mAKAP-orchestrated phosphorylation events: regulation of PDE4D3

说明（申请人提供）：cAMP是一种重要的第二信使，调节心脏中的几种生理事件，包括收缩、代谢和基因转录。最近的几项研究表明，磷酸二酯酶（PDE）不仅对控制心脏中的基础cAMP水平非常重要，而且由于PDE的细胞定位，还对cAMP的局部变化进行了调节。因此，确定决定PDE定位和活性的分子决定因素，以及归因于每个PDE的生物学过程，将进一步加深我们对心脏中cAMP信号传导的了解。我们以前已经描述了一个支架蛋白，隔离磷酸二酯酶PDE 4D 3的核膜在分化的心肌细胞。A-激酶转运蛋白mAKAP维持由cAMP依赖性蛋白激酶、PDE 4D 3、Map激酶ERK 5、鸟嘌呤核苷酸交换因子Epac、蛋白磷酸酶2A和Ryanodine受体组成的复合物。先前的数据显示mAKAP协调了ERK 5和PKA对PDE 4D 3的调节。这些磷酸化诱导的PDE 4D 3活性的变化将反过来调节复合物周围的cAMP浓度，最终调节复合物中的所有cAMP效应物，即PKA、PDE 4D 3和Epac。本提案的三个具体目标将尝试：1）阐明调节PDE 4D 3磷酸化的生理事件，调节cAMP浓度和Ryanodine受体的磷酸化状态; 2）确定PDE 4D 3磷酸化对Epac介导的ERK 5活性和ERK 5介导的心脏肥大的功能后果; 3）鉴定复合物中控制PDE去磷酸化的磷酸酶。心脏病是美国的头号死因。心脏肥大是心脏对持续增加的心输出量需求作出反应的主要代偿机制。最终，心脏肥大导致进展为心脏病和心力衰竭。然而，一些研究表明，抑制心脏肥大可降低死亡和进展为心力衰竭的风险。这项研究将进一步加深我们对心肌肥厚分子机制的理解，并有助于确定新的药物设计靶点，以帮助治疗心肌肥厚。

DESCRIPTION (provided by applicant): cAMP is an important second messenger that regulates several physiological events in the heart including contraction, metabolism and gene transcription. Several recent studies have illustrated not only the importance of phosphodiesterases (PDE) for the control of basal cAMP levels in the heart, but also in the regulation of localized changes of cAMP due to the cellular location of PDEs. Therefore, identifying the molecular determinants governing PDE localization and activity, as well as the biological processes attributed to each PDE, will further our knowledge of cAMP signaling in the heart. We have previously described a scaffolding protein that sequesters the phosphodiesterase PDE4D3 to the nuclear envelope in differentiated cardiac myocytes. The A-Kinase Anchoring Protein mAKAP maintains a complex consisting of the cAMP-dependent protein kinase, PDE4D3, the Map Kinase ERK5, the guanine nucleotide exchange factor Epac, the protein phosphatase 2A, and the Ryanodine Receptor. Previous data has shown mAKAP orchestrated both ERK5 and PKA regulation of PDE4D3. These phosphorylation-induced changes in PDE4D3 activity will in turn modulate the concentration of cAMP surrounding the complex, ultimately regulating all cAMP effectors in the complex, namely PKA, PDE4D3 and Epac. The three Specific Aims of this proposal will try to 1) elucidate the physiological events that regulate PDE4D3 phosphorylation regulate cAMP concentration and the phosphorylation state of the Ryanodine Receptor 2) determine the functional consequence of PDE4D3 phosphorylation on Epac-mediated ERK5 activity and ERK5-mediated cardiac hypertrophy, and 3) identify the phosphatase in the complex that governs dephosphorylation of the PDE. Heart disease is the number one cause of death in the United States. Cardiac hypertrophy is the major compensatory mechanism by which the heart responds to a continued, increased demand for cardiac output. Ultimately, cardiac hypertrophy leads to progression into cardiac disease and to heart failure. However, several studies have shown that inhibition of cardiac hypertrophy lowers the risk of death and progression into heart failure. This study will further our understanding of the molecular mechanism of cardiac hypertrophy and help to identify novel targets for drug design to aid in the treatment of hypertrophy.