Metastatic breast tumor Met-1 cells express CD44v(3,8-10), a major adhesion receptor that binds extracellular matrix components at its extracellular domain and interacts with the cytoskeletal protein, ankyrin, at its cytoplasmic domain. In this study, we have determined that CD44v(3,8-10) and RhoA GTPases are physically associated in vivo, and that CD44v(3,8-10)-bound RhoA displays GTPase activity, which can be inhibited by botulinum toxin C3-mediated ADP-ribosylation. In addition, we have identified a 160 kDa Rho-Kinase (ROK) as one of the downstream targets for CD44v(3,8-10)-bound RhoA GTPase. Specifically, RhoA (complexed with CD44v(3, 8-10)) stimulates ROK-mediated phosphorylation of certain cellular proteins including the cytoplasmic domain of CD44v(3,8-10). Most importantly, phosphorylation of CD44v(3,8-10) by ROK enhances its interaction with the cytoskeletal protein, ankyrin. We have also constructed two ROK cDNA constructs that encode for proteins consisting of 537 amino acids [designated as the constitutively active form of ROK containing the catalytic domain (CAT, also the kinase domain)], and 173 amino acids [designated as the dominant-negative form of ROK containing the Rho-binding domain (RB)]. Microinjection of the ROK's CAT domain into Met-1 cells promotes CD44-ankyrin associated membrane ruffling and projections. This membrane motility can be blocked by CD44 antibodies and cytochalasin D (a microfilament inhibitor). Furthermore, overexpression of a dominant-negative form of ROK by transfection of Met-1 cells with ROK's Rho-binding (RB) domain cDNA effectively inhibits CD44-ankyrin-mediated metastatic behavior (e.g., membrane motility and tumor cell migration). These findings support the hypothesis that ROK plays a pivotal role in CD44v(3,8-10)-ankyrin interaction and RhoA-mediated oncogenic signaling required for membrane-cytoskeleton function and metastatic tumor cell migration.
Copyright 1999 Wiley-Liss, Inc.