Lessons Learned from PKA: Assembly of Dynamic Macromolecular Switches

PKA 的经验教训：动态大分子开关的组装

• 批准号: 10623507
• 负责人: SUSAN S. TAYLOR
• 金额: $ 33.46万
• 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: 2019-01-01 至 2027-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10623507
• 关键词: Achievement; Active Sites; Allosteric Regulation; Biochemical; Biochemistry; Biology; Biophysics; Buffers; C-terminal; Catalytic Domain; Cells; Complex; Cryoelectron Microscopy; Crystallography; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Dedications; Dementia; Disease; Family; G-Protein-Coupled Receptors; Goals; Golgi Apparatus; Holoenzymes; Human; Image; Label; Laboratories; Learning; Length; Link; Liquid substance; Mediating; Mitochondria; Modeling; Molecular Biology; Molecular Conformation; Mutation; National Institute of General Medical Sciences; Neurons; Pathogenicity; Phase; Physical condensation; Productivity; Protein Isoforms; Protein Kinase; RNA Splicing; Recording of previous events; Retina; SHH gene; Second Messenger Systems; Signal Transduction; Site; Structure; Tail; Time; Tissue imaging; Variant; Visualization; Work; autism spectrum disorder; career; frontier; high resolution imaging; human tissue; molecular dynamics; mutant; neurodegenerative phenotype; novel; prototype; smoothened signaling pathway; tool

ABSTRACT My history with cAMP-dependent protein kinase (PKA) and NIGMS, from active site labeling to holoenzyme structures and tissue imaging, has been long and productive. My career has been guided by the fundamental principle that structure will reveal function with the ultimate goal being to elucidate how PKA signaling regulates biology and how it is altered in disease. Our tools include biochemistry, biophysics, and molecular biology to probe mechanisms as well as crystallography, cryoEM, molecular dynamics, and imaging to explore conforma- tional space and localization in cells. A hallmark of my laboratory has been to build interdisciplinary teams that reach across all of these scales. Although it has been over 30 years now since we solved that first protein kinase structure of the PKA catalytic (C) subunit, which has served ever since as the prototypical protein kinase, surprisingly we are still learning new things. PKA signaling in cells is mediated by full-length R2C2 holoenzymes that are targeted to discreet sites in the cell near dedicated substrates, and a major recent achievement was our solving the cryoEM structure of the compact full-length RII holoenzyme in 2020 where for the first time all of the domains could be visualized. During this next phase we will continue with our characterization of holoenzyme complexes focusing, in particular, on RIIβ, which is enriched in neurons and localizes to Golgi. In addition, however, we will build on two new discoveries that came from our work over the past three years. First is the discovery that Cβ subunits, a family of previously unexplored splice variants that account for ~50% of PKA signaling in neurons, are linked to a neurodegenerative phenotype that abolishes Sonic hedgehog (Shh) signaling. With imaging in human retina we then validated that Cβ is highly expressed in neurons, that it localizes differently than C, and that Cβ4/Cβ4ab are enriched at mitochondria. We are now characterizing the neuron-specific Cβ4 isoforms and the specific mutants that correlate with Shh signaling. Another new and potentially related discovery is that there is a functional PKI-like sequence embedded in the C-terminal tail of Smoothened, the GPCR that is associated with Shh signaling. A final discovery that the RI subunit undergoes liquid:liquid phase separation that contributes to cAMP buffering in cells opens another new frontier for non-canonical PKA signaling in cells. We find that RIβ also forms biomolecular condensates that are distinct from RIα, and we are now characterizing an RIβ mutant, RIβ(R335W), that is associated with dementia and autism. An essential part of our strategy is to use a multi-scale approach that includes not only biochemical characterizations and structure solutions but also high-resolution imaging in human tissues where we can hopefully correlate changes in localization and expression with pathogenic mutations in Cβ and RIβ. In parallel, we will build on our cryo-EM structure of the full length RIIβ holoenzyme where we hope to trap some of the domain dynamics that contribute to the highly allosteric and isoform-specific cAMP-mediated activation of each holoenzymes. With our exceptional team of collaborators we are poised to make rapid progress.

抽象的 我的 cAMP 依赖性蛋白激酶 (PKA) 和 NIGMS 历史，从活性位点标记到全酶 结构和组织成像，长期以来一直是我的职业生涯的指导原则。 该结构将揭示功能，最终目标是阐明 PKA 信号传导如何调节 我们的工具包括生物化学、生物物理学和分子生物学，以及它在疾病中如何改变。 探针机制以及晶体学、冷冻电镜、分子动力学和成像来探索构象 我实验室的一个标志是建立跨学科团队， 尽管距离我们解决第一个蛋白质已经过去了 30 多年。 PKA 催化 (C) 亚基的激酶结构，此后一直作为原型蛋白质 令人惊讶的是，我们仍在了解细胞中的 PKA 信号传导是由全长 R2C2 介导的。 靶向细胞中专用底物附近的离散位点的全酶，以及最近的一个主要研究 成就是我们在 2020 年解决了紧凑型全长 RII 全酶的冷冻电镜结构，其中 第一次所有领域都可以可视化，在下一阶段我们将继续我们的工作。 全酶复合物的表征，特别关注 RIIβ，它富含于神经元和 然而，此外，我们将建立在我们的工作的两项新发现的基础上。 过去三年，首先发现了 Cβ 亚基，这是一个以前未探索过的剪接变体家族。 约占神经元中 PKA 信号传导的 50%，与神经退行性表型相关，该表型消除了 通过人视网膜成像，我们验证了 Cβ 的高表达。 在神经元中，它的定位与 C 不同，并且 Cβ4/Cβ4ab 在线粒体中富集。 表征神经元特异性 Cβ4 亚型以及与 Shh 信号传导相关的特定突变体。 另一个可能相关的新发现是，有一个类似 PKI 的功能序列嵌入在 Smoothened 的 C 末端尾部，与 Shh 信号传导相关的 GPCR 最终发现是 RI。 亚基经历液：液相分离，有助于细胞中的 cAMP 缓冲，开辟了另一个新的领域 我们发现 RIβ 也形成生物分子缩合物。 与 RIα 不同，我们现在正在表征 RIβ 突变体 RIβ(R335W)，它与 我们战略的一个重要部分是采用多尺度方法，不仅包括治疗。 生化特征和结构解决方案，以及人体组织的高分辨率成像 我们希望能够将定位和表达的变化与 Cβ 和 RIβ 的致病性突变联系起来。 同时，我们将建立全长 RIIβ 全酶的冷冻电镜结构，我们希望在其中捕获一些 有助于高度变构和异构体特异性 cAMP 介导的激活的域动力学 凭借我们杰出的合作者团队，我们准备取得快速进展。