Figure 1
Transforming growth factor (TGF)-β-stimulated focal adhesion kinase (FAK) phosphorylation depends on Src and β3 integrin. (a) Fluorescently labeled phalloidin (red) and phosphorylated Y925-FAK (green) staining in quiescent (NS) NMuMG cells showed normal cortical actin distribution that colocalized with phosphorylated FAK at cell-cell junctions and borders. Administering TGF-β1 (5 ng/ml) for 18 hours promoted F-actin stress fiber formation, and reorganized phosphorylated FAK. DAPI counterstaining, shown in blue (overlay). Data are representative images from a single experiment that was repeated 3 times. (b) Immunoblot analysis of cell extracts prepared from NMuMG cells stimulated with TGF-β1 as in (a) showed that TGF-β treatment induced the phosphorylation of FAK at positions Y397, Y577, and Y925. Total FAK protein levels also were increased. Administration of the Src inhibitor, PP2 (10 μg/ml) for the duration of the TGF-β1 treatment (18 hours), prevented the phosphorylation of FAK at Y577 and Y925, but not at its autophosphorylation site, Y397. In addition, PP2 treatment prevented the induction of β3 integrin expression in NMuMG cells stimulated with TGF-β1. Differences in protein loading were monitored by reprobing stripped membranes with antibodies against β-actin. Data are representative images from a single experiment that was repeated 3 times. (c) FAK phosphorylation and its overall expression was decreased in nonadherent NMuMG cells (4 hours) expressing mutant β3 integrin (D119A), but not its wild-type counterpart (β3-WT). Protein loading was monitored by reprobing stripped membranes with antibodies against β-actin. Data are representative images from a single experiment that was repeated 3 times. (d) NMuMG cells were treated as in (c) and then replated for 4 hours in the absence or presence of TGF-β1 (5 ng/ml) before analyzing FAK phosphorylation and expression. Protein loading was monitored by reprobing stripped membranes with antibodies against β-actin. Data are representative images from a single experiment that was repeated twice with identical results. (e, f) Polyclonal populations of control (i.e., GFP), WT-β3 integrin (β3-WT)-, or D119A-β3 integrin (β3-D119A)-expressing NMuMG cells were stimulated with TGF-β1 (5 ng/ml) for 24 hours, and total RNA was isolated and analyzed with semiquantitative real-time polymerase chain reaction (PCR) to monitor FAK (e) and murine β3 integrin (f) expression. Data are expressed as the mean (± SEM; n = 3) relative FAK and β3 integrin transcript levels measured in response to TGF-β1, as compared with their unstimulated counterparts. (*P < 0.05).