α-Actinin-1 phosphorylation modulates pressure-induced colon cancer cell adhesion through regulation of focal adhesion kinase-Src interaction

Abstract

Physical forces including pressure, strain, and shear can be converted into intracellular signals that regulate diverse aspects of cell biology. Exposure to increased extracellular pressure stimulates colon cancer cell adhesion by a β1-integrin-dependent mechanism that requires an intact cytoskeleton and activation of focal adhesion kinase (FAK) and Src. α-Actinin facilitates focal adhesion formation and physically links integrin-associated focal adhesion complexes with the cytoskeleton. We therefore hypothesized that α-actinin may be necessary for the mechanical response pathway that mediates pressure-stimulated cell adhesion. We reduced α-actinin-1 and α-actinin-4 expression with isoform-specific small interfering (si)RNA. Silencing of α-actinin-1, but not α-actinin-4, blocked pressure-stimulated cell adhesion in human SW620, HT-29, and Caco-2 colon cancer cell lines. Cell exposure to increased extracellular pressure stimulated α-actinin-1 tyrosine phosphorylation and α-actinin-1 interaction with FAK and/or Src, and enhanced FAK phosphorylation at residues Y397 and Y576. The requirement for α-actinin-1 phosphorylation in the pressure response was investigated by expressing the α-actinin-1 tyrosine phosphorylation mutant Y12F in the colon cancer cells. Expression of Y12F blocked pressure-mediated adhesion and inhibited the pressure-induced association of α-actinin-1 with FAK and Src, as well as FAK activation. Furthermore, siRNA-mediated reduction of α-actinin-1 eliminated the pressure-induced association of α-actinin-1 and Src with β1-integrin receptor, as well as FAK-Src complex formation. These results suggest that α-actinin-1 phosphorylation at Y12 plays a crucial role in pressure-activated cell adhesion and mechanotransduction by facilitating Src recruitment to β1-integrin, and consequently the association of FAK with Src, to enhance FAK phosphorylation.