MAKAP FUNCTIONS OF A PKA ANCHORING PROTEIN

该提案包括为儿科医生和分子生物学家Michael Kapiloff博士申请临床科学家发展奖，他目前在俄勒冈州健康科学大学Vollum研究所进行研究。 Kapiloff博士正在寻求资金，以便在John D. Scott. Scott博士的指导应该使Kapiloff博士成功地过渡到研究心脏信号转导调节的独立首席研究员的角色。细胞外刺激的信号转导部分通过细胞内蛋白质的磷酸化发生。 在信号转导领域的一个基本问题是如何通过蛋白激酶的信号是一致的，在面对许多可能的底物，这些酶能够磷酸化。信号传导的特异性部分地通过蛋白激酶的空间分离来维持。 在分子水平上，激酶靶向细胞内结构是由锚定蛋白介导的。 这个概念在心脏中是相关的，其中cAMP介导β-肾上腺素能刺激在离子变性、变时性和肥大调节中的作用。 我最近克隆了一种称为mAKAP的A激酶锚定蛋白，它将cAMP依赖性蛋白激酶（PKA）靶向分化的心肌细胞的核膜（NM）。 我们认为mAKAP的功能是将PKA定位于NM，在NM中激酶被定位以选择性地磷酸化附近的底物。 我已经表明，mAKAP是通过含有类似于肌营养不良蛋白和血影蛋白中的重复序列的结构域靶向NM的。 在具体目标1中，我们提出确定在NM处结合mAKAP靶向结构域的蛋白质。此外，我们建议确定其他mAKAP结合伙伴，可能构成一个功能复杂。 在具体目标2中，我们建议测试mAKAP在cAMP和钙调节的转录因子激活和基因表达中的作用。 在第3个具体目标中，我们将讨论人类mAKAP基因在心脏疾病中突变的可能性，该基因与心脏疾病有遗传连锁，即致心律失常性右心室发育不良。 与Luisa Mestroni博士合作，我们正在研究mAKAP的突变是否会对心脏核功能产生直接影响，是否会导致可能导致心肌病和猝死的临床疾病。

This proposal comprises an application for a Clinical Scientist Development Award for Dr. Michael Kapiloff, a pediatrician and molecular biologist, currently performing research at the Vollum Institute of the Oregon Health Sciences University. Dr. Kapiloff is seeking funding for a period of training under Dr. John D. Scott. Mentoring by Dr. Scott should allow Dr. Kapiloff to transition successfully to the role of an independent principal investigator who studies the regulation of signal transduction in the heart. Signal transduction of extracellular stimuli occurs in part through the phosphorylation of intracellular proteins. A fundamental question in the field of signal transduction is how signaling by protein kinases is coherent in the face of many possible substrates that these enzymes are able to phosphorylate. Specificity in signaling is maintained in part by the spatial segregation of protein kinases. At the molecular level, targeting of kinases to intracellular structures is mediated by anchoring proteins. This concept is pertinent in the heart where cAMP mediates the effects of beta-adrenergic stimuli in regulation of ionotropy, chronotropy, and hypertrophy. I have recently cloned an A kinase anchoring protein called mAKAP that targets the cAMP-dependent protein kinase (PKA) to the nuclear membrane (NM) of differentiated cardiac myocytes. We propose that mAKAP functions to direct the localization of PKA to the NM where the kinase is positioned to selectively phosphorylate substrates in that vicinity. I have shown that mAKAP is targeted to the NM by a domain containing repeat sequences similar to those in dystrophin and spectrin. In Specific Aim number 1, we propose to determine what protein(s) binds the mAKAP targeting domain at the NM. Further, we propose to identify other mAKAP binding-partners which might constitute a functional complex. In Specific Aim number 2, we propose to test the role of mAKAP in cAMP and calcium-regulated transcription factor activation and gene expression. In Specific Aim number 3, we will address the possibility that the human mAKAP gene is mutated in a cardiac disorder to which it shares genetic linkage, arrhythmogenic right ventricular dysplasia. In collaboration with Dr. Luisa Mestroni, we are investigating whether mutations of mAKAP, which is poised to have a direct impact on cardiac nuclear function, are responsible for a clinical disorder which can result in cardiomyopathy and sudden death.