Rho-kinase (ROK) promotes CD44v3,8-10-ankyrin interaction and tumor cell migration in metastatic breast cancer cells

Abstract

Metastatic breast tumor Met‐1 cells express CD44v3,8–10, 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 CD44v3,8–10 and RhoA GTPases are physically associated in vivo, and that CD44v3,8–10‐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 CD44v3,8–10‐bound RhoA GTPase. Specifically, RhoA (complexed with CD44v3,8–10) stimulates ROK‐mediated phosphorylation of certain cellular proteins including the cytoplasmic domain of CD44v3,8–10. Most importantly, phosphorylation of CD44v3,8–10 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 CD44v3,8–10‐ankyrin interaction and RhoA‐mediated oncogenic signaling required for membrane‐cytoskeleton function and metastatic tumor cell migration. Cell Motil. Cytoskeleton 43:269–287, 1999. © 1999 Wiley‐Liss, Inc.

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

“…Both ankyrin1 (ANK1) and ankyrin 3 (ANK3) have been shown to be expressed in breast tumor cells (Bourguignon et al 1998b, Bourguignon et al 1999). In this study, we have carried out anti-ANK1 or anti-ANK3–mediated immunoprecipitation of SP-1 cellular proteins, followed by anti-Tiam1 immunoblot (Fig.…”

“…For example, recent studies have shown that certain metastasis-specific molecules (e.g., CD44v 3,8–10 isoform [Bourguignon et al 1998b, Bourguignon et al 1999] and its associated matrix metalloproteinase, MMP-9 [Bourguignon et al, 1998b; Yu and Stamenkovic 1999], as well as Rho kinase [Bourguignon et al 1999]) are closely associated with the cytoskeleton during tumor cell function. To further examine the regulatory mechanism(s) involved in cytoskeleton-mediated oncogenic signaling leading to tumor cell invasion and migration, we have focused on GEFs (the Dbl or DH family), such as Tiam1, which are known to display oncogenic capability and function as upstream activators of Rho-like GTPases (e.g., Rac1 or Cdc42; Woods et al, 1991; Habets et al 1994; Michiels et al 1995; Nobes and Hall 1995; Van Leeuwen et al 1995).…”

The interaction of versican with its binding partners

“…Hyaluronan-CD44 signaling can activate several pathways through activation of specific intermediates [39]. These include, but are not limited to, the Rho and Rac1 GTPases, whose activities lead to subsequent reorganization of the actin cytoskeleton [40][41][42]; erbB2 tyrosine kinase [43], which leads to cell proliferation; src-related tyrosine kinases [44], and nuclear factor-κB [NF-κB] [45,46]. Hyaluronan and CD44 interaction have also been known to affect cell adhesion and migration.…”

CD44 Variant Isoforms Promote Metastasis Formation by a Tumor Cell-Matrix Cross-talk That Supports Adhesion and Apoptosis Resistance

“…The major ligand of CD44 is hyaluronan (10,12). Hyaluronan-bound CD44 interacts with actin via ankyrin (13) or activated ERM proteins (14). The CD44 interaction with the cytoskeleton can also proceed through Src kinases, Ras, and Rho GTPases (15).…”