Lilly Y. W. Bourguignon

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).

The adhesion molecule, CD44, interacts with ankyrin within its cytoplasmic domain and binds to hyaluronic acid (HA) at its extracellular domain. In this study, we focused on the functional domain in ankyrin (in particular, the ankyrin repeat domain [ARD]) responsible for CD44 binding and its role in regulating HA‐mediated ovarian tumor cell function. Using recombinant fragments of ankyrin (e.g., ARD and subdomain 1 [S1, aa1‐aa217], subdomain 2 [S2, aa218‐aa381], subdomain 3 [S3, aa382‐aa612], and subdomain 4 [S4, aa613‐aa834]) and in vitro binding assays, we determined that the S2 but not S1, S3, or S4 of ARD is the primary ankyrin binding region for CD44. Microinjection of antiglutathione S‐transferase (GST)‐tagged S2 or GST‐tagged ARD fusion protein into CD44‐positive ovarian tumor cells (e.g., SKOV3 cell line) promotes ankyrin association with CD44 in plaque‐like structures and membrane projections. Additionally, we demonstrated that transfection of SKOV3 cells with S2cDNA or ARD cDNA results in an upregulation of HA‐mediated tumor cell migration. Taken together, we believe that the S2 of the ARD plays a pivotal role in the direct binding to CD44 and promotes the cytoskeleton activation required for HA‐mediated function such as ovarian tumor cell migration. J. Cell. Physiol. 183:182–195, 2000.