Wek S A, Zhu S, Wek R C
Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis 46202-5122, USA.
Mol Cell Biol. 1995 Aug;15(8):4497-506. doi: 10.1128/MCB.15.8.4497.
Protein kinase GCN2 is a multidomain protein that contains a region homologous to histidyl-tRNA synthetases juxtaposed to the kinase catalytic moiety. Previous studies have shown that in response to histidine starvation, GCN2 phosphorylates eukaryotic initiation factor 2 (eIF-2), to induce the translational expression of GCN4, a transcriptional activator of genes subject to the general amino acid control. It was proposed that the synthetase-related sequences of GCN2 stimulate the activity of the kinase by interacting directly with uncharged tRNA that accumulates during amino acid limitation. In addition to histidine starvation, expression of GCN4 is also regulated by a number of other amino acid limitations. Questions that we posed in this report are whether uncharged tRNA is the most direct regulator of GCN2 and whether the function of this kinase is required to recognize each of the different amino acid starvation signals. We show that GCN2 phosphorylation of eIF-2, and the resulting general amino acid control pathway, is stimulated in response to starvation for each of several different amino acids, in addition to histidine limitation. Cells containing a defective aminoacyl-tRNA synthetase also stimulated GCN2 phosphorylation of eIF-2 in the absence of amino acid starvation, indicating that uncharged tRNA levels are the most direct regulator of GCN2 kinase. Using a Northwestern blot (RNA binding) assay, we show that uncharged tRNA can bind to the synthetase-related domain of GCN2. Mutations in the motif 2 sequence conserved among class II synthetases, including histidyl-tRNA synthetases, impair the ability of this synthetase-related domain to bind tRNA and abolish GCN2 phosphorylation of eIF-2 required to stimulate the general amino acid control response. These in vivo and in vitro experiments indicate that synthetase-related sequences regulate GCN2 kinase function by monitoring the levels of multiple uncharged tRNAs that accumulate during amino acid limitations.
蛋白激酶GCN2是一种多结构域蛋白，其包含与组氨酰 - tRNA合成酶同源的区域，该区域与激酶催化部分相邻。先前的研究表明，在组氨酸饥饿的情况下，GCN2使真核起始因子2（eIF - 2）磷酸化，从而诱导GCN4的翻译表达，GCN4是受一般氨基酸控制的基因的转录激活因子。有人提出，GCN2的合成酶相关序列通过与在氨基酸限制期间积累的无电荷tRNA直接相互作用来刺激激酶的活性。除了组氨酸饥饿外，GCN4的表达还受到许多其他氨基酸限制的调节。我们在本报告中提出的问题是，无电荷tRNA是否是GCN2最直接的调节因子，以及该激酶的功能是否是识别每种不同氨基酸饥饿信号所必需的。我们表明，除了组氨酸限制外，对于几种不同氨基酸中的每一种饥饿情况，GCN2对eIF - 2的磷酸化以及由此产生的一般氨基酸控制途径都会受到刺激。含有缺陷氨酰 - tRNA合成酶的细胞在没有氨基酸饥饿的情况下也会刺激GCN2对eIF - 2的磷酸化，这表明无电荷tRNA水平是GCN2激酶最直接的调节因子。使用蛋白质印迹（RNA结合）分析，我们表明无电荷tRNA可以与GCN2的合成酶相关结构域结合。在包括组氨酰 - tRNA合成酶在内的II类合成酶中保守的基序2序列中的突变，损害了该合成酶相关结构域结合tRNA的能力，并消除了刺激一般氨基酸控制反应所需的GCN2对eIF - 2的磷酸化。这些体内和体外实验表明，合成酶相关序列通过监测在氨基酸限制期间积累的多种无电荷tRNA的水平来调节GCN2激酶的功能。
