Role of the mAKAP Complex in Cardiac Hypertrophy

mAKAP 复合物在心脏肥大中的作用

• 批准号: 6832758
• 负责人: Michael Seth Kapiloff
• 金额: $ 30.2万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-12-08 至 2008-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6832758
• 关键词: A kinase anchoring protein; RNA interference; beta adrenergic agent; biological signal transduction; calcium channel; gene expression; immunoprecipitation; laboratory rat; muscle cells; newborn animals; phosphodiesterases; phosphorylation; protein kinase A; protein localization; protein protein interaction; protein structure function; ryanodine; tissue /cell culture; transcription factor; transfection; ventricular hypertrophy; western blottings

DESCRIPTION (provided by applicant): Cardiovascular disease is the major cause of death in the United States. Regardless of the etiology the heart compensates mainly through myocyte hypertrophy in adaptation to chronic stress. Cardiac hypertrophy is induced by signaling through intracellular pathways composed of diffusable second messenger molecules such as cAMP and Ca2+, soluble enzymes, and anchored signaling complexes. The mAKAP signaling complex contains protein kinase A, a phosphodiesterase, a protein phosphatase, and the ryanodine receptor Ca2+-activated, Ca2+ channel. In this application we show that mAKAP also binds in a regulated manner the transcription factor NFATc1. Further, new data reveals that displacement of the mAKAP complex from its normal location at the nuclear envelope and inhibition of ryanodine receptors will block the induction of myocyte hypertrophy by cytokine agonists. We, therefore, propose a model in which upon agonist stimulation, ryanodine receptors in the mAKAP complex contribute to the de-phosphorylation and activation of NFATc1 by releasing Ca2+ that will activate the phosphatase calcineurin. De-phosphorylated NFATc factors will translocate to the nucleus and transactivate genes involved in hypertrophy. Upon calcineurin activation, NFATc1 will, in addition, be recruited into the mAKAP complex, where it may be rephosphorylated by protein kinase A in the complex. NFATc1 association with mAKAP may constitute a mechanism by which the induction of hypertrophy can be attenuated, preventing unrestrained hypertrophy. In this application three Specific Aims are proposed that test elements of this model and investigate the possible negative regulation of NFATc1 by the mAKAP complex. In Specific Aim #1 the functional importance of protein kinase A, phosphodiesterase 4D3, ryanodine receptor and NFATc1 binding to the mAKAP complex is examined in primary myocyte cultures by expression of mAKAP forms that lack binding sites for individual components, by RNA interference, and by assessing the induction of myocyte growth, hypertrophic gene expression, and NFATc1 activity. In Specific Aim #2, the composition of the NFATc1 bound mAKAP complex will be established and the binding domains permitting the mAKAP and NFATc1 interaction mapped. In Specific Aim #3 we propose to examine whether mAKAP-associated NFATc1 is dephosphorylated and whether NFATc1 is a substrate for protein kinase A in the complex.

描述（由申请人提供）：心血管疾病是美国的主要死亡原因。无论病因如何，心脏主要通过肌细胞肥大来适应慢性应激进行代偿。心脏肥大是通过细胞内途径的信号诱导的，该途径由可扩散的第二信使分子（例如 cAMP 和 Ca2+）、可溶性酶和锚定信号复合物组成。 mAKAP 信号复合物包含蛋白激酶 A、磷酸二酯酶、蛋白磷酸酶和兰尼碱受体 Ca2+ 激活的 Ca2+ 通道。在此应用中，我们证明 mAKAP 还以受调控的方式结合转录因子 NFATc1。此外，新数据表明，mAKAP 复合物从核膜正常位置的移位和兰尼碱受体的抑制将阻止细胞因子激动剂诱导心肌细胞肥大。因此，我们提出了一种模型，其中在激动剂刺激下，mAKAP 复合物中的兰尼碱受体通过释放 Ca2+ 来激活磷酸酶钙调神经磷酸酶，从而促进 NFATc1 的去磷酸化和激活。去磷酸化的 NFATc 因子将易位至细胞核并反式激活参与肥大的基因。此外，钙调神经磷酸酶激活后，NFATc1 将被招募到 mAKAP 复合体中，并可能被复合体中的蛋白激酶 A 重新磷酸化。 NFATc1 与 mAKAP 的结合可能构成一种机制，通过该机制可以减弱肥大的诱导，从而防止无限制的肥大。在本申请中，提出了三个具体目标来测试该模型的元素并研究 mAKAP 复合物对 NFATc1 可能的负调节。在具体目标 #1 中，通过缺乏单个成分结合位点的 mAKAP 形式的表达、RNA 干扰和评估心肌细胞生长的诱导、肥大基因表达和 NFATc1 活性。在具体目标 #2 中，将建立 NFATc1 结合的 mAKAP 复合物的组成，并映射允许 mAKAP 和 NFATc1 相互作用的结合域。在具体目标#3中，我们建议检查 mAKAP 相关的 NFATc1 是否去磷酸化以及 NFATc1 是否是复合物中蛋白激酶 A 的底物。