as bait and obtained several SAMP binding candidates including DDEF2 (development and differentiation enhancing factor 2), which is an ADP-ribosylation factor (Arf) GTPase-activating protein (GAP (ArfGAP)) involved in the regulation of focal adhesions. In vitro and in cells the Src homology 3 (SH3) domain of DDEF2 and its close homolog, DDEF1, are associated with the SAMP motif of APC competitively with Axin1. Moreover, NMR chemical shift perturbation experiments revealed that the SAMP motif interacts at the same surface of the SH3 domain of DDEF as the known SH3 binding motif, PXXP. When fluorescent protein-tagged APC and DDEF are expressed in Xenopus A6 cells, co-localization at microtubule ends is observed. Overexpression and RNA interference experiments indicate that APC and DDEFs cooperatively regulate the distributions of microtubules and focal adhesions. Our findings reveal that the SAMP motif of APC specifically binds to the SH3 domains of DDEFs, providing new insights into the functions of APC in cell migration.
Footnotes
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↵3 The abbreviations used are: MT, microtubule; APC, adenomatous polyposis coli; +TIPs, plus-end-binding proteins; DDEF, development and differentiation enhancing factor; HSQC, heteronuclear single quantum correlation; FA, focal adhesion; FAK, FA kinase; SH3, Src homology 3; RGS, regulator of G protein signaling; ArfGAP, ADP-ribosylation factor GTPase-activating protein; siRNA, small interfering RNA; Trx, thioredoxin; mAb, monoclonal antibody; pAb, polyclonal antibody; PPII, polyproline type II; aa, amino acid(s); FAP, familial adenomatous polyposis; GFP, green fluorescent protein; rAxin, rat Axin; GST, glutathione S-transferase.
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↵* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-4 and Movie 1.
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↵1 These authors contributed equally to this work.
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- Received January 16, 2008.
- Revision received August 22, 2008.
- The American Society for Biochemistry and Molecular Biology, Inc.
