Shu H K, Chang C M, Ravi L, Ling L, Castellano C M, Walter E, Pelley R J, Kung H J
Department of Molecular Biology and Microbiology, Case Western Reserve University, Cleveland, Ohio 44106.
Mol Cell Biol. 1994 Oct;14(10):6868-78. doi: 10.1128/mcb.14.10.6868-6878.1994.
Avian c-erbB is activated to a leukemia oncogene following truncation of its amino-terminal ligand-binding domain by retroviral insertion. The insertionally activated transcripts encode protein products which have constitutive tyrosine kinase activity and can induce erythroleukemia but not sarcomas. We have previously found that a valine-to-isoleucine point mutation at position 157 (V157I mutant) within the tyrosine kinase domain of this truncated erbB can dramatically activate the sarcomagenic potential of the oncogene and increase the kinase activity of this oncoprotein. This mutation lies at position 157 of the insertionally activated c-erbB product, affecting a highly conserved valine residue of the glycine loop involved in ATP binding and phosphate transfer. To investigate the functional importance of this residue in the catalytic activity of kinases, we have introduced at this position, by site-directed mutagenesis, codons representing the remaining 18 amino acid residues. Most of the mutants have diminished activity, with six of them completely devoid of kinase activity, indicating the sensitivity of this region to conformational changes. Some of these mutants displayed increased kinase activity and greater transforming potential in comparison with IA c-erbB, but none had levels as high as those of the V157I mutant. In general, the sarcomagenic potential of the various erbB mutants correlated with their autophosphorylation state and their ability to cause phosphorylation of MAP kinase. However, there are important exceptions such as the V157G mutant, which lacks enhanced autophosphorylation but is highly sarcomagenic. Studies of this and other autophosphorylation site mutants point to the existence of an autophosphorylation-independent pathway in sarcomagenesis. The requirement for leukemogenic potential is much less stringent and correlates with positivity of kinase activity. When the valine-to-isoleucine substitution was put in context of the full-length erbB protein, the mutation relaxed the ligand dependence and had a positive effect on the transforming potential of the full-length c-erbB.
禽源c-erbB基因在其氨基末端配体结合结构域被逆转录病毒插入截断后，被激活成为白血病致癌基因。插入激活的转录本编码具有组成型酪氨酸激酶活性的蛋白质产物，可诱导红白血病，但不能诱导肉瘤。我们之前发现，这种截断的erbB酪氨酸激酶结构域内第157位的缬氨酸到异亮氨酸点突变（V157I突变体）可显著激活致癌基因的肉瘤发生潜能，并增加这种癌蛋白的激酶活性。该突变位于插入激活的c-erbB产物的第157位，影响参与ATP结合和磷酸转移的甘氨酸环中一个高度保守的缬氨酸残基。为了研究该残基在激酶催化活性中的功能重要性，我们通过定点诱变在该位置引入了代表其余18个氨基酸残基的密码子。大多数突变体的活性降低，其中六个完全没有激酶活性，表明该区域对构象变化敏感。与IA c-erbB相比，其中一些突变体显示出增加的激酶活性和更大的转化潜能，但没有一个达到V157I突变体的水平。一般来说，各种erbB突变体的肉瘤发生潜能与其自身磷酸化状态以及引起MAP激酶磷酸化的能力相关。然而，也有重要的例外情况，如V157G突变体，它缺乏增强的自身磷酸化，但具有高度的肉瘤发生能力。对该突变体和其他自身磷酸化位点突变体的研究表明，肉瘤发生过程中存在一条不依赖自身磷酸化的途径。致白血病潜能的要求则宽松得多，与激酶活性的阳性相关。当缬氨酸到异亮氨酸的替换在全长erbB蛋白的背景下进行时，该突变减轻了配体依赖性，并对全长c-erbB的转化潜能产生了积极影响。
