Lowe D G, Goeddel D V
Department of Molecular Biology, Genentech, Inc., South San Francisco, California 94080.
Mol Cell Biol. 1987 Aug;7(8):2845-56. doi: 10.1128/mcb.7.8.2845-2856.1987.
We directly expressed human R-ras 23,000-dalton protein (p23) cDNA in Escherichia coli under the control of the trp promoter. GTP-dependent phosphorylation of a p23 threonine 85 substitution mutant was observed. This result is in direct analogy to the autokinase activity of H-ras and K-ras threonine 59 substitution mutants. Normal p23 protein was detected in the human fibrosarcoma cell line HT1080 by immunoprecipitation with rabbit antibodies raised against an E. coli-expressed R-ras fusion protein. The R-ras p23 protein was found to be 3H labeled in the presence of [9,10(n)-3H]palmitic acid and is associated with the P100 membrane fraction of HT1080 cells. These data suggest that human R-ras p23 has biochemical properties very similar to those of the p21 products of the H-, K-, and N-ras proto-oncogenes. We constructed an R-ras minigene and engineered the expression of normal and mutant alleles from the simian virus 40 early region promoter. Normal and mutant R-ras gene products were authenticated by transient expression in COS-7 cells and immunoprecipitation. The valine 38-substituted R-ras p23 displayed reduced electrophoretic mobility. R-ras p21-like proteins, made by eliminating the first 26 R-ras codons, displayed evident mobility differences between the pro form and mature form, along with a valine 12 substitution-dependent change in electrophoretic mobility. Rat-1 fibroblasts were transfected with normal and mutant R-ras alleles and normal and activated H-ras alleles. Unlike the human T24 bladder oncogene-encoded p21, mutant R-ras alleles do not cause monolayer focus formation or growth in soft agar of rat fibroblasts.
我们在色氨酸启动子的控制下，在大肠杆菌中直接表达人R-ras 23,000道尔顿蛋白（p23）的cDNA。观察到p23苏氨酸85替代突变体的GTP依赖性磷酸化。该结果与H-ras和K-ras苏氨酸59替代突变体的自身激酶活性直接类似。通过用针对大肠杆菌表达的R-ras融合蛋白产生的兔抗体进行免疫沉淀，在人纤维肉瘤细胞系HT1080中检测到正常的p23蛋白。发现R-ras p23蛋白在[9,10(n)-3H]棕榈酸存在下被3H标记，并与HT1080细胞的P100膜组分相关。这些数据表明，人R-ras p23具有与H-、K-和N-ras原癌基因的p21产物非常相似的生化特性。我们构建了一个R-ras小基因，并从猿猴病毒40早期区域启动子设计正常和突变等位基因的表达。通过在COS-7细胞中的瞬时表达和免疫沉淀对正常和突变的R-ras基因产物进行鉴定。缬氨酸38替代的R-ras p23显示出降低的电泳迁移率。通过去除前26个R-ras密码子产生的R-ras p21样蛋白，在原形式和成熟形式之间显示出明显的迁移率差异，以及缬氨酸12替代依赖性的电泳迁移率变化。用正常和突变的R-ras等位基因以及正常和活化的H-ras等位基因转染大鼠-1成纤维细胞。与人类T24膀胱癌基因编码的p21不同，突变的R-ras等位基因不会导致大鼠成纤维细胞在软琼脂中形成单层集落或生长。
