Kostura M, Mathews M B
Cold Spring Harbor Laboratory, New York 11724.
Mol Cell Biol. 1989 Apr;9(4):1576-86. doi: 10.1128/mcb.9.4.1576-1586.1989.
The double-stranded RNA (dsRNA)-dependent protein kinase DAI (also termed dsI and P1) possesses two kinase activities; one is an autophosphorylation activity, and the other phosphorylates initiation factor eIF-2. We purified the enzyme, in a latent form, to near homogeneity from interferon-treated human 293 cells. The purified enzyme consisted of a single polypeptide subunit of approximately 70,000 daltons, retained its dependence on dsRNA for activation, and was sensitive to inhibition by adenovirus VA RNAI. Autophosphorylation required a suitable concentration of dsRNA and was second order with respect to DAI concentration, which suggests an intermolecular mechanism in which one DAI molecule phosphorylates a neighboring molecule. Once autophosphorylated, the enzyme could phosphorylate eIF-2 but seemed unable to phosphorylate other DAI molecules, which implies a change in substrate specificity upon activation. VA RNAI blocked autophosphorylation and activation but permitted the activated enzyme to phosphorylate eIF-2. VA RNAI also blocked the binding of dsRNA to the enzyme. The data are consistent with a model in which activation requires the interaction of two molecules of DAI with dsRNA, followed by intermolecular autophosphorylation of the latent enzyme. VA RNAI would block activation by preventing the interaction between DAI and dsRNA.
双链RNA（dsRNA）依赖性蛋白激酶DAI（也称为dsI和P1）具有两种激酶活性；一种是自磷酸化活性，另一种则使起始因子eIF-2磷酸化。我们从经干扰素处理的人293细胞中以潜伏形式将该酶纯化至近乎均一。纯化后的酶由一个约70,000道尔顿的单一多肽亚基组成，保留了对dsRNA激活的依赖性，并且对腺病毒VA RNAI的抑制敏感。自磷酸化需要合适浓度的dsRNA，并且相对于DAI浓度是二级反应，这表明存在一种分子间机制，即一个DAI分子使相邻分子磷酸化。一旦自磷酸化，该酶就可以使eIF-2磷酸化，但似乎无法使其他DAI分子磷酸化，这意味着激活后底物特异性发生了变化。VA RNAI阻断自磷酸化和激活，但允许激活的酶使eIF-2磷酸化。VA RNAI还阻断dsRNA与该酶的结合。这些数据与一个模型一致，在该模型中，激活需要两个DAI分子与dsRNA相互作用，随后潜伏酶进行分子间自磷酸化。VA RNAI将通过阻止DAI与dsRNA之间的相互作用来阻断激活。
