Keratin intermediate filaments: a dynamic framework for major signaling molecules involved in epithelial cell survival and cell death

中间丝（IFs）与微管和肌动蛋白微丝是大多数哺乳动物细胞的主要细胞骨架组成部分。角蛋白是在上皮细胞中表达的IF蛋白。一种基因靶向的方法已被必要的解开角蛋白8和18在肝细胞，肝的上皮细胞的作用。例如，来自携带破坏IF网络结构或影响其磷酸化模式的K8或K18突变的小鼠的肝细胞，以及K8或K18缺失小鼠对肝毒性剂诱导的细胞凋亡高度敏感。因此，K8/18被认为在保护上皮细胞免受毒物诱导的应激中起重要作用。K8/18实现其保护作用的机制远未完全了解。* 我们研究计划的长期目标是揭示角蛋白有助于保护上皮细胞免受毒性应激的关键机制。我们假设它们在参与细胞存活和细胞死亡的主要信号通路的调节中发挥直接作用。本提案的短期目标是：1）通过使用体外模型和分子功能方法，确定K8/18如何与Akt亚型和NF-kB相互作用，这些亚型和NF-kB是许多存活过程的关键成员。我们假设角蛋白通过直接相互作用和可能的隔离在这些关键调节蛋白的细胞定位中发挥作用。2)我们将确定如何调节K8/18磷酸化，翻译后修饰已知保护上皮细胞免受毒性应激，调节信号通路的活性。我们推测K8/18磷酸化的特异性修饰将调节Akt亚型的活性。我们还将通过阐明每种Akt亚型对WT K8/K18磷酸化和动力学的影响来确定Akt是否在K8/18磷酸化中发挥作用。这项工作将在不同的上皮细胞系上实现，这些上皮细胞系是根据K8/18的存在及其抗凋亡水平而选择的。这项研究将涉及使用生物化学和分子工具以及共聚焦显微镜分析。* K8/18如何与主要的促存活信号通路如Akt和NF-kB相互作用的表征将构成我们理解角蛋白实现其保护功能的分子机制的重要一步。这将直接导致角蛋白和细胞信号之间的串扰的理解，从而更好地了解角蛋白如何影响疾病的发病机制。**

Intermediate filaments (IFs) are with microtubules and actin microfilaments the major cytoskeletal components of most mammalian cells. Keratins are the IF proteins that are expressed in epithelial cells. A gene-targeting approach has been necessary to unravel the roles of keratin 8 and 18 in hepatocytes, the epithelial cells of the liver. For instance, hepatocytes from mice carrying K8 or K18 mutations that disrupt IF network structure or affect their phosphorylation pattern, and K8- or K18-null mice are highly susceptible to apoptosis induced by hepatotoxic agents. Thus K8/18 are proposed to play an important role in the protection of epithelial cells against stress-induced by toxicants. The mechanisms by which K8/18 accomplish their protective role is far from being fully understood. ***The long-term objective of our research program is to unravel the crucial mechanisms by which keratins contribute to the protection of epithelial cells against toxic stress. We hypotheses that they play a direct role in the regulation of major signaling pathways involved in cell survival and cell death. The short term objective of this proposal is 1) by using in vitro models and molecular functional approaches, to determine how K8/18 interacts with Akt isoforms and NF-kB, key members of many survival processes. We hypotheses that keratins play a role in the cellular localization of these key regulatory proteins through direct interaction and possible sequestration. 2) We will determine how the modulation of K8/18 phosphorylation, a post-translational modification known to protect epithelial cells against toxic stress, regulates the activity of the signaling pathways. We speculate that specific modifications in K8/18 phosphorylation will modulate the activity of Akt isoforms. We will also determine if Akt play a role in K8/18 phosphorylation by unraveling the influence of each Akt isoforms on WT K8/K18 phosphorylation and dynamic. The work will be realized on different epithelial cell lines that are chosen for the presence of K8/18 and their level of resistance to apoptosis. The study will involve the use of biochemical and molecular tools as well as confocal microscopy analysis. ***The characterization of how K8/18 interact with major pro-survival signaling pathway such as Akt and NF-kB will constitute an important step in our understanding of the molecular mechanisms by which keratins accomplish their protective functions. This will directly lead to an understanding of the cross-talk between keratins and cell signaling, and consequently to a better understanding on how keratins affect disease pathogenesis. **