Identification of a Link between the SAMP Repeats of Adenomatous Polyposis Coli Tumor Suppressor and the Src Homology 3 Domain of DDEF

Chiyuki Matsui,Shuji Kaieda,Takahisa Ikegami,Yuko Mimori-Kiyosue

doi:10.1074/jbc.m800420200

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.

Highlights

To date ϩTIPs have been proven to play an important role in the polarized organization of MT networks
By a yeast two-hybrid screening system, we identified DDEF1 and DDEF2, paxillin- and focal adhesion kinase (FAK)-binding proteins, as novel SAMP repeat-interacting proteins, the interaction being mediated through their Src homology 3 (SH3) domains
Found that, in addition to the armadillo repeats associating with kinesin motor protein and the basic region binding to MTs directly, the SAMP repeats region has a MT-related function, the molecular mechanism that targets the adenomatous polyposis coli (APC) fragment retaining the SAMP repeats is not yet clear

Summary

Introduction

To date ϩTIPs have been proven to play an important role in the polarized organization of MT networks. Analysis of SAMP-SH3 Interaction by NMR Chemical Shift Perturbation—To further confirm the interactions between the SH3 domains and the SAMP motif in vitro, we acquired a series of twodimensional 1H,15N HSQC NMR spectra of 15N-labeled SH3 domains protein A-Sepharose using an anti-GFP mAb. The SAMP binding of DDEFs, ARHGAPs, and human Axin1-RGS upon the addicandidates were fused to mRFP and expressed in HEK293 cells, tion of a non-labeled SAMP peptide.

Results

Conclusion