Ogiso Y, Gutierrez L, Wrathall L S, Lu Y Y, Blair D G, Clanton D J, Hwang Y W, Shih T Y
Laboratory of Molecular Oncology, National Cancer Institute, Frederick, Maryland 21701-1013.
Cell Growth Differ. 1990 May;1(5):217-24.
Site-directed mutagenesis of the conserved sequence motifs of p21 generated a group of mutant p21s defective in GTP binding. Some of these mutants were highly transforming, whereas others were transformation defective. Among the latter group, we found two mutants, derived from the v-H-ras oncogene by substituting the asparagine-116 with tyrosine and isoleucine, that exhibited a trans-dominant activity of suppressing the transformed phenotype of NIH3T3 cells induced by a long terminal repeat-linked c-H-ras and a wild-type v-H-ras. They caused reduction of the colony-forming efficiency in soft agar (78% in c-ras-transformed cells; 55% in v-ras cells) and morphological reversion of ras transformants. Subclones of revertants expressed a great excess of mutant p21 relative to the c-ras p21 present in these cells. These mutants were not lethal to NIH3T3 cells. Apparently, defective proteins encoded by suppressor mutants sequestered vital targets for ras function. Suppressor mutants also induced morphological reversion of NIH3T3 cells transformed by src, fes/flp, sis, and fms oncogenes, suggesting that these oncogenes function upstream to ras in the signaling pathways. Cells transformed by mos and a chemical carcinogen were unaffected.
对p21保守序列基序进行定点诱变产生了一组在GTP结合方面存在缺陷的突变型p21。其中一些突变体具有高度转化能力，而另一些则存在转化缺陷。在后一组中，我们发现了两个通过将天冬酰胺-116分别替换为酪氨酸和异亮氨酸而从v-H-ras癌基因衍生而来的突变体，它们表现出反式显性活性，可抑制由长末端重复序列连接的c-H-ras和野生型v-H-ras诱导的NIH3T3细胞的转化表型。它们导致软琼脂中集落形成效率降低（在c-ras转化的细胞中降低78%；在v-ras细胞中降低55%）以及ras转化细胞的形态逆转。回复子的亚克隆相对于这些细胞中存在的c-ras p21表达了大量过量的突变型p21。这些突变体对NIH3T3细胞无致死性。显然，抑制突变体编码的缺陷蛋白隔离了ras功能的重要靶点。抑制突变体还诱导了由src、fes/flp、sis和fms癌基因转化的NIH3T3细胞的形态逆转，这表明这些癌基因在信号通路中作用于ras的上游。由mos和化学致癌物转化的细胞不受影响。
