Figure 1.

Brk sensitizes breast cancer cells to EGF stimulation by inhibiting ligand-induced EGFR degradation. (a) Brk was overexpressed more often than EGFR in breast cancer cell lines. Expression of Brk and EGFR in the indicated breast cancer cells was detected by Western blotting. β-actin was used as a loading control. (b) Brk sensitizes breast cancer cells to EGF-induced cell proliferation. SUM102-neo and SUM102-Brk cells were cultured in the presence or absence of the indicated concentrations of EGF in 0.5% FBS medium for 5 days. Relative cell growth and survival were determined by an MTT assay. (c) Brk enhances EGF-induced activation of cell signaling. Following transient transfection with Brk or vector for 24 h, SUM102 cells were treated with the indicated concentrations of EGF in 0.5% FBS medium for 10 min and then harvested for Western blotting with the indicated antibodies. (d) Knockdown of Brk expression decreases EGF-induced activation of cell signaling. Forty-eight hours after knockdown of Brk by siRNA, SUM102 cells were treated with the indicated concentrations of EGF in 0.5% FBS medium for 10 min and then harvested for Western blotting with the indicated antibodies. (e) Brk sustains activated EGFR level and signaling. SUM102 cells were treated with 10 μM cycloheximide for 30 min prior to incubation with 10 nM EGF in 0.5% FBS medium for the indicated time periods. Cell lysates were harvested for Western blotting with the indicated antibodies. EGFR-Yp = total phosphorylated EGFR. (f) Brk inhibits EGF-induced EGFR degradation. ³⁵S-labeled methionine metabolic-labeled SUM102 cells were pulse-chased for up to 120 min in the presence or absence of 10 nM EGF in 0.5% FBS medium. The levels of EGFR immunoprecipitated at 30, 60, and 120 min were quantified by densitometry and plotted against the pulse-chase time period. Note: The ratios in this figure and in other figures represent quantitative analysis of densitometric values of specific band intensities normalized to the densitometric value of the leftmost lane in the same gel, which has a densitometric value and was arbitrarily set at 1. All of the experiments in this figure and in other figures were repeated at least once with similar findings.