Cortactin is an actin-binding protein that is enriched within the lamellipodia of motile cells and in neuronal growth cones. Here, we report that cortactin is localized with the actin-related protein (Arp) 2/3 complex at sites of actin polymerization within the lamellipodia. Two distinct sequence motifs of cortactin contribute to its interaction with the cortical actin network: the fourth of six tandem repeats and the amino-terminal acidic region (NTA). Cortactin variants lacking either the fourth tandem repeat or the NTA failed to localize at the cell periphery. Tandem repeat four was necessary for cortactin to stably bind F-actin in vitro. The NTA region interacts directly with the Arp2/3 complex based on affinity chromatography, immunoprecipitation assays, and binding assays using purified components. Cortactin variants containing the NTA region were inefficient at promoting Arp2/3 actin nucleation activity. These data provide strong evidence that cortactin is specifically localized to sites of dynamic cortical actin assembly via simultaneous interaction with F-actin and the Arp2/3 complex. Cortactin interacts via its Src homology 3 (SH3) domain with ZO-1 and the SHANK family of postsynaptic density 95/dlg/ZO-1 homology (PDZ) domain–containing proteins, suggesting that cortactin contributes to the spatial organization of sites of actin polymerization coupled to selected cell surface transmembrane receptor complexes.
Cortactin Localization to Sites of Actin Assembly in Lamellipodia Requires Interactions with F-Actin and the Arp2/3 Complex
Address correspondence to J. Thomas Parsons, Department of Microbiology, Box 800734, University of Virginia Health Sciences Center, Charlottesville, VA 22908-0734. Tel.: (804) 924-5395. Fax: (804) 924-1071. E-mail: [email protected]
S.A. Weed's present address is School of Dentistry, Department of Basic Sciences and Oral Research, Box C286, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Denver, CO 80262.
Abbreviations used in this paper: Arp, actin-related protein; CMV, cytomegalovirus; CortBP-1, cortactin-binding protein 1; GFP, green fluorescent protein; GST, glutathione S-transferase; LC-MS, liquid chromatography mass spectrometry; NTA, amino-terminal acidic domain; PDZ, PSD95/dlg/ZO-1 homology; PSD, postsynaptic density; Scar, suppressor of cAr; SH, Src homology; SHANK, SH3 domain and ankyrin repeat protein; VCA, verpolin homology, cofilin, and acidic domain contained in the COOH-terminal fragment of N-WASp; WASp, Wiskott-Aldrich Syndrome protein.
Scott A. Weed, Andrei V. Karginov, Dorothy A. Schafer, Alissa M. Weaver, Andrew W. Kinley, John A. Cooper, J. Thomas Parsons; Cortactin Localization to Sites of Actin Assembly in Lamellipodia Requires Interactions with F-Actin and the Arp2/3 Complex. J Cell Biol 2 October 2000; 151 (1): 29–40. doi: https://doi.org/10.1083/jcb.151.1.29
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