Alpha-actinin-1 phosphorylation modulates pressure-induced colon cancer cell adhesion through regulation of focal adhesion kinase-Src interaction.
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 beta(1)-integrin-dependent mechanism that requires an intact cytoskeleton and activation of focal adhesion kinase (FAK) and Src. alpha-Actinin facilitates focal adhesion formation and physically links integrin-associated focal adhesion complexes with the cytoskeleton. We therefore hypothesized that alpha-actinin may be necessary for the mechanical response pathway that mediates pressure-stimulated cell adhesion. We reduced alpha-actinin-1 and alpha-actinin-4 expression with isoform-specific small interfering (si)RNA. Silencing of alpha-actinin-1, but not alpha-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 alpha-actinin-1 tyrosine phosphorylation and alpha-actinin-1 interaction with FAK and/or Src, and enhanced FAK phosphorylation at residues Y397 and Y576. The requirement for alpha-actinin-1 phosphorylation in the pressure response was investigated by expressing the alpha-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 alpha-actinin-1 with FAK and Src, as well as FAK activation. Furthermore, siRNA-mediated reduction of alpha-actinin-1 eliminated the pressure-induced association of alpha-actinin-1 and Src with beta(1)-integrin receptor, as well as FAK-Src complex formation. These results suggest that alpha-actinin-1 phosphorylation at Y12 plays a crucial role in pressure-activated cell adhesion and mechanotransduction by facilitating Src recruitment to beta(1)-integrin, and consequently the association of FAK with Src, to enhance FAK phosphorylation.
Craig
David H
DH
Department of Surgery, John D Dingell Veterans Affairs Medical Center, Wayne State University, and Karmanos Cancer Institute, Detroit, MI, USA.
Haimovich
Beatrice
B
Basson
Marc D
MD
D000185:Actinin / Q000378:metabolism*; D015153:Blotting, Western; D018938:Caco-2 Cells; D002277:Carcinoma / Q000378:metabolism* / Q000503:physiopathology; D002448:Cell Adhesion / Q000502:physiology; D003110:Colonic Neoplasms / Q000378:metabolism* / Q000503:physiopathology; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000378:metabolism*; D020868:Gene Silencing; D019073:HT29 Cells; D006801:Humans; D047468:Immunoprecipitation; D019012:Integrin beta1 / Q000378:metabolism; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D009154:Mutation; D010766:Phosphorylation; D011312:Pressure; D034741:RNA, Small Interfering; D014162:Transfection; D019061:src-Family Kinases / Q000378:metabolism*
C526905:ACTN1 protein, human; D019012:Integrin beta1; D034741:RNA, Small Interfering; D000185:Actinin; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases