Piwnica-Worms H, Kaplan D R, Whitman M, Roberts T M
Mol Cell Biol. 1986 Jun;6(6):2033-40. doi: 10.1128/mcb.6.6.2033-2040.1986.
We have constructed a recombinant murine retrovirus which efficiently transduces avian pp60c-src into murine cells and which is easily rescued from infected cells in plasmid form. To characterize the virus, several randomly selected NIH 3T3 lines were isolated after infection with recombinant retroviral stocks. All lines overproduced avian pp60c-src and appeared morphologically normal. Immunoprecipitates made from these lines with antisera specific for pp60c-src were tested for their kinase activities in vitro. We find that both autokinase and enolase kinase activities increase proportionately with the level of pp60c-src in the immunoprecipitates. To further test the authenticity of the pp60c-src encoded by the retroviral vector, these analyses were repeated in the presence of polyomavirus middle T antigen. Avian pp60c-src was activated as a protein kinase, indicating that the virally encoded pp60c-src interacts normally with middle T antigen. Interestingly, by increasing the intracellular levels of pp60c-src 15-fold over normal endogenous levels, we were unable to obtain a proportionate increase in the amount of middle-T-antigen-pp60c-src complex. Finally, using the shuttle features designed into the vector, we have isolated the first fully processed cDNA encoding functional avian pp60c-src X pp60c-src synthesized in vitro with this cDNA had intrinsic protein kinase activity and no detectable phosphatidylinositol kinase activity.
我们构建了一种重组鼠逆转录病毒，它能有效地将禽源pp60c-src转导到鼠细胞中，并且很容易以质粒形式从感染细胞中拯救出来。为了对该病毒进行特性分析，用重组逆转录病毒储备液感染后，分离出了几个随机选择的NIH 3T3细胞系。所有细胞系都过量产生禽源pp60c-src，并且形态上看起来正常。用对pp60c-src特异的抗血清从这些细胞系制备免疫沉淀物，并在体外检测其激酶活性。我们发现，免疫沉淀物中的自身激酶和烯醇酶激酶活性都与pp60c-src的水平成比例增加。为了进一步检测逆转录病毒载体编码的pp60c-src的真实性，在多瘤病毒中T抗原存在的情况下重复了这些分析。禽源pp60c-src被激活成为一种蛋白激酶，这表明病毒编码的pp60c-src与中T抗原正常相互作用。有趣的是，通过使细胞内pp60c-src的水平比正常内源性水平增加15倍，我们未能使中T抗原-pp60c-src复合物的量成比例增加。最后，利用载体中设计的穿梭特性，我们分离出了第一个完全加工的编码功能性禽源pp60c-src的cDNA。用该cDNA体外合成的pp60c-src具有内在的蛋白激酶活性，且未检测到磷脂酰肌醇激酶活性。
