Translational Control by elF-2 Kinase during ER Stress

ER 应激期间 eLF-2 激酶的翻译控制

• 批准号: 6780942
• 负责人: RONALD C WEK
• 金额: $ 22.62万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6780942
• 关键词: chemical kinetics; cofactor; dimer; electrospray ionization mass spectrometry; endoplasmic reticulum; environmental stressor; gene induction /repression; genetic regulation; immunoprecipitation; intermolecular interaction; intracellular transport; molecular chaperones; molecular site; phosphorylation; posttranslational modifications; protein biosynthesis; protein folding; protein kinase; protein structure function; protein transport; site directed mutagenesis; tissue /cell culture; translation factor; virus protein; western blottings; yeast two hybrid system

In response to different stresses, eukaryotic cells dramatically reduce protein synthesis by phosphorylation of the alpha subunit of eukaryotic translation initiation factor 2 (eIF- 2). Recently, we identified a new eIF-2alpha kinase from rat pancreas. The new protein kinase designated pancreatic eIF-2alpha kinase, PEK, is an ER transmembrane protein that is activated in response to ER stresses that impair protein folding in this organelle. PEK is expressed in all tissues examined, with highest levels in secretory tissues. PEK sequences in the ER lumen are proposed to sense ER stress, eliciting a conformation change in PEK that stimulates phosphorylation of eIF-2alpha. Reduced protein synthesis provides the cell an opportunity to remedy protein misfolding prior to introducing newly synthesized proteins into the secretory pathway. Two fundamental questions will be addressed in this proposal. First, we will address the mechanisms regulating PEK in response to ER stress. We consider two hypotheses for the regulation of PEK activity. First, the stressed ER may directly modify PEK or an associated cofactor. For example, a change in the oxidizing conditions of the ER may alter the disulfide structure of PEK or an associated cofactor, leading to an active protein conformation and enhanced autophosphorylation. The second hypothesis we consider is that ER stress is monitored by an ER resident protein, such as the chaperone GRP78/BiP, that associates with the amino terminus of PEK, maintaining it in an inactive conformation. During ER stress, GRP78 may bind to unfolded proteins that accumulates in the lumen of the ER, freeing PEK to oligomerize and trans- autophosphorylate. We also explore the possibility that proteins known to regulate the related eIF-2alpha kinase, PKR, that is involved in an antiviral defense pathway, also controls PEK function. In this fashion there would be overlapping regulatory mechanisms between these two translational control systems. The second question we will address concerns whether PEK phosphorylation of eIF-2alpha uniformly reduces protein synthesis. During ER stress there is increased transcriptional expression of many ER proteins that serve to remedy stress- mediated protein misfolding. How are these proteins expressed at elevated levels during a general reduction in translation? To answer these questions, we propose four specific aims: 1) Characterization of PEK sequences involved in the activation of eIF-2alpha kinase activity during ER stress; 2) Identification and characterization of regulatory proteins interacting with PEK; 3) Characterization of PEK control of protein synthesis in response to ER stress; and 4) Characterize the control of PEK activity by known PKR regulatory proteins. Together, these studies will further our understanding of the mechanisms regulating general and gene-specific protein synthesis during ER stress.

响应不同的应力，真核细胞通过真核翻译起始因子2（EIF-2）的α亚基的磷酸化大大降低蛋白质的合成。最近，我们从大鼠胰腺中确定了一种新的EIF-2Alpha激酶。新的蛋白激酶指定的胰腺EIF-2Alpha激酶PEK是一种ER跨膜蛋白，它因响应于ER胁迫而被激活，而ER胁迫会损害该细胞器中的蛋白质折叠。 PEK在所有检查的组织中表达，分泌组织中最高水平。 提出了ER管腔中的PEK序列，以感知ER应力，从而引起PEK的构象变化，从而刺激EIF-2Alpha的磷酸化。减少的蛋白质合成为细胞提供了弥补蛋白质错误折叠的机会，然后再将新合成的蛋白质引入分泌途径。 本提案将解决两个基本问题。 首先，我们将解决针对核心应力的调节PEK的机制。 我们考虑了调节PEK活性的两个假设。 首先，应力的ER可以直接修改PEK或相关的辅因子。例如，ER的氧化条件的变化可能会改变佩克或相关的辅助因子的二硫结构，从而导致活性蛋白质构象并增强自磷酸化。 我们认为的第二个假设是，ER应力是由ER常驻蛋白（例如伴侣GRP78/BIP）​​与PEK的氨基末端相关联的，以无效的构象保持其。 在ER应力期间，GRP78可能与积累在ER的腔内的展开的蛋白质结合，从而释放PEK以寡聚和反式自磷酸化。 我们还探讨了与抗病毒防御途径有关的已知蛋白质蛋白质PKR的可能性，也可以控制PEK功能。 以这种方式，这两个翻译控制系统之间将存在重叠的调节机制。 第二个问题我们将解决EIF-2Alpha的PEK磷酸化是否会统一减少蛋白质的合成。在ER应力期间，许多ER蛋白的转录表达增加，这些蛋白用于弥补抗应激介导的蛋白质折叠率的折叠。 这些蛋白质在翻译的总体降低期间如何以较高的水平表达？ 为了回答这些问题，我们提出了四个具体目的：1）在ER应力期间，参与EIF-2Alpha激酶活性激活的PEK序列的表征； 2）鉴定和表征与PEK相互作用的调节蛋白； 3）佩克对蛋白质合成的控制响应于ER应激； 4）表征已知的PKR调节蛋白对PEK活性的控制。 这些研究总之将进一步理解ER应激过程中调节一般和基因特异性蛋白质合成的机制。