Oncogenic signaling stimulates the dynamic remodeling of actin microfilaments and substrate adhesions, essential for cell spreading and motility. Transformation is associated with increased expression of beta1,6GlcNAc-branched N-
glycans, products of Golgi beta1,6-acetylglucosaminyltransferase V (Mgat5) and the favored
ligand for
galectins. Herein we report that
fibronectin fibrillogenesis and
fibronectin-dependent cell spreading are deficient in Mgat5(-/-) mammary epithelial
tumor cells and inhibited in Mgat5(+/+) cells by blocking Golgi N-
glycan processing with
swainsonine or by competitive inhibition of
galectin binding. At an optimum dosage, exogenous
galectin-3 added to Mgat5(+/+) cells activates
focal adhesion kinase (FAK) and
phosphatidylinositol 3-kinase (PI3K), recruits conformationally active alpha5beta1-integrin to fibrillar adhesions, and increases
F-actin turnover.
RGD peptide inhibits PI3K-dependent
fibronectin matrix remodeling and
fibronectin-dependent cell motility, while
galectin-3 stimulates and overrides the inhibitory effects of RGD.
Antibodies to the
galectin-3 N-terminal oligomerization domain stimulate alpha5beta1 activation and recruitment to fibrillar adhesions in Mgat5(+/+) cells, an effect that is blocked by disrupting
galectin-
glycan binding. Our results demonstrate that
fibronectin polymerization and
tumor cell motility are regulated by
galectin-3 binding to branched N-
glycan ligands that stimulate focal adhesion remodeling, FAK and PI3K activation, local
F-actin instability, and alpha5beta1 translocation to fibrillar adhesions.