Kim Yong-Nyun, Dam Phuongan, Bertics Paul J
Department of Biomolecular Chemistry, University of Wisconsin, Madison 53706, USA.
Exp Cell Res. 2002 Oct 15;280(1):134-47. doi: 10.1006/excr.2002.5623.
Previous studies have shown that EGF can induce the tyrosine phosphorylation of caveolin-1 in murine fibroblasts following ErbB1 (EGF receptor) mutation or overexpression, but the cell signaling events linking EGF action with caveolin phosphorylation are not fully established. In this regard, we examined multiple human carcinoma cell lines that express various ErbB family members, including A431 epidermoid carcinoma cells and several squamous carcinoma cell lines. In all cases, EGF treatment induced the tyrosine phosphorylation of caveolin-1 in a time- and EGF dose-dependent manner, and immunoblotting analysis revealed that this phosphorylation occurred at tyrosine-14. The EGF-dependent phosphorylation of caveolin-1 was observed at low temperatures (4 degrees C) and was enhanced by caveolae-disrupting agents (cyclodextrin), suggesting that this EGF-dependent system is in a low temperature-stable arrangement that allows for their interaction under conditions where mobility in the membrane is altered. To further assess the events linking EGF action with caveolin phosphorylation, we evaluated the ligand specificity of these responses and their dependence on known effectors of EGF receptor function. We observed that EGF and HB-EGF, but not heregulin, promoted caveolin-1 phosphorylation in A431 cells, suggesting that these responses are linked to EGF receptor activation and not solely occurring via the activation of other endogenous ErbB family members. In addition, the EGF-induced phosphorylation of caveolin-1 in A431 cells was blocked by the Src kinase antagonists PP1 and PP2, but not by the MEK inhibitor PD98059, the phosphoinositide 3-kinase inhibitors LY294002 and wortmannin, or cytoskeleton-disrupting agents, such as cytochalasin D, colchicine, and nocadazole. Altogether, these data indicate that multiple human carcinoma cells exhibit an EGF receptor-dependent tyrosine phosphorylation of caveolin-1 and that this process is sensitive to Src family kinase inhibitors. These observations support a role for caveolin tyrosine phosphorylation in the profile of cellular responses by which Src potentiates cancer progression following EGF receptor overexpression.
先前的研究表明，在小鼠成纤维细胞中，表皮生长因子（EGF）可在ErbB1（EGF受体）发生突变或过表达后诱导小窝蛋白-1的酪氨酸磷酸化，但将EGF作用与小窝蛋白磷酸化联系起来的细胞信号转导事件尚未完全明确。在这方面，我们检测了多种表达不同ErbB家族成员的人癌细胞系，包括A431表皮样癌细胞和几种鳞状癌细胞系。在所有情况下，EGF处理均以时间和EGF剂量依赖性方式诱导小窝蛋白-1的酪氨酸磷酸化，免疫印迹分析显示这种磷酸化发生在酪氨酸-14位点。在低温（4℃）下观察到小窝蛋白-1的EGF依赖性磷酸化，并且小窝破坏剂（环糊精）可增强这种磷酸化，这表明这种EGF依赖性系统处于低温稳定的排列状态，使得它们在膜流动性改变的条件下能够相互作用。为了进一步评估将EGF作用与小窝蛋白磷酸化联系起来的事件，我们评估了这些反应的配体特异性及其对EGF受体功能已知效应器的依赖性。我们观察到，EGF和肝素结合表皮生长因子（HB-EGF），而非这里调节蛋白，可促进A431细胞中小窝蛋白-1的磷酸化，这表明这些反应与EGF受体激活有关，并非仅通过其他内源性ErbB家族成员的激活而发生。此外，Src激酶拮抗剂PP1和PP2可阻断A431细胞中EGF诱导的小窝蛋白-1磷酸化，但MEK抑制剂PD98059、磷酸肌醇3激酶抑制剂LY294002和渥曼青霉素，或细胞骨架破坏剂，如细胞松弛素D、秋水仙碱和诺考达唑则不能。总之，这些数据表明多种人癌细胞表现出EGF受体依赖性的小窝蛋白-1酪氨酸磷酸化，并且这一过程对Src家族激酶抑制剂敏感。这些观察结果支持小窝蛋白酪氨酸磷酸化在细胞反应谱中的作用，即Src在EGF受体过表达后增强癌症进展。
