Ankyrin–Tiam1 Interaction Promotes Rac1 Signaling and Metastatic Breast Tumor Cell Invasion and Migration

Abstract

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 (717GEGTDAVKRS727L) 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.

Hyaluronan-mediated CD44 activation of RhoGTPase signaling and cytoskeleton function promotes tumor progression

“…The structural homology between the ankyrinbinding domain of Tiam1 (the sequence between aa717 and aa727 within the N-terminus PH-CC-Ex domain) and CD44 is quite striking. Most importantly, the Tiam1-ankyrin interaction promotes Rac1 activation and breast tumor cell migration [88]. These observations clearly suggest that Tiam1 contains multiple functional domains (e.g., a CD44-specific membrane localization site and a cytoskeleton binding region for ankyrin) required for the regulation of Tiam1-Rac1 signaling and cytoskeleton function.…”

“…In addition, a previous work demonstrated that the N-terminus PH-CC-Ex domain of Tiam1 also contains an ankyrin-binding site [88]. The structural homology between the ankyrinbinding domain of Tiam1 (the sequence between aa717 and aa727 within the N-terminus PH-CC-Ex domain) and CD44 is quite striking.…”

Identification of a Novel Human Tankyrase through Its Interaction with the Adaptor Protein Grb14

“…As indicated by the mode of tankyrase 2 binding to Grb14, the repeat region of the tankyrases is likely to participate in protein-protein interactions in a manner similar to that of the ankyrins themselves, which link specific integral membrane proteins to the underlying cytoskeleton (41). A precedent for the interaction of ankyrins with signaling proteins associated with the plasma membrane is the binding of the ankyrin-repeat regions of Ank1 and Ank3 to the Rho family GDP-GTP exchange factor Tiam 1 (42). However, the ankyrins are not restricted to a plasma membrane location.…”

An Alternative Transcript Derived from the Trio Locus Encodes a Guanosine Nucleotide Exchange Factor with Mouse Cell-transforming Potential

“…The coordinated activation of these Rho family proteins is thought to be a possible mechanism underlying cell motility, an obvious prerequisite for metastasis or invasion. It has also been shown that Tiam1, which is Rac1-GEF identified by retroviral insertional mutagenesis and selected for its invasive cell behavior in vitro, promotes Rac1 signaling and metastatic breast tumor cell invasion and migration by interaction with ankyrin (41). In the case of Tgat, the existence of the unique C-terminal region is indicated to be more important than Rho-GEF activity for the invasion-inducing ability, because Y-27632 treatment or GEF-dead mutants did not reduce its invasiveness completely.…”