Gallis B, Lewis A, Wignall J, Alpert A, Mochizuki D Y, Cosman D, Hopp T, Urdal D
J Biol Chem. 1986 Apr 15;261(11):5075-80.
The recombinant human interleukin-2 (IL-2) receptor was expressed in mouse mammary epithelial cells following the transfection of these cells with an expression vector containing the human IL-2 receptor cDNA. The recombinant IL-2 receptor in these cells was rapidly phosphorylated in response to phorbol myristate acetate (PMA), but its phosphorylation could not be detected in the absence of PMA or upon addition of human IL-2. The C-terminal, cytoplasmic peptide domain of the IL-2 receptor, Gln-Arg-Arg-Gln-Arg-Lys-Ser-Arg-Arg-Thr-Ile, was synthesized and used as a substrate for protein kinase C. The Km for phosphorylation of the peptide by protein kinase C was 23 microM. The stoichiometry of phosphorylation was 1 mol of phosphate/mol of peptide and serine was the predominant amino acid phosphorylated. Because this peptide was a good substrate for protein kinase C in vitro, it was possible that the same serine (serine 247) was also phosphorylated in the receptor in the cell. The IL-2 receptor gene in the expression vector was therefore altered by site-directed mutagenesis to code for an IL-2 receptor containing an alanine in the place of serine 247. The IL-2 receptor expressed by these cells was not phosphorylated in the presence of PMA. These data suggest that protein kinase C, in response to PMA, phosphorylates the C-terminal serine residue (serine 247) in the human IL-2 receptor.
用含有人白细胞介素-2（IL-2）受体cDNA的表达载体转染小鼠乳腺上皮细胞后，重组人IL-2受体在这些细胞中得以表达。这些细胞中的重组IL-2受体在佛波酯肉豆蔻酸酯（PMA）作用下迅速发生磷酸化，但在无PMA或加入人IL-2时未检测到其磷酸化。合成了IL-2受体的C末端胞质肽结构域Gln-Arg-Arg-Gln-Arg-Lys-Ser-Arg-Arg-Thr-Ile，并将其用作蛋白激酶C的底物。蛋白激酶C对该肽进行磷酸化的米氏常数（Km）为23微摩尔。磷酸化的化学计量比为1摩尔磷酸/摩尔肽，且丝氨酸是主要的被磷酸化氨基酸。由于该肽在体外是蛋白激酶C的良好底物，因此细胞中的受体中相同的丝氨酸（丝氨酸247）也可能被磷酸化。因此，通过定点诱变改变了表达载体中的IL-2受体基因，使其编码的IL-2受体中丝氨酸247被丙氨酸取代。这些细胞表达的IL-2受体在PMA存在下未发生磷酸化。这些数据表明，响应PMA时，蛋白激酶C使人IL-2受体的C末端丝氨酸残基（丝氨酸247）发生磷酸化。
