Role of the mAKAP Complex in Cardiac Hypertrophy

描述(申请人提供)：心血管疾病是美国的主要死因。无论病因如何，心脏主要通过心肌细胞肥大来适应慢性应激。心肌肥厚是通过细胞内信号通路诱导的，细胞内信号通路由cAMP和Ca~(2+)等可扩散的第二信使分子、可溶性酶和锚定信号复合体组成。MAKAP信号复合体含有蛋白激酶A、磷酸二酯酶、蛋白磷酸酶和兰尼定受体钙激活的钙通道。在本申请中，我们证明了mAKAP也以一种受调控的方式与转录因子NFATc1结合。此外，新的数据显示，mAKAP复合体从其在核膜上的正常位置移位和兰尼定受体的抑制将阻断细胞因子激动剂诱导的心肌细胞肥大。因此，我们提出了一个模型，在该模型中，在激动剂刺激下，mAKAP复合体中的ryanodine受体通过释放激活磷酸酶钙调神经磷酸酶(Calcineurin)的钙来促进NFATc1的去磷酸化和激活。去磷酸化的NFATc因子将移位到细胞核并反式激活与肥大相关的基因。一旦钙调神经磷酸酶激活，NFATc1将被招募到mAKAP复合体中，在那里它可能被复合体中的蛋白激酶A重新磷酸化。NFATc1与mAKAP的结合可能构成了一种机制，通过该机制可以减弱诱导的肥厚，防止无限制的肥厚。在这一应用中，提出了三个具体的目标，以检验该模型的元件，并研究mAKAP复合体可能对NFATc1的负调控。在特定目的#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，并通过评估对心肌细胞生长、肥大基因表达和NFATc1活性的诱导，在原代培养的心肌细胞中检测蛋白激酶A、磷酸二酯酶4D3、兰诺定受体和NFATc1结合的功能重要性。在特定目标#2中，将建立与NFATc1结合的mAKAP复合体的组成，并绘制允许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是否被去磷酸化，以及NFATc1是否是复合体中蛋白激酶A的底物。

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.