MUTATIONAL ACTIVATION OF C-RAF-1 AND DEFINITION OF THE MINIMAL TRANSFORMING SEQUENCE
MUTATIONAL ACTIVATION OF C-RAF-1 AND DEFINITION OF THE MINIMAL TRANSFORMING SEQUENCE
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DOI：
10.1128/mcb.10.6.2503
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发表时间：
1990-06-01
影响因子：
5.3
通讯作者：
RAPP, UR
中科院分区：
文献类型：
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作者：
HEIDECKER, G;HULEIHEL, M;RAPP, UR
将一系列野生型和突变型raf基因转染到NIH 3 T3细胞中并分析转化活性。全长野生型c-raf没有显示转化活性。有两种类型的突变导致了与v-raf相似的致癌活性：蛋白质氨基末端一半的截短和全长分子与gag序列的融合。一个较低水平的激活观察到的突变体与四肽插入映射到保守区域2（CR2），丝氨酸和苏氨酸丰富的域位于100个残基的氨基末端的激酶结构域。为了确定raf转化区的基本结构特征，我们分析了v-raf的点突变和缺失突变。Lys-56的取代调节转化活性，而Lys-53的突变，一个假定的ATP结合残基，取消了它。缺失分析建立了最小的转化序列正好与CR 3，保守的Raf激酶结构域。因此，Raf激酶的致癌激活可以通过去除CR 1和Cr2或通过空间畸变来实现，并且需要保留活性激酶结构域。这些发现与非刺激酶的蛋白质结构模型一致，其中活性位点被埋在蛋白质中。
A series of wild-type and mutant raf genes was transfected into NIH 3T3 cells and analyzed for transforming activity. Full-length wild-type c-raf did not show transforming activity. Two types of mutations resulted in oncogenic activity similar to that of v-raf: truncation of the amino-terminal half of the protein and fusion of the full-length molecule to gag sequences. A lower level of activation was observed for a mutant with a tetrapeptide insertion mapping to conserved region 2 (CR2), a serine- and threonine-rich domain located 100 residues amino-terminal of the kinase domain. To determine essential structural features of the transforming region of raf, we analyzed point and deletion mutants of v-raf. Substitutions of Lys-56 modulated the transforming activity, whereas mutation of Lys-53, a putative ATP binding residue, abolished it. Deletion analysis established that the minimal transforming sequence coincided precisely with CR3, the conserved Raf kinase domain. Thus, oncogenic activation of the Raf kinase can be achieved by removal of CR1 and Cr2 or by steric distortion and requires retention of an active kinase domain. These findings are consistent with a protein structure model for the nonstimulated enzyme in which the active site is buried within the protein.