Figure 2

Epidermal growth factor treatment of breast cancer cells promotes the interaction of TNK2 with EGFR. (a) Epidermal growth factor receptor (EGFR) and TNK2 have variable protein expression levels in normal and breast cancer cell lines. The levels of TNK2 are shown relative to β-actin protein levels in the following cell lines: HB2, normal human breast epithelial cells; MCF-7, MDA-MB-468, and T47D human breast cancer cells; HC11 murine mammary epithelial cells; and human breast cancer cell line, MDA-MB-231. (b) TNK2 is recruited to the EGFR following activation of the receptor in MDA-MB-231 cells, as indicated by coimmunoprecipitation in the presence and absence of epidermal growth factor (EGF) stimulation at 100 ng/ml. (c) The association of EGFR and TNK2 in MDA-MB-231 cells is not dependent on the phosphorylation status of TNK2, as indicated by the ability of wildtype (Wt), mutant kinase-deficient (Kd) and constitutively active (Ca) TNK2 to immunoprecipitate activated EGFR. (d) The percentage cell surface expression of EGFR is shown in serum-starved MDA-MB-231 cells treated with EGF (100 ng/ml) for the indicated times. Reduced numbers of basal cell surface EGFRs are seen in cells treated with targeting SiRNA (S1) relative to the nontargeting SiRNA control (N), as measured by a fluorescein isothiocyanate-conjugated anti-EGFR antibody by fluorescence-activated cell sorting analysis. On average, a 27% reduction of cell surface receptors can be seen at timepoint 0 (P = 0.0135). A representative western blot illustrating the downregulation of TNK2 achieved by SiRNA treatment relative to β-actin is shown.