Connolly D C, Toutenhoofd S L, Maihle N J
Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905.
J Virol. 1994 Oct;68(10):6804-10. doi: 10.1128/JVI.68.10.6804-6810.1994.
Expression of mutant avian c-erbB1 genes results in tissue-specific transformation in chickens. Site-directed mutagenesis was used to generate kinase-defective mutants of several tissue-specific v-erbB transforming mutants by replacement of the ATP-binding lysine residue in the kinase domain with an arginine residue. These kinase-defective v-erbB mutants were analyzed for their in vitro and in vivo transforming potentials. Specifically, kinase-defective mutants of erythroleukemogenic, hemangioma-inducing, and sarcomagenic v-erbB genes were assessed for their oncogenic potential. In vitro transformation potential was assessed by soft-agar colony formation in primary cultures of chick embryo fibroblasts (CEF). In vivo transformation potential was determined by infection of 1-day-old line 0 chicks with concentrated recombinant retrovirus and then monitoring of birds for tumor formation. These transformation assays demonstrate that kinase activity is absolutely essential for transformation by tissue-specific transforming mutants of the avian c-erbB1 gene. Since all of the tissue-specific v-erbB mutants characterized to date exhibit tyrosine kinase activity in vitro but do not transform all tissues in which they are expressed, we conclude that v-erbB-associated tyrosine kinase activity may be necessary but is not sufficient to induce tumor formation.
突变的禽源c-erbB1基因的表达导致鸡体内组织特异性转化。通过将激酶结构域中的ATP结合赖氨酸残基替换为精氨酸残基，利用定点诱变技术产生了几种组织特异性v-erbB转化突变体的激酶缺陷型突变体。对这些激酶缺陷型v-erbB突变体的体外和体内转化潜能进行了分析。具体而言，评估了致红细胞白血病、诱导血管瘤和致肉瘤的v-erbB基因的激酶缺陷型突变体的致癌潜能。通过鸡胚成纤维细胞（CEF）原代培养中的软琼脂集落形成来评估体外转化潜能。通过用浓缩重组逆转录病毒感染1日龄0系雏鸡，然后监测鸡的肿瘤形成来确定体内转化潜能。这些转化试验表明，激酶活性对于禽源c-erbB1基因的组织特异性转化突变体的转化绝对至关重要。由于迄今为止所鉴定的所有组织特异性v-erbB突变体在体外均表现出酪氨酸激酶活性，但并未转化其表达所在的所有组织，因此我们得出结论，v-erbB相关的酪氨酸激酶活性可能是必要的，但不足以诱导肿瘤形成。
