title: Disruption of Ca2+-dependent cell-matrix adhesion enhances c-Src kinase activity, but causes dissociation of the c-Src/FAK complex and dephosphorylation of tyrosine-577 of FAK in carcinoma cells.
abstract: The nonreceptor tyrosine kinase c-Src is activated in most invasive cancers. Activated c-Src binds to FAK in the focal adhesion complex, resulting in the activation of the c-Src/FAK signaling cascade, which regulates cytoskeletal functions. However, the mechanisms by which c-Src/FAK signaling is regulated during conditions of anchorage-independent growth, a hallmark of tumor progression, are not clearly known. Here, an in vivo approach to measure c-Src activity was studied using phospho-specific antibodies against phosphorylated Y418 of c-Src (Src[pY418]), an autophosphorylation site of c-Src, and phosphorylated Y577 of FAK (FAK[pY577]), a known substrate of c-Src. Using genetic and pharmacological approaches to modulate c-Src activity, we showed that the levels of Src[pY418] and FAK[pY577], and the formation of a c-Src/FAK[pY577] complex correlated with the activation state of c-Src in adherent cells. Interestingly, both the in vivo level of Src[pY418] and in vitro c-Src kinase activity were increased in carcinoma cells following disruption of Ca(2+)-dependent cell-matrix adhesion. In contrast, the level of FAK[pY577] and its association with c-Src were reduced in suspended cells. The amount of FAK[pY577] in suspended cells was recovered following attachment of rounded cells to fibronectin-coated polystyrene beads, indicating that cell spreading was not required for phosphorylation of FAK. Moreover, cells expressing activated c-Src showed sustained Src[Y418] phosphorylation, but required Ca(2+)-dependent cell adhesion for phosphorylation of FAK[Y577] and association of c-Src with FAK[pY577]. These findings indicate an important role of integrin-based cell-matrix adhesion in regulating c-Src/FAK signaling under decreased anchorage conditions.
authors.affiliation: Division of Cancer Biology and Genetics, Cancer Research Institute, Queen's University, Kingston, Ontario, Canada.
authors.forename: Eva H
authors.initials: EH
authors.lastname: Lin
authors.forename: Angela Y
authors.initials: AY
authors.lastname: Hui
authors.forename: Jalna A
authors.initials: JA
authors.lastname: Meens
authors.forename: Eric A
authors.initials: EA
authors.lastname: Tremblay
authors.forename: Erik
authors.initials: E
authors.lastname: Schaefer
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Elliott
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002277:Carcinoma / Q000235:genetics; D045744:Cell Line, Tumor; D021982:Cell-Matrix Junctions / Q000378:metabolism*; D004789:Enzyme Activation; D005353:Fibronectins / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D015674:Mammary Neoplasms, Animal / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010649:Phenylalanine / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains; D019061:src-Family Kinases
chemical_list: D005353:Fibronectins; D014443:Tyrosine; D010649:Phenylalanine; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D019061:src-Family Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases