title: Tiam1 is recruited to β1-integrin complexes by 14-3-3ζ where it mediates integrin-induced Rac1 activation and motility.
abstract: 14-3-3 is an adaptor protein that localizes to the leading edge of spreading cells, returning to the cytoplasm as spreading ceases. Previously, we showed that integrin-induced Rac1 activation and spreading were inhibited by sequestration of 14-3-3ζ and restored by its overexpression. Here, we determined whether 14-3-3 mediates integrin signaling by localizing a guanine nucleotide exchange factor (GEF) to Rac1-activating integrin complexes. We showed that GST-14-3-3ζ recruited the Rac1-GEF, Tiam1, from cell lysates through Tiam1 residues 1-182 (N(1-182) Tiam1). The physiological relevance of this interaction was examined in serum-starved Hela cells plated on fibronectin. Both Tiam1 and N(1-182) Tiam1 were recruited to 14-3-3-containing β1-integrin complexes, as shown by co-localization and co-immunoprecipitation. Integrin-induced Rac1 activation was inhibited when Tiam1 was depleted with siRNA or by overexpression of catalytically inactive N(1-182) Tiam1, which was incorporated into 14-3-3/β1-integrin complexes and inhibited spreading in a manner that was overcome by constitutively active Rac1. Integrin-induced Rac1 activation, spreading, and migration were also inhibited by overexpression of 14-3-3ζ S58D, which was unable to recruit Tiam1 from lysates, co-immunoprecipitate with Tiam1, or mediate its incorporation into β1-integrin complexes. Taken together, these findings suggest a previously unrecognized mechanism of integrin-induced Rac1 activation in which 14-3-3 dimers localize Tiam1 to integrin complexes, where it mediates integrin-dependent Rac1 activation, thus initiating motility-inducing pathways. Moreover, since Tiam1 is recruited to other sites of localized Rac1 activation through its PH-CC-EX domain, the present findings show that a mechanism involving its N-terminal 182 residues is utilized to recruit Tiam1 to motility-inducing integrin complexes.
authors.affiliation: Department of Molecular Cardiology, Joseph J. Jacobs Center for Thrombosis and Vascular Biology, The Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio 44195, USA.
authors.forename: Timothy E
authors.initials: TE
authors.lastname: O'Toole
authors.forename: Katarzyna
authors.initials: K
authors.lastname: Bialkowska
authors.forename: Xiaohong
authors.initials: X
authors.lastname: Li
authors.forename: Joan E B
authors.initials: JE
authors.lastname: Fox
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism*; D002465:Cell Movement; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D019012:Integrin beta1 / Q000378:metabolism*; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D020662:Guanine Nucleotide Exchange Factors; D019012:Integrin beta1; C107953:RAC1 protein, human; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; C087334:TIAM1 protein, human; D020830:rac1 GTP-Binding Protein