Ramaiah K V, Davies M V, Chen J J, Kaufman R J
Harvard-MIT Division of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge 02139.
Mol Cell Biol. 1994 Jul;14(7):4546-53. doi: 10.1128/mcb.14.7.4546-4553.1994.
The inhibition of protein synthesis that occurs upon phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) at serine 51 correlates with reduced guanine nucleotide exchange activity of eIF-2B in vivo and inhibition of eIF-2B activity in vitro, although it is not known if phosphorylation is the cause of the reduced eIF-2B activity in vivo. To characterize the importance of eIF-2 alpha phosphorylation in the regulation of eIF-2B activity, we studied the overexpression of mutant eIF-2 alpha subunits in which serine 48 or 51 was replaced by an alanine (48A or 51A mutant). Previous studies demonstrated that the 51A mutant was resistant to phosphorylation, whereas the 48A mutant was a substrate for phosphorylation. Additionally, expression of either mutant partially protected Chinese hamster ovary (CHO) cells from the inhibition of protein synthesis in response to heat shock treatment (P. Murtha-Riel, M. V. Davies, J. B. Scherer, S. Y. Choi, J. W. B. Hershey, and R. J. Kaufman, J. Biol. Chem. 268:12946-12951, 1993). In this study, we show that eIF-2B activity was inhibited in parental CHO cell extracts upon addition of purified reticulocyte heme-regulated inhibitor (HRI), an eIF-2 alpha kinase that phosphorylates Ser-51. Preincubation with purified HRI also reduced the eIF-2B activity in extracts from cells overexpressing wild-type eIF-2 alpha. In contrast, the eIF-2B activity was not readily inhibited in extracts from cells overexpressing either the eIF-2 alpha 48A or 51A mutant. In addition, eIF-2B activity was decreased in extracts prepared from heat-shocked cells overexpressing wild-type eIF-2 alpha, whereas the decrease in eIF-2B activity was less in heat-shocked cells overexpressing either mutant 48A or mutant 51A. While the phosphorylation at serine 51 in eIF-2 alpha impairs the eIF-2B activity, we propose that serine 48 acts to maintain a high affinity between phosphorylated eIF-2 alpha and eIF-2B, thereby inactivating eIF-2B activity. These findings support the hypothesis that phosphorylation of eIF-2 alpha inhibits protein synthesis directly through reducing eIF-2B activity and emphasize the importance of both serine 48 and serine 51 in the interaction with eIF-2B and regulation of eIF-2B activity.
真核起始因子2（eIF-2α）的α亚基在丝氨酸51位点磷酸化后发生的蛋白质合成抑制，与体内eIF-2B的鸟嘌呤核苷酸交换活性降低以及体外eIF-2B活性受抑制相关，尽管尚不清楚磷酸化是否是体内eIF-2B活性降低的原因。为了表征eIF-2α磷酸化在调节eIF-2B活性中的重要性，我们研究了丝氨酸48或51被丙氨酸取代的突变型eIF-2α亚基的过表达（48A或51A突变体）。先前的研究表明，51A突变体对磷酸化具有抗性，而48A突变体是磷酸化的底物。此外，两种突变体的表达都能部分保护中国仓鼠卵巢（CHO）细胞免受热休克处理引起的蛋白质合成抑制（P. Murtha-Riel、M. V. Davies、J. B. Scherer、S. Y. Choi、J. W. B. Hershey和R. J. Kaufman，《生物化学杂志》268:12946 - 12951，1993）。在本研究中，我们发现，添加纯化的网织红细胞血红素调节抑制剂（HRI，一种使Ser-51磷酸化的eIF-2α激酶）后，亲本CHO细胞提取物中的eIF-2B活性受到抑制。用纯化的HRI预孵育也降低了过表达野生型eIF-2α的细胞提取物中的eIF-2B活性。相比之下，过表达eIF-2α 48A或51A突变体的细胞提取物中的eIF-2B活性不容易被抑制。此外，过表达野生型eIF-2α的热休克细胞制备的提取物中eIF-2B活性降低，而过表达突变体48A或突变体51A的热休克细胞中eIF-2B活性的降低幅度较小。虽然eIF-2α中丝氨酸51位点的磷酸化会损害eIF-2B活性，但我们提出丝氨酸48起到维持磷酸化的eIF-2α与eIF-2B之间高亲和力的作用，从而使eIF-2B活性失活。这些发现支持了eIF-2α磷酸化通过降低eIF-2B活性直接抑制蛋白质合成的假说，并强调了丝氨酸48和丝氨酸51在与eIF-2B相互作用及调节eIF-2B活性中的重要性。
