Epidermal Growth Factor (EGF) Regulates α5β1 Integrin Activation State in Human Cancer Cell Lines through the p90RSK-dependent Phosphorylation of Filamin A

Cell adhesion, motility, and invasion are regulated by the ligand-binding activity of integrin receptors, transmembrane proteins that bind to the extracellular matrix. Integrins whose conformation allows for ligand binding and appropriate functional activity are said to be in an active state. Integrin activation and subsequent ligand binding are dynamically regulated by the association of cytoplasmic proteins with integrin intracellular domains. In this study, we evaluated the role of EGF in the regulation of the activation state of the α5β1 integrin receptor for fibronectin. The addition of EGF to either A431 squamous carcinoma cells or DiFi colon cancer cells resulted in loss of α5β1-dependent adhesion to fibronectin but no loss of integrin from the cell surface. EGF activated the EGF receptor/ERK/p90RSK and Rho/Rho kinase signaling pathways. Blocking either pathway inhibited EGF-mediated loss of adhesion, suggesting that they work in parallel to regulate integrin function. EGF treatment also resulted in phosphorylation of filamin A (FLNa), which binds and inactivates β1 integrins. EGF-mediated FLNa phosphorylation was completely blocked by an inhibitor of p90RSK and partially attenuated by an inhibitor of Rho kinase, suggesting that both pathways converge on FLNa to regulate integrin function. A431 clonal cell lines expressing non-phosphorylated dominant-negative FLNa were resistant to the inhibitory effects of EGF on integrin function, whereas clonal cell lines overexpressing wild-type FLNa were more sensitive to the inhibitory effect of EGF. These data suggest that EGF-dependent inactivation of α5β1 integrin is regulated through FLNa phosphorylation and cellular contractility.