Translational Control by elF-2 Kinase during ER Stress

在不同的应激反应中，真核细胞通过真核翻译起始因子2（eIF- 2）的α亚基的磷酸化显著减少蛋白质合成。最近，我们从大鼠胰腺中鉴定了一种新的eIF-2 α激酶。新的蛋白激酶命名为胰腺eIF-2 α激酶，PEK，是一种ER跨膜蛋白，其响应于损害该细胞器中蛋白质折叠的ER应激而被激活。 PEK在检查的所有组织中表达，在分泌组织中表达水平最高。 ER腔中的PEK序列被认为可以感知ER应激，引起PEK的构象变化，从而刺激eIF-2 α的磷酸化。减少的蛋白质合成为细胞提供了在将新合成的蛋白质引入分泌途径之前补救蛋白质错误折叠的机会。 本提案将涉及两个基本问题。 首先，我们将解决的机制，调节PEK响应ER压力。 我们考虑两个假设PEK活性的调节。 首先，应激的ER可以直接修饰PEK或相关的辅因子。例如，ER氧化条件的变化可能改变PEK或相关辅因子的二硫键结构，导致活性蛋白构象和增强的自磷酸化。 我们考虑的第二个假设是，ER应激是由ER常驻蛋白，如伴侣GRP 78/BiP，与PEK的氨基末端，保持它在一个非活性构象相关联的监测。 在ER应激期间，GRP 78可以结合在ER腔中积累的未折叠蛋白，释放PEK以寡聚化和反式自磷酸化。 我们还探讨了已知调节相关eIF-2 α激酶（PKR）的蛋白质也控制PEK功能的可能性，PKR参与抗病毒防御途径。 以这种方式，在这两个翻译控制系统之间存在重叠的调节机制。 第二个问题是，PEK对eIF-2 α的磷酸化是否会均匀地减少蛋白质的合成。在ER应激期间，许多ER蛋白的转录表达增加，其用于补救应激介导的蛋白质错误折叠。 这些蛋白质是如何在翻译普遍减少的过程中以升高的水平表达的？ 为了回答这些问题，我们提出了四个具体的目标：1）表征PEK序列参与激活的eIF-2 α激酶活性在ER压力; 2）识别和表征的调节蛋白与PEK相互作用; 3）表征PEK控制蛋白质合成的响应ER压力;和4）表征PEK活性的控制已知PKR调节蛋白。 总之，这些研究将进一步加深我们对ER应激过程中调节一般和基因特异性蛋白质合成的机制的理解。

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.