Structural requirements for double-stranded RNA binding, dimerization, and activation of the human eIF-2 alpha kinase DAI in Saccharomyces cerevisiae.
Structural requirements for double-stranded RNA binding, dimerization, and activation of the human eIF-2 alpha kinase DAI in Saccharomyces cerevisiae.
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酿酒酵母中人 eIF-2 α 激酶 DAI 的双链 RNA 结合、二聚化和激活的结构要求。
DOI：
10.1128/mcb.15.1.365
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发表时间：
1995
影响因子：
5.3
通讯作者：
Hinnebusch,AG
中科院分区：
文献类型：
--
作者：
Romano,PR;Green,SR;Barber,GN;Mathews,MB;Hinnebusch,AG
蛋白激酶DAI在病毒感染哺乳动物细胞后被激活，并通过翻译起始因子2 α亚基（eIF-2α）的磷酸化抑制蛋白质合成。DAI在体外被双链RNA（dsRNA）激活，并且dsRNA的结合依赖于位于DAI中激酶结构域N末端的保守序列基序的两个拷贝。DAI在酵母细胞中的高水平表达是致命的，因为eIF-2α的过度磷酸化;在较低水平下，DAI可以在功能上取代蛋白激酶GCN 2并刺激GCN 4 mRNA的翻译。这两种表型用于表征体内DAI功能的结构要求，通过检查两个dsRNA结合基序中匹配位置处的氨基酸取代以及用另一个取代基序的一个拷贝的影响。我们发现dsRNA结合基序的两个拷贝都是高水平激酶功能所需的，并且N-末端拷贝比C-末端拷贝对酿酒酵母中DAI的激活更重要。在这些发现的基础上，我们得出结论，dsRNA的体外结合和DAI激酶功能在体内的激活的要求密切一致。当在酵母细胞中共表达时，含有第一或第二结合基序的缺失的两个突变等位基因在功能上互补，强烈表明DAI的活性形式是二聚体。与此结论雅阁，在蛋白激酶结构域中含有不同缺失的四个催化失活等位基因的过表达干扰了在相同细胞中产生的野生型DAI。有趣的是，激酶结构域中的三个失活点突变都是隐性的，这表明显性干扰涉及缺陷异二聚体的形成，而不是突变酶对dsRNA激活剂的螯合。我们认为，激酶结构域的大的结构改变损害了DAI二聚体中两个原聚体之间的相互作用，这是dsRNA激活或催化eIF-2α磷酸化所必需的。
The protein kinase DAI is activated upon viral infection of mammalian cells and inhibits protein synthesis by phosphorylation of the α subunit of translation initiation factor 2 (eIF-2α). DAI is activated in vitro by double-stranded RNAs (dsRNAs), and binding of dsRNA is dependent on two copies of a conserved sequence motif located N terminal to the kinase domain in DAI. High-level expression of DAI inSaccharomyces cerevisiaecells is lethal because of hyperphosphorylation of eIF-2α; at lower levels, DAI can functionally replace the protein kinase GCN2 and stimulate translation ofGCN4mRNA. These two phenotypes were used to charac-terize structural requirements for DAI function in vivo, by examining the effects of amino acid substitutions at matching positions in the two dsRNA-binding motifs and of replacing one copy of the motif with the other. We found that both copies of the dsRNA-binding motif are required for high-level kinase function and that the N-terminal copy is more important than the C-terminal copy for activation of DAI inS. cerevisiae. On the basis of these findings, we conclude that the requirements for dsRNA binding in vitro and for activation of DAI kinase function in vivo closely coincide. Two mutant alleles containing deletions of the first or second binding motif functionally complemented when coexpressed in yeast cells, strongly suggesting that the active form of DAI is a dimer. In accord with this conclusion, overexpression of four catalytically inactive alleles containing different deletions in the protein kinase domain interfered with wild-type DAI produced in the same cells. Interestingly, three inactivating point mutations in the kinase domain were all recessive, suggesting that dominant interference involves the formation of defective heterodimers rather than sequestration of dsRNA activators by mutant enzymes. We suggest that large structural alterations in the kinase domain impair an interaction between the two protomers in a DAI dimer that is necessary for activation by dsRNA or for catalysis of eIF-2α phosphorylation.