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J. Biol. Chem., Vol. 282, Issue 49, 36024-36036, December 7, 2007

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The Intercalated Disc Protein, mXin, Is Capable of Interacting with β-Catenin and Bundling Actin Filaments^*

From the Department of Biological Sciences, University of Iowa, Iowa City, Iowa 52242-1324

Targeted deletion of mXin results in cardiac hypertrophy and cardiomyopathy with conduction defects (Gustafson-Wagner, E., Sinn, H. W., Chen, Y.-L., Wang, D.-Z., Reiter, R. S., Lin, J. L.-C., Yang, B., Williamson, R. A., Chen, J. N., Lin, C.-I., and Lin, J. J.-C. (2007) Am. J. Physiol. 293, H2680-H2692). To understand the underlying mechanisms leading to such cardiac defects, the functional domains of mXin and its interacting proteins were investigated. Interaction studies using co-immunoprecipitation, pull-down, and yeast two-hybrid assays revealed that mXin directly interacts with β-catenin. The β-catenin-binding site on mXin was mapped to amino acids 535-636, which overlaps with the known actin-binding domains composed of the Xin repeats. The overlapping nature of these domains provides insight into the molecular mechanism for mXin localization and function. Purified recombinant glutathione S-transferase- or His-tagged mXin proteins are capable of binding and bundling actin filaments, as determined by co-sedimentation and electron microscopic studies. The binding to actin was saturated at an approximate stoichiometry of nine actin monomers to one mXin. A stronger interaction was observed between mXin C-terminal deletion and actin as compared with the interaction between full-length mXin and actin. Furthermore, force expression of green fluorescent protein fused to an mXin C-terminal deletion in cultured cells showed greater stress fiber localization compared with force-expressed GFP-mXin. These results suggest a model whereby the C terminus of mXin may prevent the full-length molecule from binding to actin, until the β-catenin-binding domain is occupied by β-catenin. The binding of mXin to β-catenin at the adherens junction would then facilitate actin binding. In support of this model, we found that the actin binding and bundling activity of mXin was enhanced in the presence of β-catenin.

Received for publication, September 11, 2007

^* This work was supported by National Institutes of Health Grant HL075015. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2.

¹ These authors contributed equally to this work.

² Supported by a predoctoral fellowship from the American Heart Association Heartland Affiliate.

³ To whom correspondence should be addressed: Dept. of Biological Sciences, University of Iowa, 340 Biology Bldg. East, 210 E. Iowa Ave., Iowa City, IA 52242-1324. Tel.: 319-335-1075; Fax: 319-353-2275; E-mail: jim-lin{at}uiowa.edu.

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