AKAP Integration of Phosphorylation and Ubiquitin Signaling

项目总结 每一次心跳都涉及局部cAMP和钙信号事件的协调，这些信号事件导致心肌细胞收缩 心肌细胞。这种反复发生的过程需要细胞质中的钙离子通过 肌浆/内质网钙三磷酸腺苷酶(SERCA2)。SERCA2蛋白的病理生理变化 心肌梗死后的表达与泛素介导的蛋白酶体降解有关 频道。因此，从SERCA2中去除泛素的分子机制有可能是 保护心脏。斯科特实验室发现了一种含有这种蛋白质酶的大分子复合体。 磷酸化和蛋白质泛素化机制。这个新的信令单元可以调制SERCA2水平 肌浆网促进健康的心肌细胞功能。对这些问题进行更详细的调查 有证据表明，三分之一的死亡是由心血管疾病引起的，这是分子和细胞研究的结果。 在美国。 我的初步发现表明，A-激酶锚定蛋白AKAP18隔离了cAMP依赖的 蛋白激酶(PKA)和泛素特异蛋白酶USP4与SERCA2。转基因细胞的初步研究 提示锚定的PKA磷酸化USP4以刺激其脱泛素酶活性。这导致了一种工作 假设这两种酶，当与AKAP18相关时，协同作用，防止泛素- 介导SERCA2从肌浆网中移除。这一令人兴奋的新心脏保护范例将 通过两个目标的实验方法进行测试。目标1将使用最先进的生化和生物物理 研究USP4-AKAP18-PKA复合体分子组织的方法。机械学研究 在Aim 2中，将结合先进的成像和体内遗传策略来了解这些基因是如何锚定的 酶协调控制心脏生理的重要方面，如钙的处理。 我的研究将在华盛顿大学药理学系进行。这 环境为学术轨道的博士后研究人员提供了极好的培训。培训的好处包括 部门内部和部门之间的强大合作，平易近人的主要调查人员进行创新 工作，以及来自多个部门的频繁研讨会，有各自领域的世界专家参加。 此外，这些研究的培训潜力很高。我要学习尖端扩展显微成像技术 技术，并将被引入到测量活的心肌细胞中的钙瞬变。

PROJECT SUMMARY Every heartbeat involves the coordination of local cAMP and Ca2+ signaling events that elicit the contraction of cardiomyocytes. This recurrent process necessitates the reuptake of cytoplasmic Ca2+ through the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA2). Pathophysiological changes in SERCA2 protein expression following myocardial infarction have been linked to ubiquitin-mediated proteasomal degradation of the channel. Thus, molecular mechanisms that remove ubiquitin from SERCA2 have the potential to be cardioprotective. The Scott lab have discovered a macromolecular complex containing enzymes of the protein phosphorylation and protein ubiquitination machinery. This new signaling unit may modulate SERCA2 levels at the sarcoplasmic reticulum to facilitate healthy cardiomyocyte function. A more detailed investigation of these molecular and cellular findings is mandated by evidence that cardiovascular diseases account for 1 in 3 deaths in the US. My preliminary findings show that the A-Kinase Anchoring protein AKAP18 sequesters the cAMP-dependent protein kinase (PKA) and the ubiquitin-specific proteinase USP4 with SERCA2. Pilot studies in transfected cells suggest that anchored PKA phosphorylates USP4 to stimulate its deubiquitinase activity. This led to a working hypothesis that both enzymes, when associated with AKAP18, act cooperatively to protect against ubiquitin- mediated removal of SERCA2 from the sarcoplasmic reticulum. This exciting new cardioprotective paradigm will be tested by an experimental approach of two aims. Aim 1 will use state of the art biochemical and biophysical approaches to investigate the molecular organization of the USP4-AKAP18-PKA complex. Mechanistic studies in aim 2 will combine advanced imaging and in vivo genetic strategies to understand how these anchored enzymes coordinately control vital aspects of cardiac physiology, such as calcium handling. My research will be conducted at the University of Washington in the Department of Pharmacology. This environment provides excellent training for academic-track postdoctoral researchers. Training benefits include strong collaboration within and among departments, approachable principal investigators performing innovative work, and frequent seminars from multiple departments featuring world experts in their respective fields. Moreover, the training potential of these studies is high. I will learn cutting edge expansion microscopy imaging techniques and will be introduced to measurement of calcium transients in live cardiomyocytes.