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© The Rockefeller University Press,
0021-9525/2000//177 $5.00
The Journal of Cell Biology, Volume 150, Number 1,
, 2000 177-192
Original Article |
Ankyrin–Tiam1 Interaction Promotes Rac1 Signaling and Metastatic Breast Tumor Cell Invasion and Migration
Lbourgui{at}med.miami.edu
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.
Key Words: Tiam1 • ankyrin • Rac1 signaling • invasion/migration • metastatic breast tumor cells
© 2000 The Rockefeller University Press
Abbreviations used in this paper: ARD, ankyrin repeat domain; CBP, calmodulin-binding peptide; DH, Dbl homology; GFP, green fluorescent protein; GFP-SBD, GFP-tagged spectrin binding domain; GEF, guanine nucleotide exchange factor; HA, hemagglutinin; PH, pleckstrin homology; PHn, NH2-terminal PH; Rh, rhodamine; S1P, sphingosine-1-phosphate; SBD, spectrin binding domain; Tiam1, T lymphoma invasion and metastasis 1.
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