Lessons Learned from PKA: Assembly of Dynamic Macromolecular Switches

摘要 我的cAMP依赖性蛋白激酶（PKA）和NIGMS的历史，从活性位点标记到全酶 结构和组织成像，已经是长期和富有成效的。我的职业生涯一直遵循着 结构将揭示功能的原则，最终目标是阐明PKA信号转导如何调节 以及它在疾病中是如何改变的。我们的工具包括生物化学、生物物理学和分子生物学， 探针机制以及晶体学，cryoEM，分子动力学和成像，以探索共形， 在细胞中的功能空间和定位。我的实验室的一个特点是建立跨学科的团队， 达到所有这些尺度。尽管我们解决第一个蛋白质问题已经过去了30多年 PKA催化（C）亚基的激酶结构，从那时起一直作为原型蛋白 令人惊讶的是，我们仍然在学习新的东西。细胞中PKA信号传导由全长R2C2介导 全酶是针对离散的网站在细胞附近的专用基板，和一个主要的最近 我们的成就是在2020年解决了紧凑型全长RII全酶的cryoEM结构， 第一次所有的领域都可以被可视化。在下一阶段，我们将继续我们的 全酶复合物的表征，特别是集中在RII β上，其在神经元中富集， 定位于高尔基体。然而，除此之外，我们将建立在两个新发现的基础上，这两个新发现来自我们在2000年的工作。 近三年首先是发现C β亚基，一个以前未探索的剪接变体家族， 约占神经元PKA信号的50%，与神经退行性表型有关， Sonic Hedgehog（Shh）信号。通过对人视网膜的成像，我们验证了C β在视网膜中的高表达， 在神经元中，它的定位不同于C β 4/C β 4ab，并且C β 4/C β 4ab在线粒体中富集。我们现在 表征神经元特异性C β 4亚型和与Shh信号相关的特异性突变体。 另一个新的和潜在的相关发现是，在细胞中嵌入了一个功能性的PKI样序列。 Smoothened的C末端尾部，与Shh信号相关的GPCR。最后的发现是， 亚基经历液：液相分离，有助于细胞中cAMP缓冲，打开了另一个新的 细胞中非经典PKA信号传导的前沿。我们发现RI β还形成生物分子凝聚物， 与RI α不同，我们现在正在描述一种RI β突变体RI β（R335W）， 痴呆症和自闭症。我们战略的一个重要部分是使用多尺度方法，不仅包括 生物化学表征和结构解决方案，以及人体组织中的高分辨率成像， 我们有希望将定位和表达的变化与C β和RI β的致病性突变联系起来。在 平行，我们将建立在我们的全长RII β全酶的冷冻-EM结构上，我们希望在那里捕获一些 的结构域动力学，有助于高度变构和异构体特异性cAMP介导的激活 每一种全酶。凭借我们出色的合作者团队，我们准备取得快速进展。

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.