Cherkasova Vera A, Hinnebusch Alan G
Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Genes Dev. 2003 Apr 1;17(7):859-72. doi: 10.1101/gad.1069003. Epub 2003 Mar 21.
Yeast protein kinase GCN2 stimulates the translation of transcriptional activator GCN4 by phosphorylating eIF2alpha in response to amino acid starvation. Kinase activation requires binding of uncharged tRNA to a histidyl tRNA synthetase-related domain in GCN2. Phosphorylation of serine 577 (Ser 577) in GCN2 by another kinase in vivo inhibits GCN2 function in rich medium by reducing tRNA binding activity. We show that rapamycin stimulates eIF2alpha phosphorylation by GCN2, with attendant induction of GCN4 translation, while reducing Ser 577 phosphorylation in nonstarved cells. The alanine 577 (Ala 577) mutation in GCN2 (S577A) dampened the effects of rapamycin on eIF2alpha phosphorylation and GCN4 translation, suggesting that GCN2 activation by rapamycin involves Ser 577 dephosphorylation. Rapamycin regulates the phosphorylation of Ser 577 and eIF2alpha by inhibiting the TOR pathway. Rapamycin-induced dephosphorylation of Ser 577, eIF2alpha phosphorylation, and induction of GCN4 all involve TAP42, a regulator of type 2A-related protein phosphatases. Our results add a new dimension to the regulation of protein synthesis by TOR proteins and demonstrate cross-talk between two major pathways for nutrient control of gene expression in yeast.
酵母蛋白激酶GCN2在氨基酸饥饿时通过磷酸化真核生物翻译起始因子2α（eIF2α）来刺激转录激活因子GCN4的翻译。激酶激活需要空载tRNA与GCN2中与组氨酰tRNA合成酶相关的结构域结合。在体内，另一种激酶对GCN2中丝氨酸577（Ser 577）的磷酸化通过降低tRNA结合活性来抑制丰富培养基中GCN2的功能。我们发现雷帕霉素可刺激GCN2介导的eIF2α磷酸化，伴随GCN4翻译的诱导，同时减少非饥饿细胞中Ser 577的磷酸化。GCN2中的丙氨酸577（Ala 577）突变（S577A）减弱了雷帕霉素对eIF2α磷酸化和GCN4翻译的影响，表明雷帕霉素对GCN2的激活涉及Ser 577的去磷酸化。雷帕霉素通过抑制TOR途径调节Ser 577和eIF2α的磷酸化。雷帕霉素诱导的Ser 577去磷酸化、eIF2α磷酸化以及GCN4的诱导均涉及TAP42，一种2A型相关蛋白磷酸酶的调节因子。我们的结果为TOR蛋白对蛋白质合成的调控增添了新的维度，并证明了酵母中基因表达营养控制的两条主要途径之间存在相互作用。
