Defective PKA Signaling in Cushing's Syndrome

库欣综合征中 PKA 信号传导缺陷

• 批准号: 9789863
• 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/9789863
• 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; Structure; 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反应的动员。这 基本过程是通过隔离的信号传导酶的大分子组件进行的 首选底物。通过这些固态信号单元的信息继电器可确保主动性（或 终止分子事件在定义的细胞内位置。最近的证据表明中断 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。该建议将测试是否 影响蛋白激酶A（PKA）催化（C）亚基的亚细胞分布的突变有助于 独立于ACTH的库欣综合症的病因。 库欣综合症是一种由血液中过量皮质醇水平诊断出的内分泌疾病 体重增加，糖尿病和高血压。依赖ACTH的库欣氏病经常出现 垂体肿瘤过量产生肾上腺皮质性马酮（ACTH），刺激多余的皮质醇 从肾上腺释放。但是，疾病的非ACTH独立形式与突变有关 编码蛋白激酶A催化亚基的基因 PKA全酶是由两个调节（R）和两个催化（C）亚基组成的异驱精。一个 传统视图Ind Inds PKA的R和C亚基在第二信使训练营激活后解离。 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 结果表明，当细胞刺激细胞时，活性C亚基未从II型PKA全酶释放 激素。在此新范式下，活动C亚基与RII亚基保持相关，并受到约束 在亚细胞“信号岛”中，A-激酶锚定蛋白（AKAP）。因此，活性激酶 在几微米的底物中保持隔离。 这些发现忘记了一个可检验的假设，即主动PKA的错误定位是造成ACTH-的原因 独立的库欣综合症。初步研究暗示1）招募到AKAP信号岛是 II型PKA底物选择性的关键确定剂，以及2）禁止C亚基相互作用的突变 锚定的R亚基导致库欣综合症中的非定位和不受管制的激酶。两个具体 提出了目标。 AIM1将将结构性，活细胞成像和化学生物学策略整合到 建立I型PKA同工酶的空间参数。 AIM 2将结合CRISPR/CAS9基因编辑 具有活细胞成像和皮质醇分析的肾上腺细胞，研究是否最近在PKA C中鉴定出突变 与Cushing综合征相关的亚基排除了AKAP信号岛的招募，以驱动此内分泌 紊乱。