MAPK signaling: gates, oscillators and circadian timing

项目总结/摘要 事实上，人类生理和行为的每个方面都受到固有的24小时（昼夜节律）的调节。 定时过程在这个时钟计时系统的中心是视交叉上核（SCN）的 下丘脑SCN时钟的一个关键特征是MAPK的紧密的、一天中的时间依赖性调节。 (p44/42丝裂原活化蛋白激酶）途径。这一现象的两个例子是， MAPK通路激活状态的振荡，以及光信号的时钟门控调节， 路径的响应性。重要的是，MAPK的时钟产生的，时间限定的，调节 信号传导似乎在SCN定时和夹带中起中心作用。此外，MAPK的每日门控 信号传导可能是所有振荡器群体的潜在设计原则，因此，MAPK节律 可能会对一系列生理过程产生深远的影响。考虑到这些影响， 令人惊讶的是，我们仍然对细胞机制和突触回路知之甚少， 赋予对MAPK活性的昼夜节律控制。在这里，我们假设MAPK的昼夜调节 信号传导是SCN细胞振荡器的固有（细胞自主）特征，并且这种MAPK节律是一种关键的 生物钟调节基本和复杂生理状态的机制构建块。测试 根据这一假设，我们提出了以下一组实验目标。在目标1中，我们将识别细胞和 SCN的网络特性引起MAPK途径的节律调节。为此，委员会建议， 我们将，A）确定MAPK节律是否是细胞自主的，或者它们是否是由一个细胞周期引起的。 细胞间SCN网络，和B）确定产生MAPK活性的细胞内信号传导事件 节奏在目标2中，我们提出了分子，细胞和系统为基础的机制， SCN时钟门控光诱发的MAPK途径激活。为了解决这一基本上未被探索的现象， 我们将，A）确定分子门何时以及如何打开，和B）测试细胞质ERK是否 支架蛋白PEA-15充当MAPK信号传导的主要昼夜节律门。值得注意的是，我们最近 鉴定PEA-15作为SCN中MAPK信号传导的调节剂，及其动态调节ERK的能力 信号转导使其成为MAPK信号转导门控的有吸引力的候选者。在目标3中，我们建议采用 选择性靶向方法以转基因方式破坏SCN核心和外壳区域内的MAPK信号传导 为了阐明MAPK信号传导在A）昼夜节律的产生和B） 生物钟此外，将对条件PEA-15 KO和点突变PEA-15转基因小鼠系进行筛选。 用于测试PEA-15磷酸化导致ERK快速解离的模型，我们认为这是一种新的方法。 光诱发相移的关键步骤。总之，这些数据将提供基本的新的 深入了解MAPK信号传导和生物钟之间的关系，并指出潜在的方式， 其中，时钟门控MAPK信号转导的失调可能导致CNS的紊乱。

Project Summary/Abstract Virtually every aspect of human physiology and behavior is modulated by an inherent 24 hour (circadian) timing process. At the center of this clock timing system is the suprachiasmatic nucleus (SCN) of the hypothalamus. A key feature of the SCN clock is the tight, time-of-day, dependent regulation of the MAPK (p44/42 mitogen-activated protein kinase) pathway. Two examples of this phenomenon are the daily oscillations in the activation state of the MAPK pathway, and the clock-gated regulation of the photic responsiveness of the pathway. Importantly, the clock-generated, temporally-delimited, regulation of MAPK signaling appears to play a central role in SCN timing and entrainment. Further, the daily gating of MAPK signaling may be an underlying design principal of all oscillator populations, and as such, MAPK rhythms could have profound and far-reaching effects on a range of physiological processes. Given these implications, it is surprising that we still know relatively little about the cellular mechanisms and synaptic circuits that confer circadian control over MAPK activity. Here, we hypothesize that the circadian regulation of MAPK signaling is an inherent (cell autonomous) feature of SCN cellular oscillators and that this MAPK rhythm is a key mechanistic building-block by which the circadian clock modulates both basic and complex physiological states. To test this hypothesis, we propose the following set of experimental goals. In Aim 1, we will identify the cellular and network properties of the SCN that give rise to the rhythmic regulation of the MAPK pathway. To this end, we will, A) Determine whether MAPK rhythms are cell autonomous or whether they result from an intercellular SCN network, and B) Determine the intracellular signaling events that generate MAPK activity rhythms. In Aim 2 we propose to characterize the molecular, cellular and systems-based mechanisms by which the SCN clock gates light-evoked MAPK pathway activation. To address this largely unexplored phenomenon, we will, A) determine when and how the molecular gate opens, and B), test whether the cytoplasmic ERK scaffold protein PEA-15 serves as the principal circadian gate on MAPK signaling. Of note, we recently identified PEA-15 as a modulator of MAPK signaling in the SCN, and its capacity to dynamically regulate ERK signaling makes it an attractive candidate for the gating of MAPK signaling. In Aim 3 we propose to employ a selective targeting approach to transgenically disrupt MAPK signaling within the SCN core and shell regions to address the roles of MAPK signaling in A) the generation of circadian rhythms, and B) the entrainment of the circadian clock. Further, conditional PEA-15 KO and point mutant PEA-15 transgenic mouse lines will be used to test a model in which PEA-15 phosphorylation leads to rapid ERK dissociation, which we posit to be a key step in the initiation of light-evoked phase-shifting. Together, these data will provide fundamental new insights into the relationship between MAPK signaling and the circadian clock, and point to potential ways in which the dysregulation of clock-gated MAPK signaling could contribute to disorders of the CNS.