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常常引起信号转导级联的动员。 基本过程是通过隔离的信号酶的大分子组装进行的 通过这些固态信号单元传递信息可确保启动（或 最近的证据表明，细胞内特定位置的分子事件的破坏。 该提案将测试维持局部信号复合物的蛋白质-蛋白质相互作用是否与疾病有关。 影响蛋白激酶 A (PKA) 催化 (C) 亚基亚细胞分布的突变有助于 不依赖ACTH的库欣综合征的病因学。 库欣综合征是一种内分泌疾病，诊断为血液中皮质醇水平过高 体重增加、糖尿病和高血压通常会因此发生。 垂体肿瘤过度产生促肾上腺皮质激素 (ACTH)，刺激过量的皮质醇 然而，该疾病的非 ACTH 形式与突变有关。 编码蛋白激酶 A 催化亚基的基因 PKA 全酶以异四聚体形式存在，由两个调节 (R) 亚基和两个催化 (C) 亚基组成。 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。 结果表明，当细胞受到刺激时，II 型 PKA 全酶不会释放活性 C 亚基。 在这种新的范式下，活性 C 亚基仍然与 RII 亚基相关，并受到限制。 位于亚细胞“信号岛”内，由经过测试的 A 激酶锚定蛋白 (AKAP) 激活。 仍然隔离在基材的几微米内。 这些发现形成了一个可检验的假设，即活性 PKA 的错误定位是导致 ACTH- 独立库欣综合征的初步研究表明 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) 禁止 C 亚基相互作用的突变 锚定的 R 亚基会导致库欣综合征中两种特定的激酶定位错误和不受调节。 提出的目标是整合结构、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，目标 2 将结合 CRISPR/Cas9 基因编辑。 通过活细胞成像和皮质醇分析来研究肾上腺细胞是否最近发现了 PKA C 突变 与库欣综合征相关的亚基阻止招募到 AKAP 信号岛来驱动这种内分泌 紊乱。