Qiu H, Garcia-Barrio M T, Hinnebusch A G
Laboratory of Eukaryotic Gene Regulation, National Institute of Child Health and Human Development, Bethesda, Maryland 20892-2716, USA.
Mol Cell Biol. 1998 May;18(5):2697-711. doi: 10.1128/MCB.18.5.2697.
The protein kinase GCN2 stimulates translation of the transcriptional activator GCN4 in yeast cells starved for amino acids by phosphorylating translation initiation factor 2. Several regulatory domains, including a pseudokinase domain, a histidyl-tRNA synthetase (HisRS)-related region, and a C-terminal (C-term) segment required for ribosome association, have been identified in GCN2. We used the yeast two-hybrid assay, coimmunoprecipitation analysis, and in vitro binding assays to investigate physical interactions between the different functional domains of GCN2. A segment containing about two thirds of the protein kinase (PK) catalytic domain and another containing the C-term region of GCN2 interacted with themselves in the two-hybrid assay, and both the PK and the C-term domains could be coimmunoprecipitated with wild-type GCN2 from yeast cell extracts. In addition, in vitro-translated PK and C-term segments showed specific binding in vitro to recombinant glutathione S-transferase (GST)-PK and GST-C-term fusion proteins, respectively. Wild-type GCN2 could be coimmunoprecipitated with a full-length LexA-GCN2 fusion protein from cell extracts, providing direct evidence for dimerization by full-length GCN2 molecules. Deleting the C-term or PK segments abolished or reduced, respectively, the yield of GCN2-LexA-GCN2 complexes. These results provide in vivo and in vitro evidence that GCN2 dimerizes through self-interactions involving the C-term and PK domains. The PK domain showed pairwise in vitro binding interactions with the pseudokinase, HisRS, and C-term domains; additionally, the HisRS domain interacted with the C-term region. We propose that physical interactions between the PK domain and its flanking regulatory regions and dimerization through the PK and C-term domains both play important roles in restricting GCN2 kinase activity to amino acid-starved cells.
蛋白激酶GCN2通过磷酸化翻译起始因子2来刺激氨基酸饥饿的酵母细胞中转录激活因子GCN4的翻译。在GCN2中已鉴定出几个调节结构域，包括一个假激酶结构域、一个组氨酰-tRNA合成酶（HisRS）相关区域以及核糖体结合所需的C末端（C-term）片段。我们使用酵母双杂交试验、免疫共沉淀分析和体外结合试验来研究GCN2不同功能结构域之间的物理相互作用。在双杂交试验中，包含约三分之二蛋白激酶（PK）催化结构域的片段和另一个包含GCN2 C末端区域的片段发生了自身相互作用，并且PK结构域和C末端结构域都可以从酵母细胞提取物中与野生型GCN2进行免疫共沉淀。此外，体外翻译的PK片段和C末端片段分别在体外与重组谷胱甘肽S-转移酶（GST）-PK和GST-C末端融合蛋白显示出特异性结合。野生型GCN2可以从细胞提取物中与全长LexA-GCN2融合蛋白进行免疫共沉淀，为全长GCN2分子的二聚化提供了直接证据。删除C末端或PK片段分别消除或降低了GCN2-LexA-GCN2复合物的产量。这些结果提供了体内和体外证据，表明GCN2通过涉及C末端和PK结构域的自身相互作用形成二聚体。PK结构域在体外与假激酶、HisRS和C末端结构域表现出两两结合相互作用；此外，HisRS结构域与C末端区域相互作用。我们提出，PK结构域与其侧翼调节区域之间的物理相互作用以及通过PK和C末端结构域的二聚化在将GCN2激酶活性限制于氨基酸饥饿细胞中均起重要作用。
