Brown D J, Gordon J A
J Biol Chem. 1984 Aug 10;259(15):9580-6.
We have observed that intact wild type Rous sarcoma virus-transformed chicken embryo fibroblasts (CEF), when incubated with micromolar sodium orthovanadate for as little as 4 h, results in a 2-3-fold increase in pp60v-src kinase activity as judged by the IgG kinase assay. The addition of sodium vanadate or vanadyl sulfate to transformed cell lysates or the immunoprecipitate assay was without effect on the activity of isolated pp60v-src kinase activity. The increased kinase activity is reflected intracellularly by an increased phosphotyrosine content of a known substrate of pp60v-src, a 36-kDa phosphoprotein, without a change in total phosphate incorporation into cellular proteins. Pulse and pulse-chase experiments with [35S]methionine indicated that there was no change in the net or rate of biosynthesis of pp60v-src which might have accounted for the elevated kinase activity. Along with this stimulation of pp60v-src kinase activity, we found a parallel increase in the phosphate content of the enzyme and the appearance of an electrophoretic variant after vanadate treatment. The increased phosphorylation of the kinase is accounted for by an increase in phosphotyrosine content without a change in phosphoserine content. The increased phosphotyrosine content is shown to be resident in the carboxyl-terminal fragment and, unexpectedly, in the amino-terminal fragment of the kinase. The phosphotyrosine present in the amino-terminal portion may be a de novo phosphorylation, but could also represent a tyrosine site(s) not easily detected under unstimulated conditions in the absence of vanadate.
我们观察到，完整的野生型劳氏肉瘤病毒转化的鸡胚成纤维细胞（CEF），当与微摩尔浓度的原钒酸钠孵育仅4小时时，通过IgG激酶测定法判断，pp60v-src激酶活性会增加2至3倍。向转化细胞裂解物或免疫沉淀测定中添加钒酸钠或硫酸氧钒对分离的pp60v-src激酶活性没有影响。激酶活性的增加在细胞内表现为pp60v-src的已知底物（一种36 kDa的磷蛋白）的磷酸酪氨酸含量增加，而细胞蛋白质中总磷酸盐掺入量没有变化。用[35S]甲硫氨酸进行的脉冲和脉冲追踪实验表明，pp60v-src的生物合成净量或速率没有变化，这可能是激酶活性升高的原因。伴随着pp60v-src激酶活性的这种刺激，我们发现钒酸盐处理后该酶的磷酸盐含量平行增加，并且出现了一种电泳变体。激酶磷酸化增加是由于磷酸酪氨酸含量增加，而磷酸丝氨酸含量没有变化。增加的磷酸酪氨酸含量显示存在于激酶的羧基末端片段中，出乎意料的是，也存在于氨基末端片段中。氨基末端部分存在的磷酸酪氨酸可能是从头磷酸化，但也可能代表在没有钒酸盐的未刺激条件下不易检测到的酪氨酸位点。
