Linkage of rapid estrogen action to MAPK activation by ERalpha-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 (Src-homology 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 ERalpha and Src are upstream regulators of Shc. A rapid physical association between ERalpha and Shc upon E2 stimulation further evidenced the role of ERalpha 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 ERalpha. Using a glutathione-S-transferase-Shc pull-down assay, we demonstrated that this ERalpha-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 ERalpha membrane translocation. The E2-induced morphological changes were prevented by antiestrogen. Together our results demonstrate that ERalpha can mediate the rapid effects of E2 on Shc, MAPK, Elk-1, and morphological changes in breast cancer cells
Song
Robert X-D
RX
Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA. rs5wf@virginia.edu
McPherson
Robert A
RA
Adam
Liana
L
Bao
Yongde
Y
Shupnik
Margaret
M
Kumar
Rakesh
R
Santen
Richard J
RJ
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D000818:Animals; D001665:Binding Sites; D001943:Breast Neoplasms / Q000378:metabolism; D019556:COS Cells; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004958:Estradiol / Q000378:metabolism / Q000494:pharmacology; D047628:Estrogen Receptor alpha; D005419:Flavonoids / Q000494:pharmacology; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D011960:Receptors, Estrogen / Q000378:metabolism*; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014157:Transcription Factors; D014407:Tumor Cells, Cultured; D050807:ets-Domain Protein Elk-1
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D004268:DNA-Binding Proteins; C494112:ELK1 protein, human; D004791:Enzyme Inhibitors; D047628:Estrogen Receptor alpha; D005419:Flavonoids; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011960:Receptors, Estrogen; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D014157:Transcription Factors; D050807:ets-Domain Protein Elk-1; D019000:Phosphotyrosine; D004958:Estradiol; D020928:Mitogen-Activated Protein Kinases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one