Decker S J, Dorai B, Russell S
Rockefeller University, New York, New York 10021-6399.
J Virol. 1988 Oct;62(10):3649-54. doi: 10.1128/JVI.62.10.3649-3654.1988.
Tumor promoter-stimulated phosphorylation of threonine 98 of the erbB protein of avian erythroblastosis virus (AEV) correlates with inhibition of erbB-dependent mitogenesis. To more clearly define the role of phosphorylation of this residue in regulation of the activity of the erbB protein, we have constructed erbB mutations which encode alanine (Ala-98), tyrosine (Tyr-98), or serine (Ser-98) at position 98. The biosynthesis and stability of the three mutant proteins were similar to those of the wild-type erbB protein, and all three retained the ability to transform chicken embryo fibroblasts. Treatment of transformed CEF with 12-tetradecanoylphorbol-13-acetate (TPA) stimulated incorporation of 32Pi into wild-type and mutant erbB proteins and resulted in a slight decrease in the electrophoretic mobilities of all the erbB proteins. Tryptic maps of erbB phosphopeptides showed no endogenous or TPA-stimulated phosphorylation of alanine 98 or tyrosine 98 in cells transformed by the Ala-98 and Tyr-98 mutants. Analysis of tryptic phosphopeptides by high-pressure liquid chromatography revealed that TPA treatment of cells stimulated phosphorylation of other sites of the erbB protein in addition to threonine 98. A high endogenous level of phosphorylation of serine 98 of the Ser-98 mutant protein was found, and TPA treatment of cells did not result in further phosphorylation of this residue. Cells transformed by wild-type and mutant AEV were equally sensitive to TPA-dependent inhibition of growth in soft agar and TPA-dependent inhibition of [3H]thymidine incorporation. TPA treatment inhibited tyrosine phosphorylation to a similar extent in cells transformed by wild-type or Ala-98 AEV. These data indicate that phosphorylation of threonine 98 of the erbB protein is not responsible for TPA-dependent inhibition of growth of AEV-transformed cells or TPA-induced inhibition of erbB-dependent tyrosine phosphorylation. TPA-stimulated phosphorylation of the erbB protein at other sites may mediate these effects. The data also show that subtle changes in a phosphorylation site (i.e., changing threonine to serine) can drastically alter recognition by protein kinases.
肿瘤启动子刺激的禽成红细胞增多症病毒（AEV）erbB蛋白苏氨酸98位点的磷酸化与erbB依赖的有丝分裂抑制相关。为了更清楚地界定该位点磷酸化在erbB蛋白活性调节中的作用，我们构建了在98位编码丙氨酸（Ala - 98）、酪氨酸（Tyr - 98）或丝氨酸（Ser - 98）的erbB突变体。这三种突变蛋白的生物合成和稳定性与野生型erbB蛋白相似，并且都保留了转化鸡胚成纤维细胞的能力。用12 - 十四酰佛波醇 - 13 - 乙酸酯（TPA）处理转化的鸡胚成纤维细胞可刺激32Pi掺入野生型和突变型erbB蛋白，并导致所有erbB蛋白的电泳迁移率略有下降。erbB磷酸肽的胰蛋白酶图谱显示，在由Ala - 98和Tyr - 98突变体转化的细胞中，丙氨酸98或酪氨酸98没有内源性或TPA刺激的磷酸化。通过高压液相色谱分析胰蛋白酶磷酸肽发现，TPA处理细胞除了刺激苏氨酸98位点磷酸化外，还刺激erbB蛋白其他位点的磷酸化。发现Ser - 98突变蛋白的丝氨酸98位点有较高的内源性磷酸化水平，TPA处理细胞并未导致该位点的进一步磷酸化。野生型和突变型AEV转化的细胞对TPA依赖的软琼脂中生长抑制和TPA依赖的[3H]胸腺嘧啶核苷掺入抑制同样敏感。TPA处理在野生型或Ala - 98 AEV转化的细胞中对酪氨酸磷酸化的抑制程度相似。这些数据表明，erbB蛋白苏氨酸98位点的磷酸化与TPA依赖的AEV转化细胞生长抑制或TPA诱导的erbB依赖的酪氨酸磷酸化抑制无关。TPA刺激erbB蛋白其他位点的磷酸化可能介导了这些效应。数据还表明，磷酸化位点的细微变化（即将苏氨酸变为丝氨酸）可显著改变蛋白激酶的识别。
