Background and aims: The epidermal growth factor receptor (EGFR) is activated by extracellular ligands of the epidermal growth factor (EGF) family, resulting in a cascade of cytoplasmic signaling events. Emerging evidence indicates a mode of EGF signaling in which growth factor signals are transmitted via EGFR nuclear transport. The aim of this study was to determine whether EGF promotes EGFR nuclear accumulation and the role of clathrin-coated pits, EGFR kinase activity, caveolae microdomains and cytoskeleton integrity in breast cancer cells.
Methods: MCF-7 cells were treated without or with 100 ng/ml EGF for various times and nuclear extracts were obtained. Nuclear accumulation of EGFR was analyzed by SDS-PAGE followed by Western blotting of nuclear extracts using an anti-EGFR Ab or with a phosphospecific Ab against the Tyr-1068 of EGFR and with anti-Rb Ab as the loading control. DNA binding activity of EGFR was analyzed by EMSA using nuclear extracts and a radiolabeled oligonucleotide probe representing the AT-rich minimal sequence (ATRS).
Results: EGF induces the nuclear accumulation of EGFR, an increase in EGFR phosphorylation at Tyr-1068 and the formation of the complex EGFR-DNA in MCF-7 and MDA-MB-231 breast cancer cells. In addition, EGFR nuclear accumulation is dependent of clathrin-coated pits, EGFR kinase activity, caveolae microdomains and cytoskeleton integrity.
Conclusions: This study demonstrates that in breast cancer cells EGF promotes nuclear accumulation of EGFR and is dependent on clathrin-coated pits, EGFR kinase activity, caveolae microdomains and cytoskeleton integrity.