Bolen J B, Israel M A
J Biol Chem. 1984 Oct 10;259(19):11686-94.
We have observed increased phosphorylation of tyrosine residues on the polyoma virus middle tumor antigen (MTAg) in in vitro kinase assays of the immune complexes immunoprecipitated from lysates of polyoma virus-infected mouse embryo cells to which increasing amounts of uninfected mouse embryo cell lysate had been added. The components from uninfected mouse cells responsible for increased MTAg phosphorylation were localized by subcellular fractionation to the plasma membrane and found to be sensitive to protease digestion, N-ethylmaleimide, and 5'-p-fluorosulfonylbenzoyladenosine inactivation. The majority of the membrane-associated activity responsible for the increased MTAg phosphorylation in these assays could be cleared from lysates of uninfected mouse cell lysates by centrifugation after reaction with Sepharose-bound monoclonal antibodies which recognize pp60c-src. These results suggest that MTAg can associate with cellular tyrosyl kinases in vitro and be phosphorylated by these enzymes in immune-complex kinase assays. The identity of at least one of these cellular tryosyl kinases which can associate with MTAg in vitro is likely to be pp60c-src.
在体外激酶分析中，我们观察到，从多瘤病毒感染的小鼠胚胎细胞裂解物中免疫沉淀的免疫复合物，在加入越来越多未感染的小鼠胚胎细胞裂解物后，多瘤病毒中间肿瘤抗原（MTAg）上酪氨酸残基的磷酸化增加。通过亚细胞分级分离，将未感染小鼠细胞中负责MTAg磷酸化增加的成分定位到质膜，并发现其对蛋白酶消化、N-乙基马来酰亚胺和5'-对氟磺酰苯甲酰腺苷失活敏感。在这些分析中，负责MTAg磷酸化增加的大部分膜相关活性，在与识别pp60c-src的琼脂糖结合单克隆抗体反应后，可通过离心从未感染小鼠细胞裂解物的裂解物中清除。这些结果表明，MTAg在体外可与细胞酪氨酸激酶结合，并在免疫复合物激酶分析中被这些酶磷酸化。这些能在体外与MTAg结合的细胞酪氨酸激酶中，至少有一种可能是pp60c-src。
