Ankyrin-Tiam1 interaction promotes Rac1 signaling and metastatic breast tumor cell invasion and migration.
Tiam1 (T-lymphoma invasion and metastasis 1) is one of the known guanine nucleotide (GDP/GTP) exchange factors (GEFs) for Rho GTPases (e.g., Rac1) and is expressed in breast tumor cells (e.g., SP-1 cell line). Immunoprecipitation and immunoblot analyses indicate that Tiam1 and the cytoskeletal protein, ankyrin, are physically associated as a complex in vivo. In particular, the ankyrin repeat domain (ARD) of ankyrin is responsible for Tiam1 binding. Biochemical studies and deletion mutation analyses indicate that the 11-amino acid sequence between amino acids 717 and 727 of Tiam1 ((717)GEGTDAVKRS(727)L) is the ankyrin-binding domain. Most importantly, ankyrin binding to Tiam1 activates GDP/GTP exchange on Rho GTPases (e.g., Rac1). Using an Escherichia coli-derived calmodulin-binding peptide (CBP)-tagged recombinant Tiam1 (amino acids 393-728) fragment that contains the ankyrin-binding domain, we have detected a specific binding interaction between the Tiam1 (amino acids 393-738) fragment and ankyrin in vitro. This Tiam1 fragment also acts as a potent competitive inhibitor for Tiam1 binding to ankyrin. Transfection of SP-1 cell with Tiam1 cDNAs stimulates all of the following: (1) Tiam1-ankyrin association in the membrane projection; (2) Rac1 activation; and (3) breast tumor cell invasion and migration. Cotransfection of SP1 cells with green fluorescent protein (GFP)-tagged Tiam1 fragment cDNA and Tiam1 cDNA effectively blocks Tiam1-ankyrin colocalization in the cell membrane, and inhibits GDP/GTP exchange on Rac1 by ankyrin-associated Tiam1 and tumor-specific phenotypes. These findings suggest that ankyrin-Tiam1 interaction plays a pivotal role in regulating Rac1 signaling and cytoskeleton function required for oncogenic signaling and metastatic breast tumor cell progression.
Bourguignon
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LY
Department of Cell Biology and Anatomy, School of Medicine, University of Miami, Miami, Florida 33136, USA. Lbourgui@med.miami.edu
Zhu
H
H
Shao
L
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Chen
Y W
YW
D000818:Animals; D017487:Ankyrins / Q000378:metabolism*; D001665:Binding Sites; D001667:Binding, Competitive / Q000187:drug effects; D019556:COS Cells; D002465:Cell Movement / Q000187:drug effects; D003598:Cytoskeletal Proteins / Q000378:metabolism; D005260:Female; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D008325:Mammary Neoplasms, Experimental / Q000378:metabolism* / Q000473:pathology*; D051379:Mice; D009361:Neoplasm Invasiveness / Q000235:genetics; D010446:Peptide Fragments / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; D014162:Transfection; D014407:Tumor Cells, Cultured; D020830:rac1 GTP-Binding Protein / Q000378:metabolism*
D017487:Ankyrins; D003598:Cytoskeletal Proteins; D020662:Guanine Nucleotide Exchange Factors; D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; D000075684:T-Lymphoma Invasion and Metastasis-inducing Protein 1; C513260:Tiam1 protein, mouse; D020830:rac1 GTP-Binding Protein