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 nu cleotide (GDP/GTP) exchange factors (GEFs) for Rho GTPases (e.g., Rad) and is expressed in breast tumor cells (e.g., SP-1 cell line). Immunoprecipitat ion and immunoblot analyses indicate that Tiam1 and the cytoskeletal protei n, 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 Il-a mino acid sequence between amino acids 717 and 727 of Tiam1 ((717)GEGTDAVKR S(727)L) is the ankyrinbinding domain. Most importantly, ankyrin binding to Tiam1 activates GDP/GTP exchange on Rho GTPases (e.g., Rad). Using an Escherichia coli-derived calmodulin-binding peptide (CBP)-tagged r ecombinant Tiam1 (amino acids 393-728) fragment that contains the ankyrinbi nding domain, we have detected a specific binding interaction between the T iam1 (amino acids 393-738) fragment and ankyrin in vitro. This Tiam1 fragme nt also acts as a potent competitive inhibitor for Tiam1 binding to ankyrin . Transfection of SP-1 cell with Tiam1 cDNAs stimulates all of the followin g: (1) Tiam1-ankyrin association in the membrane projection; (2) Rad activa tion; and (3) breast tumor cell invasion and migration. Cotransfection of S P1 cells with green fluorescent protein (GFP)-tagged Tiam1 fragment cDNA an d Tiam1 cDNA effectively blocks Tiam1-ankyrin colocalization in the cell me mbrane, and inhibits GDP/GTP exchange on Rad by ankyrin-associated Tiam1 an d tumor-specific phenotypes. These findings suggest that ankyrin-Tiam1 inte raction plays a pivotal role in regulating Rad signaling and cytoskeleton f unction required for oncogenic signaling and metastatic breast tumor cell p rogression.