Koch W, Carbone A, Walter G
Mol Cell Biol. 1986 Jun;6(6):1866-74. doi: 10.1128/mcb.6.6.1866-1874.1986.
Medium T antigen, the transforming protein of polyoma virus, is associated with pp60c-src and strongly activates its tyrosine-specific protein kinase activity. We investigated whether the medium T-pp60c-src complex is also associated with an activity that phosphorylates the membrane phospholipid phosphatidylinositol, as shown for pp60v-src and p68v-ros, the transforming proteins of Rous sarcoma virus and avian sarcoma virus UR2, respectively. Medium T was purified by affinity chromatography from extracts of polyoma virus-infected mouse fibroblasts. It was bound to antibodies against a peptide corresponding to the carboxy terminus of medium T and released from the immune complex with an excess of the same peptide. In a second step, the partially purified medium T was bound to antibodies against another peptide corresponding to an internal region of medium T and released with excess peptide. Further purification was carried out with a monoclonal antibody against pp60c-src. Samples from each purification step were examined for protein kinase and phosphatidylinositol kinase activity. The highly purified preparations of the medium T-pp60c-src complex showed very low levels of phosphatidylinositol kinase activity, and no difference between medium T from transforming viruses and nontransforming hr-t mutants was detected. In contrast, protein kinase activity was associated with medium T purified from transforming viruses but not from hr-t mutants.
中T抗原是多瘤病毒的转化蛋白，与pp60c-src相关，并强烈激活其酪氨酸特异性蛋白激酶活性。我们研究了中T-pp60c-src复合物是否也与一种使膜磷脂磷脂酰肌醇磷酸化的活性相关，就像劳氏肉瘤病毒和禽肉瘤病毒UR2的转化蛋白pp60v-src和p68v-ros分别显示的那样。通过亲和层析从多瘤病毒感染的小鼠成纤维细胞提取物中纯化中T。它与针对对应于中T羧基末端的肽的抗体结合，并用过量的相同肽从免疫复合物中释放出来。在第二步中，部分纯化的中T与针对对应于中T内部区域的另一种肽的抗体结合，并用过量肽释放。用抗pp60c-src的单克隆抗体进行进一步纯化。检查每个纯化步骤的样品的蛋白激酶和磷脂酰肌醇激酶活性。中T-pp60c-src复合物的高度纯化制剂显示出非常低水平的磷脂酰肌醇激酶活性，并且未检测到来自转化病毒的中T与非转化hr-t突变体之间的差异。相比之下，蛋白激酶活性与从转化病毒纯化的中T相关，而与hr-t突变体纯化的中T无关。
