Sudol M
Laboratory of Viral Oncology, Rockefeller University, New York, NY 10021-6399.
J Neurosci Res. 1989 Sep;24(1):1-8. doi: 10.1002/jnr.490240103.
Using monospecific antibodies raised against the amino terminal domain of viral-yes protein, we precipitated a 58 kD protein-tyrosine kinase from the brain of Xenopus laevis frog. By a number of criteria, including pattern of expression in various tissues and one-dimensional peptide mapping, we concluded that pp58 is very likely the authentic amphibian yes protein because it is more similar to the chicken yes protein than to any other known tyrosine kinases. The pp58c-yes is expressed in adult brain at elevated levels. In contrast, its level of expression in follicular and denuded oocytes is 30-50 times lower. Because of the low endogenous expression of the oocyte-associated yes kinase, we were able to transiently express and analyze pp62c-yes by injecting oocytes with 27S size fraction of poly(A)+ mRNA isolated from chicken cerebella. The pp62c-yes expressed in oocytes from the exogenous message comigrated on SDS polyacrylamide gel with pp62c-yes from cerebellum and was indistinguishable from it by one-dimensional peptide mapping. The pp62c-yes expressed in oocytes was enzymatically active. A number of phosphotyrosine-containing proteins were detected specifically in the c-yes mRNA-injected oocytes. The usefulness of the Xenopus oocyte expression system to study the functional aspects of c-yes protein and other tyrosine kinases was evaluated.
利用针对病毒原癌基因yes蛋白氨基末端结构域产生的单特异性抗体，我们从小爪蟾蛙脑中沉淀出一种58 kD的蛋白酪氨酸激酶。通过包括在各种组织中的表达模式和一维肽图谱分析等多种标准，我们得出结论，pp58很可能是真正的两栖类yes蛋白，因为它与鸡yes蛋白的相似性高于任何其他已知的酪氨酸激酶。pp58c-yes在成年蛙脑中高水平表达。相比之下，其在卵泡和裸卵母细胞中的表达水平低30 - 50倍。由于卵母细胞相关yes激酶的内源性表达较低，我们能够通过向卵母细胞注射从小鸡小脑分离的27S大小的聚腺苷酸加尾mRNA来瞬时表达和分析pp62c-yes。在外源信使RNA注射的卵母细胞中表达的pp62c-yes在SDS聚丙烯酰胺凝胶上与来自小脑的pp62c-yes共迁移，并且通过一维肽图谱分析与之一致。在卵母细胞中表达的pp62c-yes具有酶活性。在注射了c-yes mRNA的卵母细胞中特异性检测到了许多含磷酸酪氨酸的蛋白质。评估了非洲爪蟾卵母细胞表达系统在研究c-yes蛋白和其他酪氨酸激酶功能方面的实用性。
