Translational Control by elF-2 Kinase during ER Stress

在不同的胁迫下，真核细胞通过真核翻译起始因子2 (eIF- 2) α亚基的磷酸化显著减少蛋白质的合成。最近，我们从大鼠胰腺中发现了一种新的eif -2 α激酶。这种新的蛋白激酶被称为胰腺eif -2 α激酶（PEK），是一种内质网跨膜蛋白，在内质网应激损害该细胞器中的蛋白质折叠时被激活。PEK在所有组织中均有表达，在分泌组织中表达水平最高。内质网管中的PEK序列被认为可以感知内质网应激，引发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在将新合成的蛋白质引入分泌途径之前纠正蛋白质错误折叠的机会。这项建议将处理两个基本问题。首先，我们将探讨PEK在内质网应激下的调节机制。我们考虑PEK活性调控的两种假设。首先，受压的内质网可以直接修饰PEK或相关的辅助因子。例如，内质网氧化条件的改变可能改变PEK或相关辅因子的二硫结构，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内质网应激是由内质网驻留蛋白监测的，如伴侣蛋白GRP78/BiP，它与PEK的氨基末端相关联，使其保持非活性构象。在内质网应激期间，GRP78可能与内质网管腔中积累的未折叠蛋白结合，释放PEK寡聚和反式自磷酸化。我们还探索了已知调节相关eif -2 α激酶PKR的蛋白质的可能性，PKR参与抗病毒防御途径，也控制PEK功能。在这种情况下，这两个转译控制系统之间会有重叠的调节机制。我们将解决的第二个问题是PEK磷酸化eIF-2alpha是否会均匀地减少蛋白质合成。在内质网应激期间，许多内质网蛋白的转录表达增加，这些蛋白用于补救应激介导的蛋白质错误折叠。在翻译普遍减少的过程中，这些蛋白是如何以高水平表达的？为了回答这些问题，我们提出了四个具体目标：1)表征内质网应激下参与eif - 2α激酶活性激活的PEK序列；2)与PEK相互作用的调控蛋白的鉴定与表征；3)内质网胁迫下PEK调控蛋白合成的研究；4)表征已知PKR调控蛋白对PEK活性的控制。总之，这些研究将进一步加深我们对内质网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.