Moon S O, Palfrey H C, King A C
Proc Natl Acad Sci U S A. 1984 Apr;81(8):2298-302. doi: 10.1073/pnas.81.8.2298.
Incubation of membranes prepared from A431 cells with either epidermal growth factor (EGF) or phorbol 12-myristate 13-acetate (PMA) stimulates the transfer of 32phosphate from [gamma-32P]ATP into 8-10 membrane proteins. The major phosphorylated protein migrates on NaDodSO4/polyacrylamide gels with an apparent Mr of 180,000, corresponding to the previously identified EGF receptor. Stimulation of EGF receptor phosphorylation by PMA does not require Ca2+, suggesting that prior activation of protein kinase C is not a prerequisite for phosphate transfer. PMA-enhanced phosphorylation proceeds at 4 degrees C and requires Mn2+, both properties of tyrosine-specific protein kinases. Phospho amino acid analysis of the Mr 180,000 receptor band shows that only tyrosine residues are phosphorylated when A431 membranes are treated with either EGF or PMA. Moreover, proteolysis reveals that these residues are located in the same peptides of the receptor. These results demonstrate that a potent tumor-promoting phorbol ester can mimic a critical early response usually elicited by EGF.
用表皮生长因子（EGF）或佛波醇12 - 肉豆蔻酸酯13 - 乙酸酯（PMA）处理从A431细胞制备的细胞膜，可刺激32磷酸从[γ-32P]ATP转移到8 - 10种膜蛋白中。主要的磷酸化蛋白在十二烷基硫酸钠/聚丙烯酰胺凝胶上迁移，表观分子量为180,000，与先前鉴定的EGF受体相对应。PMA对EGF受体磷酸化的刺激不需要Ca2 +，这表明蛋白激酶C的预先激活不是磷酸转移的先决条件。PMA增强的磷酸化在4℃下进行，并且需要Mn2 +，这两者都是酪氨酸特异性蛋白激酶的特性。对分子量为180,000的受体条带进行磷酸氨基酸分析表明，当用EGF或PMA处理A431细胞膜时，只有酪氨酸残基被磷酸化。此外，蛋白水解显示这些残基位于受体的相同肽段中。这些结果表明，一种强效的促肿瘤佛波酯可以模拟通常由EGF引发的关键早期反应。
