Davis R J, Czech M P
J Biol Chem. 1984 Jul 10;259(13):8545-9.
4 beta-Phorbol 12 beta-myristate 13 alpha-acetate (PMA) markedly inhibited the binding of low concentrations (less than 10(-9 m) of 125I-epidermal growth factor (EGF) to A431 human epidermoid carcinoma cells. However, very little change in the binding of 125-I-EGF at high concentrations (greater than 10(-8) M) was observed in response to PMA. Affinity labeling of the 170,000-dalton EGF receptor with 125I-EGF and disuccinimidyl suberate was also decreased by the tumor promoter at low, but not high, concentrations of 125I-EGF. In order to examine this action of PMA on the EGF receptor, the receptor phosphorylation state was evaluated in A431 cells that had been incubated with [32P]phosphate for 3 h prior to the addition of PMA. The 32P content of the EGF receptor purified with EGF-Sepharose was increased by 38% compared with the same amount of receptor isolated from control cells. The increase in EGF receptor phosphorylation was dose-dependent with a half-maximal effect between 0.1 and 1 nM PMA and was specific for tumor promoting analogues of phorbol diesters. Phosphoamino acid analysis indicated that the increase in the 32P content of the EGF receptor was mainly due to phosphoserine. These results demonstrate that the EGF receptor is a target for PMA action and suggest that the mechanism of PMA action on the response of cells to epidermal growth factor may be mediated in part by phosphorylation of the EGF receptor.
4β-佛波醇12β-肉豆蔻酸酯13α-乙酸酯（PMA）显著抑制低浓度（小于10⁻⁹ M）的¹²⁵I-表皮生长因子（EGF）与A431人表皮样癌细胞的结合。然而，在高浓度（大于10⁻⁸ M）的¹²⁵I-EGF存在下，未观察到PMA对其结合有显著影响。在低浓度而非高浓度的¹²⁵I-EGF条件下，肿瘤促进剂也降低了用¹²⁵I-EGF和辛二酸二琥珀酰亚胺酯对170,000道尔顿EGF受体进行的亲和标记。为了研究PMA对EGF受体的这种作用，在添加PMA之前，对用[³²P]磷酸盐孵育3小时的A431细胞中的受体磷酸化状态进行了评估。与从对照细胞中分离的等量受体相比，用EGF-琼脂糖纯化的EGF受体的³²P含量增加了38%。EGF受体磷酸化的增加呈剂量依赖性，在0.1至1 nM PMA之间达到半数最大效应，并且对佛波醇二酯的肿瘤促进类似物具有特异性。磷酸氨基酸分析表明，EGF受体³²P含量的增加主要归因于磷酸丝氨酸。这些结果表明，EGF受体是PMA作用的靶点，并提示PMA对细胞对表皮生长因子反应的作用机制可能部分是由EGF受体的磷酸化介导的。
