Role of the mAKAP Complex in Cardiac Hypertrophy

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，心脏主要通过心肌细胞肥大来适应慢性应激。心肌肥大是由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信号通路诱导的。mAKAP信号复合体包含蛋白激酶A、磷酸二酯酶、蛋白磷酸酶和ryanodine受体Ca2+激活的Ca2+通道。在这个应用中，我们发现mAKAP也以一种受调节的方式结合转录因子NFATc1。此外，新的数据显示，将mAKAP复合物从其正常位置的核膜移位和抑制ryanodine受体将阻止细胞因子激动剂诱导心肌细胞肥大。因此，我们提出了一个模型，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磷酸酶的Ca2+来促进NFATc1的去磷酸化和激活。去磷酸化的NFATc因子会转移到细胞核并反激活与肥大有关的基因。在钙调磷酸酶激活后，NFATc1还会被招募到mAKAP复合体中，在那里它可能被复合体中的蛋白激酶A再磷酸化。NFATc1与mAKAP的关联可能构成了一种机制，通过这种机制可以减弱肥厚的诱导，防止无限制的肥厚。在本应用中，提出了三个具体目标来测试该模型的元素，并研究mAKAP复合体对NFATc1可能的负调控。在Specific Aim #1中，蛋白激酶A、磷酸二酯酶4D3、ryanodine受体和NFATc1与mAKAP复合物结合的功能重要性在原代肌细胞培养中通过表达缺乏单个成分结合位点的mAKAP形式、通过RNA干扰、通过评估肌细胞生长、肥厚基因表达和NFATc1活性的诱导来检查。在Specific Aim #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Specific Aim #3中，我们建议检查makap相关的NFATc1是否被去磷酸化，以及NFATc1是否是复合物中蛋白激酶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.