Heidecker G, Huleihel M, Cleveland J L, Kolch W, Beck T W, Lloyd P, Pawson T, Rapp U R
Section of Viral Pathology, National Cancer Institute-Frederick Cancer Research Facility, Maryland 21701.
Mol Cell Biol. 1990 Jun;10(6):2503-12. doi: 10.1128/mcb.10.6.2503-2512.1990.
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.
将一系列野生型和突变型raf基因转染到NIH 3T3细胞中，并分析其转化活性。全长野生型c-raf未显示出转化活性。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以及全长分子与gag序列的融合。对于一个在保守区域2（CR2）有四肽插入的突变体，观察到较低水平的激活，CR2是位于激酶结构域氨基末端100个残基处的富含丝氨酸和苏氨酸的结构域。为了确定raf转化区域的基本结构特征，我们分析了v-raf的点突变和缺失突变体。赖氨酸-56的取代调节了转化活性，而假定的ATP结合残基赖氨酸-53的突变则消除了转化活性。缺失分析确定最小转化序列与CR3精确重合，CR3是保守的Raf激酶结构域。因此，Raf激酶的致癌激活可以通过去除CR1和CR2或通过空间扭曲来实现，并且需要保留一个活性激酶结构域。这些发现与非刺激状态下酶的蛋白质结构模型一致，在该模型中活性位点被埋在蛋白质内部。
