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。心脏肥大是由通过细胞内途径的信号传导诱导的，所述细胞内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Ca 2+激活的Ca 2+通道。在本申请中，我们表明mAKAP也以受调节的方式结合转录因子NFATc1。此外，新的数据表明，mAKAP复合物从其在核膜的正常位置的位移和ryanodine受体的抑制将阻断细胞因子激动剂诱导的肌细胞肥大。因此，我们提出了一个模型，在激动剂刺激后，mAKAP复合物中的ryanodine受体通过释放Ca2+激活磷酸酶钙调磷酸酶，从而促进NFATc1的去磷酸化和激活。去磷酸化的NFATc因子将易位到细胞核并反式激活参与肥大的基因。在钙调磷酸酶激活后，NFATc1将另外被募集到mAKAP复合物中，在那里它可以被复合物中的蛋白激酶A再磷酸化。NFATc1与mAKAP的相关性可能构成了一种机制，通过该机制可以减弱肥大的诱导，防止不受限制的肥大。在本申请中，提出了三个特定目的，以测试该模型的元素并研究mAKAP复合物对NFATc1的可能负调控。在具体目标#1中，通过表达缺乏单个组分结合位点的mAKAP形式，通过RNA干扰，并通过评估诱导肌细胞生长、肥大基因表达和NFATc1活性，在原代肌细胞培养物中检查蛋白激酶A、磷酸二酯酶4D3、兰尼碱受体和NFATc1与mAKAP复合物结合的功能重要性。在具体目标#2中，将确定NFATc1结合mAKAP复合物的组成，并绘制允许mAKAP和NFATc1相互作用的结合结构域。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。