In this study we have examined CD44 (a
hyaluronan (HA) receptor) interaction with a RhoA-specific
guanine nucleotide exchange factor (
p115RhoGEF) in human metastatic
breast tumor cells (MDA-MB-231 cell line). Immunoprecipitation and immunoblot analyses indicate that both CD44 and
p115RhoGEF are expressed in MDA-MB-231 cells and that these two
proteins are physically associated as a complex in vivo. The binding of HA to MDA-MB-231 cells stimulates p115RhoGEF-mediated RhoA signaling and
Rho kinase (ROK) activity, which, in turn, increases
serine/
threonine phosphorylation of the adaptor
protein, Gab-1 (Grb2-associated binder-1). Phosphorylated Gab-1 promotes
PI 3-kinase recruitment to CD44v3. Subsequently,
PI 3-kinase is activated (in particular, alpha, beta, gamma forms but not the delta form of the p110 catalytic subunit), AKT signaling occurs, the
cytokine (
macrophage-colony stimulating factor (
M-CSF)) is produced, and
tumor cell-specific phenotypes (e.g.
tumor cell growth, survival and invasion) are up-regulated. Our results also demonstrate that HA/CD44-mediated oncogenic events (e.g. AKT activation,
M-CSF production and
breast tumor cell-specific phenotypes) can be effectively blocked by a
PI 3-kinase inhibitor (
LY294002). Finally, we have found that overexpression of a dominant-negative form of ROK (by transfection of MBA-MD-231 cells with the Rho-binding domain
cDNA of ROK) not only inhibits HA/CD44-mediated RhoA-ROK activation and Gab-1 phosphorylation but also down-regulates oncogenic signaling events (e.g. Gab-1.
PI 3-kinase-CD44v3 association,
PI 3-kinase-mediated AKT activation, and
M-CSF production) and
tumor cell behaviors (e.g. cell growth, survival, and invasion). Taken together, these findings strongly suggest that CD44 interaction with
p115RhoGEF and ROK plays a pivotal role in promoting Gab-1 phosphorylation leading to Gab-1.PI 3-kinase membrane localization, AKT signaling, and
cytokine (
M-CSF) production during HA-mediated
breast cancer progression.