Defective PKA Signaling in Cushing's Syndrome

库欣综合征中 PKA 信号传导缺陷

• 批准号: 10453810
• 负责人: John D Scott
• 金额: $ 37.85万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-24 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10453810
• 关键词: A kinase anchoring protein; Adrenal Glands; Affect; Articular Range of Motion; Biology; Blood; CRISPR/Cas technology; Ca(2+)-Calmodulin Dependent Protein Kinase; Catalytic Domain; Cells; Chemicals; Complex; Corticotropin; Cushing Syndrome; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Diabetes Mellitus; Diagnosis; Diffusion; Disease; Elderly; Endocrine; Endocrine Diagnostic Techniques; Endocrine System Diseases; Ensure; Enzymes; Etiology; Event; Genes; Holoenzymes; Hormones; Hydrocortisone; Hypertension; Island; Isoenzymes; Lead; Link; Location; Modeling; Molecular; Mutation; Pathologic; Pathology; Pharmacology; Phosphotransferases; Physiology; Pituitary Neoplasms; Pituitary-dependent Cushing' s disease; Process; Production; Second Messenger Systems; Signal Transduction; Signaling Protein; Testing; Weight Gain; in vivo; live cell imaging; macromolecular assembly; mouse model; mutant; protein protein interaction; recruit; response; solid state; translational study

Summary/Abstract Molecular and cellular endocrine responses often evoke the mobilization of signal transduction cascades. This fundamental process proceeds through macromolecular assemblies of signaling enzymes sequestered with preferred substrates. The relay of information through these solid-state signaling units ensures initiation (or termination) of molecular events at defined intracellular locations. Recent evidence suggests that disruption of protein-protein interactions that sustain local signal complexes is linked to disease. This proposal will test if mutations affecting the subcellular distribution of the catalytic (C) subunit of protein kinase A (PKA) contribute to the etiology of ACTH-independent Cushing's syndrome. Cushing's syndrome is an endocrine disorder diagnosed by excessive cortisol levels in the blood, mid-section weight gain, diabetes and hypertension. ACTH-dependent Cushing's disease often occurs as a consequence of pituitary tumors that overproduce adrenocorticotropic hormone (ACTH), which stimulates excess cortisol release from the adrenal glands. However, ACTH-independent forms of the disease are linked to mutations in genes encoding the catalytic subunits of protein kinase A PKA holoenzymes exist as heterotetramers consisting of two regulatory (R) and two catalytic (C) subunits. A traditional view infers R and C subunits of PKA dissociate upon activation by the second messenger cAMP. Recent discoveries have redefined our understanding of how this configuration operates. Last year we showed that active C subunits are not released from type II PKA holoenzymes when cells are stimulated with hormones. Under this new paradigm, active C subunits remain associated with RII subunits, and constrained within subcellular “signaling islands” by A-kinase anchoring proteins (AKAPs). Consequently, active kinase remains sequestered within a few microns of substrates. These findings have forged a testable hypothesis that mislocalization of active PKA is responsible for ACTH- independent Cushing's syndrome. Preliminary studies imply that 1) recruitment to AKAP signaling islands is the key determinant for type II PKA substrate selectivity, and 2) mutations that prohibit C subunit interaction with anchored R subunits lead to mislocalized and unregulated kinase in Cushing's syndrome. Two specific aims are proposed. Aim1 will integrate structural, live-cell imaging and chemical-biology strategies to establish the spatial parameters of the type I PKA isozyme. Aim 2 will combine CRISPR/Cas9 gene-editing in adrenal cells with live-cell imaging and cortisol profiling to investigate if recently identified mutations in PKA C subunits linked to Cushing's syndrome preclude recruitment into AKAP signaling islands to drive this endocrine disorder.

摘要/摘要 分子和细胞内分泌反应经常引起信号转导级联的动员。这 基本过程通过信号酶的大分子组装进行， 优选的基质。通过这些固态信号单元的信息中继确保了初始化（或 终止）分子事件在确定的细胞内位置。最近的证据表明， 维持局部信号复合物的蛋白质-蛋白质相互作用与疾病有关。该提案将测试， 影响蛋白激酶A（PKA）催化（C）亚基亚细胞分布的突变有助于 促肾上腺皮质激素非依赖性库欣综合征的病因。 库欣氏综合征是一种内分泌紊乱，诊断为血液中皮质醇水平过高， 体重增加、糖尿病和高血压。促肾上腺皮质激素依赖性库欣氏病经常发生的后果 垂体瘤过度产生促肾上腺皮质激素（ACTH），刺激过量的皮质醇 从肾上腺中释放出来然而，ACTH非依赖性形式的疾病与基因突变有关。 编码蛋白激酶A催化亚基的基因 PKA全酶以由两个调节亚基（R）和两个催化亚基（C）组成的异源四聚体存在。一 传统观点认为PKA的R和C亚基在被第二信使cAMP激活后解离。 最近的发现重新定义了我们对这种配置如何运作的理解。去年我们 表明，当细胞被刺激时，活性C亚基不会从II型PKA全酶中释放出来。 荷尔蒙在这种新的范式下，活性C亚基仍然与RII亚基相关联，并且受到限制。 在亚细胞“信号岛”内通过A-激酶锚定蛋白（AKAP）。因此，活性激酶 保持在基底的几微米内。 这些发现形成了一个可验证的假设，即活性PKA的错误定位是导致ACTH- 独立性库欣综合征。初步研究表明：1）AKAP信号岛的募集是 II型PKA底物选择性的关键决定因素，以及2）阻止C亚基相互作用的突变 与锚定R亚基的结合导致库欣综合征中的错误定位和不受调节的激酶。两个具体 提出了目标。aim 1将整合结构，活细胞成像和化学生物学策略， 建立Ⅰ型PKA同工酶的空间参数。Aim 2将联合收割机CRISPR/Cas9基因编辑技术结合在 肾上腺细胞活细胞成像和皮质醇分析，以调查最近发现的PKA C突变 与库欣综合征相关的亚基阻止了AKAP信号岛的募集， disorder.