Myers J N, LeVea C M, Smith J E, Kallen R G, Tung L, Greene M I
Department of Pathology, University of Pennsylvania, Philadelphia.
Receptor. 1992 Spring;2(1):1-16.
To further characterize the structure and regulation of the tyrosine kinase encoded by the rodent neu oncogene, its cytoplasmic tyrosine kinase domain has been expressed as a soluble protein, called Bacneu, in Sf9 insect cells, using the baculovirus expression system. Expression of Bacneu was detected by immunoblotting with anti p185neu antisera and in vitro autophosphorylation analysis as early as 24 h postinfection. Maximal expression was observed at 48 h postinfection. The soluble kinase was purified to near homogeneity by sequential chromatography on DEAE-Sepharose, phosphocellulose, poly-L-lysine, and Sephacryl 300, yielding 0.55 mg Bacneu per L of Sf9 cells (4% yield). The kinase is more active in the presence of Mn2+ compared to Mg2+ ions. The specific activity of the kinase using poly(Glu4Tyr1) as a substrate is 179 nmol/min/mg. Maximal incorporation of 1.4 mol of phosphate per mol of enzyme by autophosphorylation was found to increase the activity of the enzyme 1.5- to twofold. These results indicate that the Bacneu kinase is activated by phosphorylation. Therefore, it will be a useful reagent for characterizing the effects that phosphorylation by other cellular kinases and dephosphorylation by phosphatases have on its activity.
为了进一步表征由啮齿动物neu癌基因编码的酪氨酸激酶的结构和调控，利用杆状病毒表达系统，其细胞质酪氨酸激酶结构域已在Sf9昆虫细胞中表达为一种可溶性蛋白，称为Bacneu。早在感染后24小时，就通过用抗p185neu抗血清进行免疫印迹和体外自磷酸化分析检测到了Bacneu的表达。在感染后48小时观察到最大表达。通过在DEAE-琼脂糖、磷酸纤维素、聚-L-赖氨酸和Sephacryl 300上进行连续层析，将可溶性激酶纯化至接近均一，每升Sf9细胞产生0.55毫克Bacneu（产率4%）。与Mg2+离子相比，该激酶在Mn2+存在下更具活性。以聚（Glu4Tyr1）为底物时，该激酶的比活性为179 nmol/分钟/毫克。发现每摩尔酶通过自磷酸化最大掺入1.4摩尔磷酸盐可使酶的活性提高1.5至两倍。这些结果表明Bacneu激酶通过磷酸化被激活。因此，它将是一种有用的试剂，用于表征其他细胞激酶的磷酸化和磷酸酶的去磷酸化对其活性的影响。
