The histidine-221 to tyrosine substitution in v-mos abolishes its biological function and its protein kinase activity.
The histidine-221 to tyrosine substitution in v-mos abolishes its biological function and its protein kinase activity.
复制标题
v-mos 中的组氨酸 221 被酪氨酸取代消除了其生物学功能及其蛋白激酶活性。
DOI：
10.1016/0042-6822(88)90626-5
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发表时间：
1988
期刊：
影响因子：
3.7
通讯作者：
Arlinghaus,RB
中科院分区：
文献类型：
--
作者：
Singh,B;Wittenberg,C;Hannink,M;Reed,SI;Donoghue,DJ;Arlinghaus,RB
该病毒基因编码一种名为p37mos的细胞质转化蛋白。从一些实验方法收集的证据与p37蛋白具有丝氨酸/苏氨酸蛋白激酶活性是一致的。为了进一步了解p37mos相关的生化活性，我们通过寡核苷酸定向突变构建了v-Mos基因的突变，在p37mos的221位残基产生组氨酸到酪氨酸的替代。根据核苷酸序列，相应位置的组氨酸残基在从酵母到人的所有丝氨酸/苏氨酸蛋白激酶中都是保守的，而在蛋白酪氨酸激酶中是不存在的。将突变体p37mos(Tyr-221)在酵母中表达，并对其进行了活性测定。该突变蛋白在体外失去了自身磷酸化活性，从而进一步支持了p37mos是一种蛋白激酶的结论。将突变的v-Mosgene导入逆转录病毒载体pDD102，检测其在NIH/3T3细胞上的成灶能力，发现在37°和30°时均无作用。相反，野生型v-mos具有转化能力和蛋白质温度。这些结果扩展了我们早先关于转化能力和蛋白激酶活性之间的相关性的发现。V-mos蛋白和酵母CDC28基因产物相应位置上的组氨酸到酪氨酸的替换会对激酶活性产生类似的影响。因此，这个残基和/或保守的预测磷酸转移结构域N端附近的序列，靠近完整催化域的中间，可能特异性地参与了丝氨酸/苏氨酸蛋白激酶的催化活性。
The viralmosgene encodes a cytoplasmic transforming protein termed p37mos. Evidence gathered from a number of experimental approaches is consistent with p37moshaving a serine/threonine protein kinase activity. To gain further understanding of the p37mos-associated biochemical activity, we constructed a mutation in the v-mosgene by oligonucleotide-directed mutagenesis yielding a histidine to tryosine substitution at residue 221 in p37mos. Based upon nucleotide sequences, the histidine residue at the corresponding position is conserved in all the serine/threonine protein kinases from yeast to man, and is absent in protein-tyrosine kinases. The mutant p37mos(Tyr-221) was expressed in yeast and assayed for kinase activity. The mutant protein was inactive as judged by a loss of autophosphorylation activityin vitro, thus providing further support for the conclusion that p37mosis a protein kinase. When the mutant v-mosgene was introduced into a retroviral vector, pDD102, and assayed for focus-forming ability on NIH/3T3 cells, it was found to be inactice at both 37 and 30°. In contrast, the wild-type v-moshad transforming ability and protein temperatures. These results extend our earlier findings on the correlation between transforming ability and protein kinase activity. A histidine to tyrosine substitution at the corresponding position of the v-mosprotein and the yeastCDC28gene product causes a similar effect on the kinase activity. Therefore, this residue and/or the sequence near the N-terminal side of the conserved predicted phosphate transfer domain, near the middle of the complete catalytic domain, might be specifically involved in the catalytic activity of serine/threonine protein kinases in general.