Illuminating the Role of understudied PRKACB Splice Variants in PKA Signaling

阐明正在研究的 PRKACB 剪接变体在 PKA 信号传导中的作用

• 批准号: 9813753
• 负责人: SUSAN S. TAYLOR
• 金额: $ 15.75万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9813753
• 关键词: Ablation; Activated Lymphocyte; Antibodies; Atrial myxoma with lentigines; Binding; Biochemistry; Biological; Biological Assay; Biological Models; Biological Process; Biology; Brain; CNR1 gene; CNR2 gene; Cancer Patient; Cells; Complement; Complex; Coupled; Crystallization; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Deuterium; Development; Disease; Docking; Escherichia coli; Exons; Family; Fluorescence Polarization; Functional disorder; Gene Family; Genes; Goals; Hela Cells; Holoenzymes; Human; Hydrocortisone; Hydrogen; Image; Immune; Immunosuppression; In Vitro; Kidney; Kinetics; Knock-out; Knowledge; Link; Lobe; Luciferases; Lymphocyte; Malignant neoplasm of prostate; Mammalian Cell; Maps; Mass Spectrum Analysis; Measures; Memory; Metabolism; Modeling; Mosaicism; Mutation; N-terminal; Norway; PRKACB gene; Phenotype; Phosphotransferases; Physiological; Pituitary-dependent Cushing' s disease; Probability; Protein Isoforms; Protein Kinase; Protein Kinase C; Proteins; Proteomics; RNA Splicing; Regulation; Resolution; Rest; Role; Signal Transduction; Site; Solvents; Specificity; Spleen; Structure; Substrate Specificity; Surface; Tail; Thymus Gland; Tissues; Variant; Work; adenoma; alpha helix; biophysical properties; disease phenotype; experimental study; inhibitor/antagonist; innovation; interest; novel; response; thyroid neoplasm; tissue culture

PROJECT SUMMARY. Protein kinases represent one of the largest gene families and regulate much of biology. Kinase dysfunction is also associated with a plethora of diseases. Although the protein kinase gene families have been mapped onto the kinome, the details of specificity and regulation are buried within each family. These families include not only isoforms but also splice variants for many genes. Splice variants are not represented the kinome, but enormously expand the complexity of signaling networks and specificity. Knockout experiments tell us repeatedly that the isoforms and splice variants are functionally non-redundant, highlighting that the assembly of highly specific complexes within cells and tissues is an essential feature of kinase signaling. Increasingly we are also coming to appreciate the importance of these isoforms and splice variants from disease phenotypes, which further highlights that biology is controlled by finely tuned regulatory networks. cAMP-dependent protein kinase (PKA), expressed in every mammalian cell, regulates fundamental biological processes that include metabolism, development/differentiation, memory, and immune responsiveness. While the PKA Cα1 subunit has served in so many ways as the prototypical protein kinase, surprisingly almost nothing is known about the Cβ isoforms, which include multiple splice variants. While Cα1 is ubiquitous in all human cells, expression of the Cβ isoforms is more tissue-specific, and disease phenotypes suggest that they also are likely to be functionally non-redundant. Our goal here is to characterize three of the Cβ splice variants that differ only in their first exon. These Cβ isoforms correlate with several diseases. Cβ1 leads to cortisol producing adenomas in Cushing’s Disease, ablation of Cβ2 in immune cells leads to immuno- suppression, and increased Cβ2 correlates with survival in prostate cancer patients and can cause Carney Complex Disease (CNC) and thyroid tumors. This emphasizes the importance of Cβ signaling and suggests that our work will have important and previously unappreciated biological and disease relevance. Our innovation lies in the fact that we can easily cross so many scales that extend from basic biochemistry and atomic resolution of the molecules to their isoform-specific distribution in cells and tissues. We will use this multi-scale approach to characterize the structure, function and regulation of three Cβ isoforms. In parallel we will map the tissue-specific localization of these isoforms in kidney, spleen, thymus and brain using isoform- specific antibodies. Finally we will use a proteomic strategy to identify isoform-specific binding partners. Our broad knowledge of PKA signaling, coupled with our deep understanding of the four functionally non-redundant PKA holoenzymes, provides us with a unique opportunity to explore a wide swath of previously untapped cAMP signaling space. Given the global importance of PKA signaling in all cells, the probability that the Cβ isoforms will have important physiological as well as disease relevance is high. Our studies will allow us to move forward creatively with developing novel isoform-specific therapies.

项目摘要。蛋白激酶代表最大的基因家族之一，并调节许多 生物学激酶功能障碍也与多种疾病有关。虽然蛋白激酶基因 家族已经被定位到激酶组上，特异性和调节的细节被隐藏在每个家族中。 家人这些家族不仅包括同种型，而且还包括许多基因的剪接变体。剪接变体不是 代表了激酶组，但极大地扩展了信号网络的复杂性和特异性。敲除 实验反复告诉我们，同种型和剪接变体在功能上是非冗余的， 细胞和组织内高度特异性复合物的组装是激酶的基本特征， 信号我们也越来越认识到这些异构体和剪接变异体的重要性 这进一步强调了生物学是由微调的调控网络控制的。 cAMP依赖性蛋白激酶（PKA），在每一个哺乳动物细胞中表达，调节基本的 包括新陈代谢、发育/分化、记忆和免疫在内的生物学过程 响应能力。虽然PKA Cα1亚基在许多方面作为原型蛋白激酶发挥作用， 令人惊讶的是，几乎对Cβ同种型一无所知，它包括多种剪接变体。而Cα1 在所有人类细胞中普遍存在，Cβ亚型的表达更具组织特异性，疾病表型 这表明它们在功能上也可能是非冗余。我们的目标是描述三个 Cβ剪接变体仅在第一个外显子上不同。这些Cβ亚型与几种疾病相关。Cβ1 在库欣氏病中导致皮质醇产生腺瘤，免疫细胞中Cβ2的消融导致免疫- 抑制，Cβ2增加与前列腺癌患者的生存相关，并可导致Carney 复杂疾病（CNC）和甲状腺肿瘤。这强调了Cβ信号的重要性，并表明 我们的工作将具有重要的、以前未被重视的生物学和疾病相关性。我们 创新在于，我们可以很容易地跨越这么多的尺度，从基础生物化学， 分子的原子分辨率与其在细胞和组织中的异构体特异性分布。我们将使用这个 多尺度方法表征三种Cβ亚型的结构、功能和调节。与此同时，我们 将使用同种型- 特异性抗体最后，我们将使用蛋白质组学的策略，以确定异构体特异性结合伙伴。我们 广泛的PKA信号传导知识，加上我们对四个功能非冗余的 PKA全酶，为我们提供了一个独特的机会，探索广泛的以前未开发的 cAMP信号传导空间。考虑到PKA信号在所有细胞中的全局重要性，Cβ 同种型将具有重要的生理以及疾病相关性高。我们的研究将使我们能够 通过开发新型亚型特异性疗法创造性地向前迈进。