STRUCTURAL SIGNIFICANCE OF THE GTP-BINDING DOMAIN OF RAS P21 STUDIED BY SITE-DIRECTED MUTAGENESIS
STRUCTURAL SIGNIFICANCE OF THE GTP-BINDING DOMAIN OF RAS P21 STUDIED BY SITE-DIRECTED MUTAGENESIS
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DOI：
10.1128/mcb.7.9.3092
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发表时间：
1987-09-01
影响因子：
5.3
通讯作者：
SHIH, TY
中科院分区：
文献类型：
--
作者：
CLANTON, DJ;LU, YY;SHIH, TY
p21蛋白的点突变是通过v-rasH癌基因的寡核苷酸定向突变构建的，其取代了核苷酸结合共有序列GXG GXGK内的氨基酸残基。当10、13或15位的甘氨酸残基被缬氨酸取代时，病毒rasH产物p21失去了其GTP结合和自激酶活性。在位置33、51、59处的其它取代不损害其结合活性。G418抗性NIH 3 T3细胞系通过用通过将突变体前病毒DNA插入pSV 2neo质粒中获得的构建体转染而得到。在位置13或15处具有缬氨酸突变的克隆不能转化细胞，而具有GTP结合活性的所有其它突变体是胜任的。在第10位用缬氨酸取代甘氨酸的突变体丧失了其结合GTP的能力和其自身激酶活性，完全能够转化NIH 3 T3细胞。这些细胞在软琼脂中生长，并在裸鼠体内迅速形成肿瘤。肿瘤组织来源的细胞系p21仍缺乏自激酶活性。这些研究结果表明，富含甘氨酸的共识序列是重要的，在控制p21的活动，某些突变可能会赋予p21的活性构象，而不参与配体结合。
Point mutations of p21 proteins were constructed by oligonucleotide-directed mutagenesis of the v-rasH oncogene, which substituted amino acid residues within the nucleotide-binding consensus sequence, GXG GXGK. When the glycine residue at position 10, 13, or 15 was substituted with valine, the viral rasH product p21 lost its GTP-binding and autokinase activities. Other substitutions at position 33, 51, 59 did not impair its binding activity. G418-resistant NIH 3T3 cell lines were derived by transfection with constructs obtained by inserting the mutant proviral DNA into the pSV2neo plasmid. Clones with a valine mutation at position 13 or 15 were incapable of transforming cells, while all other mutants with GTP-binding activity were competent. A mutant with a substitution of valine for glycine at position 10 which had lost its ability to bind GTP and its autokinase activity was fully capable of transforming NIH 3T3 cells. These cells grew in soft agar and rapidly formed tumors in nude mice. The p21 of cell lines derived from tumor explants still lacked the autokinase activity. These findings suggest that the glycine-rich consensus sequence is important in controlling p21 activities and that certain mutations may confer to p21 its active conformation without participation of ligand binding.