FIG. 5.

Active Src kinase is required for the endocytic uptake of Tf. (a and b) WB analysis and corresponding quantitation of activated Src precipitated from MEFs that were treated with Tf (10 μg/ml) over 0 to 20 min. Ligand addition induced a 7- to 10-fold activation of Src by 5 to 10 min. This activation exceeded that observed by EGF addition and occurs at a time corresponding to endocytic vesicle formation, and an increased physical interaction between the TfR1, Dyn2, and Cort. (c and d) Src inhibitory drugs reduce Tf internalization in MEF or Clone 9 cells. Serum-starved cells were incubated in vehicle alone or various increasing concentrations of PP2 or SU6656 for 2 h prior to addition of 10 μg/ml Alexa-594 Tf for 20 min. Cells were then fixed, and fluorescence was quantitated for Tf internalization. A marked reduction of Tf internalization was observed at low or moderate concentrations of either drug. Data are presented as means ± the standard deviation. (e and f) IF images of MEF control cells (e) or MEFs cultured from SYF^−/− mice (f) that were incubated with Alexa-594-labeled Tf at 37°C for 20 min. While the control MEFs internalized substantial amounts of ligand, the SYF^−/− cells internalized 80% less (g). Expression of an exogenous, active c-Src (Y530F) in the SYF^−/− cells (h) rescued over 60% of the cells to internalize Alexa-594 Tf (h′ and i) compared to the untransfected cells. (j to m) Clone 9 cells that were incubated with Alexa-594-labeled Tf at 37°C for 20 min and then fixed and viewed by IF. Control cells (j) internalized substantially more Tf than did cells expressing inactive forms of c-Src, such as the c-Src Y419F mutant (k) or the c-Src K297M mutant (l) that were reduced by 50% and 70%, respectively (m). Asterisks represent transfected cells. Results represent the averages ± SE of >100 cells measured in each of 3 independent experiments. Bars = 10 μm.