Winston J T, Olashaw N E, Pledger W J
Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
J Cell Biochem. 1991 Sep;47(1):79-89. doi: 10.1002/jcb.240470110.
Addition of tumor promoting phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), to many cell lines results in a decrease of 125I-epidermal growth factor (EGF) binding and increased serine/threonine phosphorylation of the EGF receptor in a process termed transmodulation. It is, however, unclear whether or not receptor phosphorylation is causally related to the inhibition of high affinity EGF binding. We have investigated the significance of phosphorylation/dephosphorylation events in the mechanism of PMA-induced transmodulation using the adenylate cyclase activator cholera toxin and the serine/threonine protein phosphatase inhibitor okadaic acid. In Rat-1 fibroblasts treated at 37 degrees C, PMA induced a rapid decrease in EGF binding which persisted for 3 hours. In contrast, cells exposed to PMA in the presence of cholera toxin exhibited a marked recovery of binding within 60 minutes. The PMA-stimulated decrease in binding correlated with a rapid increase in the phosphorylation state of the EGF receptor. While phosphorylation of the receptor was sustained at an elevated level for at least three hours in cells receiving PMA alone, EGF receptor phosphorylation decreased between 1 and 3 hours in cells treated with PMA and cholera toxin. Furthermore, the cholera toxin-stimulated return of EGF binding was inhibited by treatment with the phosphatase inhibitor okadaic acid. These results suggest that a cholera toxin-activated phosphatase can increase binding capacity of the transmodulated EGF receptor in Rat-1 cells. Cholera toxin treatment elicited a qualitatively similar response in cells transmodulated by platelet-derived growth factor (PDGF). Okadaic acid antagonized the natural return of binding observed in cells stimulated with PDGF alone, indicating that a dephosphorylation event may be required for the recovery of normal EGF binding after receptor transmodulation.
向许多细胞系中添加促肿瘤佛波酯，如佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA），会导致125I - 表皮生长因子（EGF）结合减少，并在一个称为转调节的过程中增加EGF受体的丝氨酸/苏氨酸磷酸化。然而，尚不清楚受体磷酸化是否与高亲和力EGF结合的抑制存在因果关系。我们使用腺苷酸环化酶激活剂霍乱毒素和丝氨酸/苏氨酸蛋白磷酸酶抑制剂冈田酸，研究了磷酸化/去磷酸化事件在PMA诱导的转调节机制中的意义。在37℃处理的大鼠-1成纤维细胞中，PMA诱导EGF结合迅速减少，并持续3小时。相比之下，在霍乱毒素存在下暴露于PMA的细胞在60分钟内显示出结合的显著恢复。PMA刺激的结合减少与EGF受体磷酸化状态的快速增加相关。在仅接受PMA的细胞中，受体磷酸化至少持续3小时维持在升高水平，而在用PMA和霍乱毒素处理的细胞中，EGF受体磷酸化在1至3小时之间下降。此外，磷酸酶抑制剂冈田酸处理抑制了霍乱毒素刺激的EGF结合恢复。这些结果表明，霍乱毒素激活的磷酸酶可以增加大鼠-1细胞中转调节的EGF受体的结合能力。霍乱毒素处理在由血小板衍生生长因子（PDGF）转调节的细胞中引发了定性相似的反应。冈田酸拮抗了仅用PDGF刺激的细胞中观察到的结合自然恢复，表明去磷酸化事件可能是受体转调节后正常EGF结合恢复所必需的。
