MAKAP FUNCTIONS OF A PKA ANCHORING PROTEIN

该提案包括为目前在俄勒冈健康科学大学Vollum研究所从事研究的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。卡皮洛夫博士正在为约翰·D·斯科特博士的一段时间的培训寻求资金。斯科特博士的指导应该会让卡皮洛夫成功地过渡到独立首席研究员的角色，研究心脏信号转导的调节。细胞外刺激的信号转导部分是通过细胞内蛋白的磷酸化进行的。在信号转导领域的一个基本问题是，面对这些酶能够磷酸化的许多可能的底物，蛋白激酶的信号是如何连贯的。信号的特异性在一定程度上是通过蛋白激酶的空间分离来维持的。在分子水平上，以细胞内结构为靶点的蛋白是由锚定蛋白介导的。这一概念在心脏是相关的，cAMP介导了β-肾上腺素能刺激在调节变离子、变时和肥厚中的作用。我最近克隆了一种名为mAKAP的A激酶锚定蛋白，它将cAMP依赖的蛋白激酶(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，使其附近的底物选择性磷酸化。我已经证明，mAKAP是通过包含与dystrophin和Spectrin中的重复序列类似的重复序列来靶向NM的。在特定的目标1中，我们建议确定什么蛋白(S)与mAKAP靶向结构域结合在NM。此外，我们建议确定其他可能构成功能复合体的mAKAP结合伙伴。在特定的目标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。在特定的目标3中，我们将讨论人类mAKAP基因突变导致心脏疾病的可能性，该疾病与其有遗传联系，即致心律失常的右室发育不良。与路易莎·梅斯特罗尼博士合作，我们正在调查mAKAP的突变是否会对心脏核功能产生直接影响，从而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.