Hyaluronan-mediated CD44 Interaction with RhoGEF and Rho Kinase Promotes Grb2-associated Binder-1 Phosphorylation and Phosphatidylinositol 3-Kinase Signaling Leading to Cytokine (Macrophage-Colony Stimulating Factor) Production and Breast Tumor Progression

Lilly Y.W Bourguignon,Patrick A Singleton,Hongbo Zhu,Falko Diedrich

doi:10.1074/jbc.m301885200

Lilly Y.W Bourguignon, Patrick A Singleton + Show 2 more

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https://doi.org/10.1074/jbc.m301885200

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Abstract

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.

Highlights

Hyaluronan-mediated CD44 Interaction with RhoA-specific guanine nucleotide exchange factors (RhoGEFs) and Rho Kinase Promotes Grb2-associated Binder-1 Phosphorylation and Phosphatidylinositol 3-Kinase Signaling Leading to Cytokine (Macrophage-Colony Stimulating Factor) Production and Breast Tumor Progression*
MDA-MB-231 cells were solubilized by 1.0% Nonidet P-40 and immunoprecipitated with rabbit anti-CD44v3 antibody followed by anti-p115RhoGEF-mediated or antip115RhoGEF-free serum-mediated immunoblot or immunoprecipitated with anti-p115RhoGEF antibody followed by anti-CD44v3mediated or anti-CD44v3-free serum-mediated immunoblot, respectively
Several RhoA-specific guanine nucleotide exchange factors (RhoGEFs) have been shown to be involved in RhoA signaling [33, 34, 58], identification of the specific RhoGEF protein that plays a direct role in regulating CD44v3-specific metastatic behaviors in breast tumor cells is addressed in this study

Summary

Introduction

Hyaluronan-mediated CD44 Interaction with RhoGEF and Rho Kinase Promotes Grb2-associated Binder-1 Phosphorylation and Phosphatidylinositol 3-Kinase Signaling Leading to Cytokine (Macrophage-Colony Stimulating Factor) Production and Breast Tumor Progression*. Several mechanisms for the regulation of HA/CD44-mediated function have been suggested These include modifications by an additional exon-coded structure (via an alternative splicing process) [1,2,3,4,5,6], variable N/O-linked glycosylation on the extracellular domain of the CD44 [17, 18], and selective interactions of the cytoplasmic domain of the CD44 with ankyrin [11, 15] and various signaling molecules (e.g. the Src family tyrosine kinases [14, 19], p185HER2 [20, 21], Rho kinase (ROK) [22], transforming growth factor-␤ receptor kinases [23], and the guanine nucleotide exchange factors Tiam1 [24] and Vav2 [21]). CD44 has been shown to be involved in the production of cytokines

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