Song Robert X-D, McPherson Robert A, Adam Liana, Bao Yongde, Shupnik Margaret, Kumar Rakesh, Santen Richard J
Department of Internal Medicine, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.
Mol Endocrinol. 2002 Jan;16(1):116-27. doi: 10.1210/mend.16.1.0748.
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
E2能迅速激活乳腺癌细胞中的丝裂原活化蛋白激酶（MAPK），但其作用机制尚未完全明确。由于生长因子诱导的MAPK激活涉及衔接蛋白Shc（Src同源和胶原同源）介导的信号传导及其与膜受体的结合，我们推测乳腺癌细胞对E2的反应利用了类似的信号传导机制。在本研究中，我们证明E2能迅速诱导MCF-7细胞中Shc磷酸化以及Shc-Grb2（生长因子受体结合蛋白2）-Sos（七号染色体失活蛋白的儿子）复合物形成。显性负性Shc的过表达阻断了E2对MAPK的作用，表明Shc在E2作用中起关键作用。使用选择性抑制剂，我们还证明雌激素受体α（ERα）和Src是Shc的上游调节因子。E2刺激后ERα与Shc迅速发生物理结合，进一步证明了ERα对Shc激活的作用。诱变研究表明，Shc的磷酸酪氨酸结合域和SH2结构域与ERα的激活功能1相互作用是必需的，但与激活功能2结构域无关。通过谷胱甘肽-S-转移酶-Shc下拉试验，我们证明这种ERα-Shc结合是直接的。在基因组和非基因组水平上进一步研究了该信号通路的生物学后果。E2刺激了MAPK介导的Elk-1转录活性。共聚焦显微镜研究表明，E2能迅速诱导膜皱褶、伪足形成以及ERα膜转位。抗雌激素可阻止E2诱导的形态学变化。我们的结果共同表明，ERα可介导E2对乳腺癌细胞中Shc、MAPK、Elk-1和形态学变化的快速作用
