Fukui Y, Hanafusa H
Rockefeller University, New York, New York 10021.
Mol Cell Biol. 1989 Apr;9(4):1651-8. doi: 10.1128/mcb.9.4.1651-1658.1989.
Immunoprecipitates of p60v-src proteins from chicken embryo fibroblasts infected with Rous sarcoma virus were assayed for phosphatidylinositol (PI) kinase activity in the absence of detergents. The product of the PI kinase reaction, phosphatidylinositol monophosphate (PIP), migrated slightly slower than did the authentic phosphatidylinositol-4-monophosphate marker in thin-layer chromatography and was indistinguishable from phosphatidylinositol-3-monophosphate produced by PI kinase type I. Furthermore, the deacylated product comigrated with glycerophosphoinositol-3-phosphate in high-performance liquid chromatography. Both sucrose gradient fractionation and the heat stability of PI kinase activity from cells infected with temperature-sensitive mutants suggest that the PI kinase activity is not intrinsic to p60v-src but is a property of another molecule complexed with p60v-src. All transforming variants of p60src were associated with PI kinase activity, whereas this enzyme activity was hardly detectable in immunoprecipitates from cells infected with nontransforming viruses encoding p60c-src or an enzymatically inactive variant. However, PI kinase activity was found in p60src immunoprecipitates from cells infected with nonmyristylated, nontransforming mutants as well as temperature-sensitive mutants at the nonpermissive temperature, which indicated that simple association of PI kinase activity with p60src is not sufficient for cell transformation.
在无去污剂的情况下，对感染劳氏肉瘤病毒的鸡胚成纤维细胞中p60v-src蛋白的免疫沉淀物进行磷脂酰肌醇（PI）激酶活性检测。PI激酶反应的产物磷脂酰肌醇单磷酸（PIP）在薄层色谱中的迁移速度略慢于 authentic磷脂酰肌醇-4-单磷酸标记物，且与I型PI激酶产生的磷脂酰肌醇-3-单磷酸无法区分。此外，去酰化产物在高效液相色谱中与甘油磷酸肌醇-3-磷酸迁移位置相同。蔗糖梯度分级分离以及来自感染温度敏感突变体的细胞中PI激酶活性的热稳定性均表明，PI激酶活性并非p60v-src所固有，而是与p60v-src复合的另一种分子的特性。p60src的所有转化变体均与PI激酶活性相关，而在感染编码p60c-src或无酶活性变体的非转化病毒的细胞免疫沉淀物中几乎检测不到这种酶活性。然而，在感染非肉豆蔻酰化、非转化突变体以及处于非允许温度的温度敏感突变体的细胞的p60src免疫沉淀物中发现了PI激酶活性，这表明PI激酶活性与p60src的简单结合不足以实现细胞转化。
