Illuminating the Role of understudied PRKACB Splice Variants in PKA Signaling

项目总结。蛋白激酶是最大的基因家族之一，它调节许多 生物学。激酶功能障碍也与过多的疾病有关。虽然蛋白激酶基因 家族已经被映射到亲属组上，特异性和调节的细节被隐藏在每个家族中 一家人。这些家族不仅包括异构体，还包括许多基因的剪接变体。剪接变体不是 代表了基因组，但极大地扩展了信号网络的复杂性和专一性。淘汰赛 实验反复告诉我们，异构体和剪接变体在功能上是非冗余的，突出了 在细胞和组织内组装高度特异的复合体是激酶的一个基本特征 发信号。我们也逐渐意识到这些异构体和剪接变体的重要性 这进一步强调了生物学是由微调的调控网络控制的。 CAMP依赖的蛋白激酶(PKA)在每个哺乳动物细胞中都有表达，它调节基本的 生物过程，包括新陈代谢、发育/分化、记忆和免疫 响应性。虽然PKA Cα1亚单位在许多方面都是典型的蛋白激酶， 令人惊讶的是，几乎对包括多种剪接变体的Cβ亚型一无所知。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，并且疾病表型 这表明它们在功能上也可能是非冗余的。我们在这里的目标是描述其中三个 Cβ剪接变异体，仅在其第一个外显子上不同。这些C-β亚型与多种疾病相关。Cβ1 库欣病中产生皮质醇的腺瘤，切除免疫细胞中的Cβ2导致免疫 C-β-2的抑制和增加与前列腺癌患者的生存相关，并可导致卡尼 复杂疾病(CNC)和甲状腺肿瘤。这强调了Cβ信号的重要性，并建议 我们的工作将具有重要的、以前未被认识到的生物学和疾病相关性。我们的 创新在于，我们可以很容易地跨越从基础生物化学和 分子在细胞和组织中对其特定异构体分布的原子分辨率。我们将使用这个 用多尺度方法研究三种C-β异构体的结构、功能和调控同时，我们 将使用异构体在肾、脾、胸腺和脑中定位这些异构体的组织特异性- 特定的抗体。最后，我们将使用蛋白质组学策略来确定异构体特定的结合伙伴。我们的 广泛的PKA信令知识，再加上我们对四种功能非冗余的深刻理解 PKA全酶，为我们提供了一个独特的机会来探索以前未开发的大片区域 营地信号空间。鉴于所有小区中PKA信令的全球重要性，Cβ 异构体将具有重要的生理学意义以及与疾病的高度相关性。我们的研究将使我们能够 创造性地向前发展，开发新的针对异构体的疗法。

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.