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J. Biol. Chem., Vol. 281, Issue 45, 34640-34650, November 10, 2006

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Involvement of the Conserved Adaptor Protein Alix in Actin Cytoskeleton Assembly^*

Shujuan Pan¹, Ruoning Wang¹, Xi Zhou, Guangan He, John Koomen, Ryuji Kobayashi, Le Sun^¶, Joe Corvera^¶, Gary E. Gallick^||, and Jian Kuang²

From the Departments of Experimental Therapeutics, Molecular Pathology, and ^||Cancer Biology, the University of Texas, M. D. Anderson Cancer Center, Houston, Texas 77030 and ^¶A & G Pharmaceuticals, Inc., Baltimore, Maryland 21202

The conserved adaptor protein Alix, also called AIP1 or Hp95, promotes flattening and alignment of cultured mammalian fibroblasts; however, the mechanism by which Alix regulates fibroblast morphology is not understood. Here we demonstrate that Alix in WI38 cells, which require Alix expression for maintaining typical fibroblast morphology, associates with filamentous actin (F-actin) and F-actin-based structures lamellipodia and stress fibers. Reducing Alix expression by small interfering RNA (siRNA) decreases F-actin content and inhibits stress fiber assembly. In cell-free systems, Alix directly interacts with F-actin at both the N-terminal Bro1 domain and the C-terminal proline-rich domain. In Alix immunoprecipitates from WI38 cell lysates, actin is the most abundant partner protein of Alix. In addition, the N-terminal half of the middle region of Alix binds cortactin, an activator of the ARP2/3 complex-mediated initiation of actin polymerization. Alix is required for lamellipodial localization of cortactin. The C-terminal half of the middle region of Alix interacts with -actinin, a key factor that bundles F-actin in stress fibers. Alix knockdown decreases the amount of -actinin that associates with F-actin. These findings establish crucial involvement of Alix in actin cytoskeleton assembly.

Received for publication, March 10, 2006 , and in revised form, July 10, 2006.

^* This work was supported by American Cancer Society Grant RPG-00-071-01-DDC and NCI Grant 1 RO1 CA93941 from the National Institutes of Health (to J. K.). 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 Table S1 and Figs. S1-S4.

¹ Both authors should be considered as first authors.

² To whom correspondence should be addressed: Dept. of Experimental Therapeutics, the University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd., Box 019, Houston, TX 77030. Tel.: 713-792-8505; Fax: 713-792-3754; E-mail: jkuang{at}mdanderson.org.

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