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J. Biol. Chem., Vol. 281, Issue 47, 36454-36465, November 24, 2006

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Tropomodulin 3 Binds to Actin Monomers^*

Robert S. Fischer¹, Elena G. Yarmola, Kari L. Weber, Kaye D. Speicher^¶, David W. Speicher^¶, Michael R. Bubb, and Velia M. Fowler

From the Department of Cell Biology, The Scripps Research Institute, La, Jolla, California 92037, the Department of Medicine, University of Florida College of Medicine, Gainesville, Florida 32610, and the ^¶Systems Biology Division, The Wistar Institute, Philadelphia, Pennsylvania 19104

Regulation of the actin cytoskeleton by filament capping proteins is critical to myriad dynamic cellular functions. The ability of these proteins to bind both filaments as well as monomers is often central to their cellular functions. The ubiquitous pointed end capping protein Tmod3 (tropomodulin 3) acts as a negative regulator of cell migration, yet mechanisms behind its cellular functions are not understood. Analysis of Tmod3 effects on kinetics of actin polymerization and steady state monomer levels revealed that Tmod3, unlike previously characterized tropomodulins, sequesters actin monomers with an affinity similar to its affinity for capping pointed ends. Furthermore, Tmod3 is found bound to actin in high speed supernatant cytosolic extracts, suggesting that Tmod3 can bind to monomers in the context of other cytosolic monomer binding proteins. The Tmod3-actin complex can be efficiently cross-linked with 1-ethyl-3-(dimethylaminopropyl)carbodiimide/N-hydroxylsulfosuccinimide in a 1:1 complex. Subsequent tryptic digestion and liquid chromatography/tandem mass spectrometry revealed two binding interfaces on actin, one distinct from other actin monomer binding proteins, and two potential binding sites in Tmod3, which are independent of the previously characterized leucine-rich repeat structure involved in pointed end capping. These data suggest that the Tmod3 isoform may regulate actin dynamics differently in cells than the previously described tropomodulin isoforms.

Received for publication, July 3, 2006 , and in revised form, September 11, 2006.

^* This work was supported by National Institutes of Health Grants EY014972 (to R. S. F.), 5K25AR048918 (to M. R. B., and E. G. Y.), GM34225/HL083464 (to V. M. F.), and HL38794 (to D. W. S.). 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.

¹ To whom correspondence should be addressed: Dept. of Cell Biology, The Scripps Research Institute, CB163, La Jolla, CA 92037. Tel.: 858-784-9839; Fax: 858-784-8753; E-mail: bfischer{at}scripps.edu.

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