van den Heuvel J, Lang V, Richter G, Price N, Peacock L, Proud C, McCarthy J E
Department of Gene Expression, GBF-National Biotechnology Research Centre, Braunschweig, Germany.
Biochim Biophys Acta. 1995 Apr 26;1261(3):337-48. doi: 10.1016/0167-4781(95)00026-d.
Regulation of the effective activity of eukaryotic initiation factor 2 (eIF-2) in protein synthesis is known to involve phosphorylation of its alpha subunit. Two mammalian enzymes, the haem-controlled repressor (HCR) and the double-stranded RNA-activated inhibitor (dsI), phosphorylate Ser-51 of the alpha subunit, thereby inhibiting the exchange of bound nucleotides on, and thus the recycling of, eIF-2. In Saccharomyces cerevisiae, the equivalent serine seems to be phosphorylated by the GCN2 protein kinase, which is activated by amino acid starvation. However, in the present paper we show that this is not the only site of phosphorylation in yeast eIF-2 alpha. We report the preparation of recombinant yeast eIF-2 alpha from Escherichia coli and its use in in vitro phosphorylation studies. Mammalian HCR and dsI are shown to phosphorylate specifically Ser-51 of yeast eIF-2 alpha, whereas extracts from yeast cells do not. Instead, at least one of three serine residue in the acidic C-terminal region of this protein is phosphorylated by fractions of yeast possessing casein kinase activities 1 and 2. A triple Ser-->Ala mutant form of yeast eIF-2 alpha was found to be no longer phosphorylated by either of the yeast (or mammalian) casein kinase activities in vitro. Isoelectric focusing of yeast extracts confirmed that the mutated sites normally act as sites of phosphorylation in vivo. The same mutant was used to show that the three sites have no essential function under normal physiological conditions in yeast. In contrast, deletion of the 13 amino acid long C-terminal region of eIF-2 alpha, including the three phosphorylation sites, led to derepression of GCN4 in vivo. Thus removal of the short, highly acidic C-terminal region of eIF-2 alpha has the same regulatory effect on translational (re)initiation as phosphorylation of the Ser-51 residue of the wild-type protein. This result provides new insight into the role of eIF-2 alpha activity in the regulation of translational (re-) initiation.
已知真核生物起始因子2（eIF - 2）在蛋白质合成中的有效活性调节涉及其α亚基的磷酸化。两种哺乳动物酶，即血红素控制的阻遏物（HCR）和双链RNA激活的抑制剂（dsI），可使α亚基的丝氨酸 - 51磷酸化，从而抑制eIF - 2上结合核苷酸的交换，进而抑制其循环利用。在酿酒酵母中，等效的丝氨酸似乎由GCN2蛋白激酶磷酸化，该激酶由氨基酸饥饿激活。然而，在本文中我们表明这不是酵母eIF - 2α中唯一的磷酸化位点。我们报道了从大肠杆菌制备重组酵母eIF - 2α及其在体外磷酸化研究中的应用。结果表明，哺乳动物HCR和dsI特异性地使酵母eIF - 2α的丝氨酸 - 51磷酸化，而酵母细胞提取物则不能。相反，该蛋白酸性C末端区域的三个丝氨酸残基中至少有一个被具有酪蛋白激酶活性1和2的酵母组分磷酸化。发现酵母eIF - 2α的三重丝氨酸→丙氨酸突变体形式在体外不再被酵母（或哺乳动物）酪蛋白激酶活性磷酸化。酵母提取物的等电聚焦证实，突变位点在体内通常作为磷酸化位点起作用。相同的突变体用于表明这三个位点在酵母正常生理条件下没有基本功能。相比之下，缺失eIF - 2α的13个氨基酸长的C末端区域（包括三个磷酸化位点）导致体内GCN4的去阻遏。因此，去除eIF - 2α短的、高度酸性的C末端区域对翻译（再）起始具有与野生型蛋白丝氨酸 - 51残基磷酸化相同的调节作用。这一结果为eIF - 2α活性在翻译（再）起始调节中的作用提供了新的见解。
