INHIBITION OF NIH-3T3 CELL-PROLIFERATION BY A MUTANT RAS PROTEIN WITH PREFERENTIAL AFFINITY FOR GDP
INHIBITION OF NIH-3T3 CELL-PROLIFERATION BY A MUTANT RAS PROTEIN WITH PREFERENTIAL AFFINITY FOR GDP
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DOI：
10.1128/mcb.8.8.3235
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发表时间：
1988-08-01
影响因子：
5.3
通讯作者：
COOPER, GM
中科院分区：
文献类型：
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作者：
FEIG, LA;COOPER, GM
用天冬酰胺取代17号位置的丝氨酸，使rasH p21对GTP的亲和力降低了20- 40倍，但对GDP的亲和力没有显著影响。用含有Asn-17 rasH基因和Neor基因的哺乳动物表达载体转染NIH 3T3细胞，在相同启动子的控制下，只产生了预期g418抗性菌落数量的一小部分，表明表达Asn-17 p21抑制了细胞增殖。Asn-17 p21的抑制作用需要定位到质膜上，并通过激活ras基因的共表达被逆转，这表明突变体p21阻断了NIH 3T3细胞增殖所需的内源性大鼠功能。由v-mos和v-raf转化的NIH 3T3细胞，但v-src对Asn-17 p21的抑制具有抗性，这表明这些细胞质癌基因可以绕过对正常ras功能的需求。Asn-17突变体在体内具有抑制细胞ras活性的能力，为研究ras功能提供了一种新的试剂。由于这种表型可能与突变蛋白对GDP的优先亲和力有关，类似的突变也可能产生其他蛋白质的抑制剂，其活性受鸟嘌呤核苷酸结合的调节。
Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitor effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous rats function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.