Defective PKA Signaling in Cushing's Syndrome

摘要/摘要 分子和细胞内分泌反应常常引起信号转导通路的动员。这 基本过程是通过信号酶的大分子组装进行的，这些大分子组装与 首选底物。通过这些固态信令单元的信息中继确保启动(或 在确定的细胞内位置的分子事件的终止)。最近的证据表明， 维持局部信号复合体的蛋白质-蛋白质相互作用与疾病有关。这项提案将测试是否 影响蛋白激酶A(PKA)催化(C)亚单位亚细胞分布的突变 ACTH非依赖性库欣综合征的病因学。 库欣综合征是一种内分泌疾病，诊断为中段血液皮质醇水平过高。 体重增加、糖尿病和高血压。ACTH依赖型库欣病常因此而发生 过度产生促肾上腺皮质激素(ACTH)刺激过量皮质醇的垂体肿瘤 从肾上腺释放。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全酶是由两个调节亚基(R)和两个催化亚基(C)组成的异构体。一个 传统观点认为，PKA的R和C亚基被第二信使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行的理解。去年我们 结果表明，当细胞受到刺激时，II型PKA全酶不释放活性C亚基 荷尔蒙。在这种新的模式下，活性C亚基仍然与RII亚基相关联，并受到限制 通过A-激酶锚定蛋白(AKAP)在亚细胞内的“信号孤岛”。因此，活性激酶 仍然被隔离在几微米的底物内。 这些发现形成了一个可检验的假说，即活性PKA的错误定位是ACTH的原因。 独立库欣综合征。初步研究表明，1)AKAP信令岛的招募 II型PKA底物选择性的关键决定因素，以及2)阻止C亚单位相互作用的突变 与锚定的R亚基导致库欣综合征中的错误定位和未调控的激酶。两个具体的 提出了目标。AIM1将整合结构、活细胞成像和化学生物学策略，以 建立I型PKA同工酶的空间参数。AIM 2将结合CRISPR/Cas9基因编辑 肾上腺细胞活细胞成像和皮质醇图谱研究最近发现的PKA C突变 与库欣综合征相关的亚基排除了重新进入AKAP信号岛来驱动内分泌的可能性 无序。

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.