Shimizu K, Nakatsu Y, Nomoto S, Sekiguchi M
Molecular Genetics Section, Faculty of Science, Kyushu University, Fukuoka, Japan.
Princess Takamatsu Symp. 1986;17:85-91.
We previously isolated a novel human transforming gene from a primary stomach cancer and identified it as an activated version of the c-raf-1 gene which is the human homologue of v-raf, a viral oncogene encoding a serine/threonine-specific protein kinase. Analyses of cDNA and genomic clones of this gene revealed that it was generated by substitution of 5'-sequence (exons 1-5) of the normal c-raf-1 gene with an unrelated human sequence. We identified the region in the genomic clone where the rearrangement had occurred. The rearranged EcoRI fragment was detected in all the primary transformants obtained from two independent transfections, suggesting that the recombination had occurred in the primary cancer. By sequence analysis of cDNA, the putative product of the transforming gene was inferred to have a hydrophobic stretch ahead of the ser/thr-protein kinase domain of the c-raf-1 gene product. We introduced one of the cDNA which contains the 1.6-kb open reading frame into the pUC9 vector. An autophosphorylating, 58 kd protein was induced in Escherichia coli cells bearing the plasmid upon induction. Since ser/thr-protein kinase activity of the normal c-raf protein has not been evidenced, these results suggest that the truncation/replacement of the amino-terminal domain of the c-raf-1 protein leads to constitutive activation of the protein kinase probably residing on the downstream domain.
我们先前从一例原发性胃癌中分离出一个新的人类转化基因，并将其鉴定为c-raf-1基因的激活形式，该基因是v-raf的人类同源物，v-raf是一种编码丝氨酸/苏氨酸特异性蛋白激酶的病毒癌基因。对该基因的cDNA和基因组克隆进行分析后发现，它是由正常c-raf-1基因的5'端序列（外显子1-5）被一段不相关的人类序列取代而产生的。我们确定了基因组克隆中发生重排的区域。在两次独立转染获得的所有原发性转化体中均检测到重排的EcoRI片段，这表明重组发生在原发性癌症中。通过对cDNA的序列分析，推测转化基因的推定产物在c-raf-1基因产物的丝氨酸/苏氨酸蛋白激酶结构域之前有一段疏水序列。我们将其中一个包含1.6kb开放阅读框的cDNA导入pUC9载体。携带该质粒的大肠杆菌细胞在诱导后可诱导产生一种自磷酸化的58kd蛋白。由于尚未证实正常c-raf蛋白具有丝氨酸/苏氨酸蛋白激酶活性，这些结果表明c-raf-1蛋白氨基末端结构域的截短/替换可能导致可能位于下游结构域的蛋白激酶的组成性激活。
