Linkage of rapid estrogen action to MAPK activation by ERα-Shc association and Shc pathway activation

E2 rapidly activates MAPK in breast cancer cells, and the mechanism for this effect has not been fully identified. Since growth factor-induced MAPK activation involves signaling via the adapter protein Shc [Srchomology and collagen homology) and its association with membrane receptors, we hypothesized that breast cancer cells utilize similar signaling mechanisms in response to E2. In the present study, we demonstrated that E2 rapidly induced Shc phosphorylation and Shc-Grb2 (growth factor receptor binding protein 2)-Sos (son of sevenless) complex formation in MCF-7 cells. Overexpression of dominant negative Shc blocked the effect of E2 on MAPK, indicating a critical role of Shc in E2 action. Using selective inhibitors, we also demonstrated that ER alpha and Src are upstream regulators of Shc. A rapid physical association between ERa and Shc upon E2 stimulation further evidenced the role of ERa on Shc activation. Mutagenesis studies showed that the phosphotyrosine binding and SH2 domains of Shc are required to interact with the activation function 1, but not activation function 2, domain of ERa. Using a glutathione-S-transferanse-Shc pull-down assay, we demonstrated that this ER alpha -Shc association was direct. Biological consequences of this pathway were further investigated at the genomic and nongenomic levels. E2 stimulated MAPK-mediated Elk-1 transcriptional activity. Confocal microscopy studies showed that E2 rapidly induced formation of membrane ruffles, pseudopodia, and ERa membrane translocation. The E2-induced morphological changes were prevented by antiestrogen. Together our results demonstrate that ERa can mediate the rapid effects of E2 on Shc, MAPK, Elk-1, and morphological changes in breast cancer cells.