Spatiotemporal Regulation of PKA in Response to beta-Adrenergic Stimulation

描述(申请人提供)：这项研究的总体目标是阐明参与调节cAMP依赖的蛋白激酶(PKA)活性的机制，这些PKA活性可引起对Padrenerable Receptor(P-AR)刺激的高特异性信号转导。鉴于该激酶控制的信号事件的广谱，为了获得选择性和特异性的细胞反应，PKA在正确的时间在正确的细胞内发生适当的磷酸化是至关重要的。因此，心肌细胞中PKA的解除调控通常与心力衰竭有关。研究活细胞PKA活动所需的工具最近已经可用，并将允许有效地探索这些机制。本项目的具体目标1将比较P-AR通过刺激和/或抑制P-AR与受体亚型特异性激动剂和拮抗剂而刺激的PKA活性的时空动力学。具体目标2将试图确定膜微域和空间区划的p-AR通过破坏膜微域结构(通过胆固醇消耗)和监测局部PKA活性来调节PKA动态的作用。具体目标3将试图研究A-激酶锚定蛋白(AKAPs)通过破坏AKAPs，然后监测亚细胞位置的PKA活性，在调节P-AR刺激的PKA动态中所起的作用。这些目标中的每一个都是在心肌细胞中实现的，试图填补目前我们对PKA调控机制的了解空白，PKA调控将受体激活与特定的心输出量(即。心率、收缩力、松弛)。此外，每个目标都需要对PKA活动进行活细胞测量，这将利用基于FRET的探针来时空解析PKA活动。这项研究将对心肌细胞的基本信号机制提供更深入的了解，并有助于心力衰竭的治疗和诊断的发展。

DESCRIPTION (provided by applicant): The overall goal of this research is to elucidate the mechanisms involved in regulating cAMP dependent protein kinase (PKA) activity that give rise to high specificity signaling in response to Padrenergic receptor (P-AR) stimulation. Given the broad spectrum of signaling events that are controlled by this kinase, it is crucial that proper phosphorylation by PKA occur at the right time in the right location within a cell, in order to attain selective and specific cellular responses. As such, deregulation of PKA in cardiomyocytes is often associated with heart failure. The tools needed to study live-cell PKA activity have recently become available and will allow for effective exploration of these mechanisms. Specific aim 1 of this project will compare P-AR stimulated spatiotemporal dynamics of PKA activity by stimulating and/or inhibiting p-ARs with receptor subtype specific agonists and antagonists. Specific aim 2 will attempt to determine the roles that membrane microdomains and spatially compartmentalized p-ARs play in the regulation of PKA dynamics by disrupting membrane microdomain structure (via cholesterol depletion) and monitoring local PKA activity. Specific aim 3 will attempt to investigate the roles that A-kinase anchoring proteins (AKAPs) play in regulating P-AR stimulated PKA dynamics by disrupting AKAPs and then monitoring PKA activity at subcellular locations. Each of these aims is to be carried out in cardiomyocytes in an attempt to fill in the current gaps in our knowledge about mechanisms underlying PKA regulation that links receptor activation to specific cardiac output (ie. heart rate, contraction force, relaxation). Additionally, each aim requires live-cell measurement of PKA activity, which will utilize a FRET-based probe for spatiotemporal resolution of PKA activity. This research should provide more in-depth understanding about basic cardiomyocyte signaling mechanisms and aid in the development of treatment and diagnosis of heart failure.