Role of the mAKAP Complex in Cardiac Hypertrophy

mAKAP 复合物在心脏肥大中的作用

• 批准号: 6710560
• 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/6710560
• 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，心脏都主要通过肌细胞肥大来适应慢性应激。心脏肥大是通过通过可扩散的第二信使分子（如CAMP和CA2+，可溶性酶和锚定信号复合物）组成的细胞内途径来诱导的。 MAKAP信号复合物包含蛋白激酶A，磷酸二酯酶，蛋白质磷酸酶和ryanodine受体CA2+激活的Ca2+通道。在此应用中，我们表明MAKAP还以调节的方式结合转录因子NFATC1。此外，新的数据表明，MAKAP复合物从其在核膜上的正常位置和抑制ryanodine受体的置换将阻止细胞因子激动剂诱导肌细胞肥大。因此，我们提出了一个模型，在这种模型中，在激动剂刺激下，MAKAP复合物中的ryanodine受体通过释放Ca2+来促进NFATC1的去磷酸化和激活，从而激活磷酸酶钙调蛋白。去磷酸化的NFATC因子将转移到核并涉及肥大的基因。钙调蛋白激活后，NFATC1还将募集到Makap复合物中，在该复合物中可能会在络合物中被蛋白激酶A重新磷酸化。 NFATC1与MAKAP的关联可能构成一种机制，通过该机制可以减弱肥大的诱导，从而防止肥大。在此应用程序中，提出了三个特定的目的，该目标是测试该模型的元素，并研究了Makap复合物对NFATC1的负面调节。在特定的目标1中，蛋白激酶A，磷酸二酯酶4D3，ryanodine受体和NFATC1与MAKAP复合物的结合的功能重要性在原代肌细胞培养中通过表达通过RNA干扰的单个组件的结合位点的表达来表达在原代肌细胞培养中，并通过RNA Intivercriention和Entivercy grontic andc grontuction gene gene and and and gene ant and and and and and and and and and and and and ant ant and and and and and and and and and and ant and and and and and and。在特定的目标＃2中，将建立NFATC1结合MAKAP复合物的组成，并允许映射MAKAP和NFATC1相互作用的结合域。在特定的目标＃3中，我们建议检查MAKAP相关的NFATC1是否被脱磷酸化，以及NFATC1是否是络合物中蛋白激酶A的底物。