Lessons Learned from PKA: Assembly of Dynamic Macromolecular Switches

摘要 我对cAMP依赖的蛋白激酶(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。我的职业生涯一直以基本的 结构揭示功能的原理，最终目的是阐明PKA信号如何调节 生物学及其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 探索构象的探测机制以及结晶学、低温电子显微镜、分子动力学和成像 细胞中的空间和定位。我的实验室的一个特点是建立跨学科的团队 覆盖所有这些标尺。虽然从我们解决第一个蛋白质到现在已经有30多年了 PKA催化(C)亚单位的激酶结构，自那以来一直是原型蛋白 令人惊讶的是，我们还在学习新的东西。细胞内的PKA信号是由全长R2C2介导的 全酶定位于细胞中靠近专用底物的谨慎位置，以及最近一个主要的 我们的成就是在2020年解决了紧凑的全长RII全酶的低温EM结构，其中 这是第一次所有的域都可以被可视化。在下一阶段，我们将继续我们的 全酶复合体的特性，特别是集中在RIIβ上，它富含神经元和 本地化到高尔基山脉。此外，然而，我们将建立在两个新发现的基础上，这两个发现来自我们在 过去三年。首先是发现Cβ亚基，一系列以前未被发现的剪接变体， 占神经元中~50%的PKA信号，与一种神经退行性表型有关，这种表型 Sonic Hedgehog(嘘)信号。通过在人的视网膜中进行成像，我们随后验证了C-β的高表达 在神经元中，它的定位与C不同，Cβ4/Cβ4AB在线粒体中丰富。我们现在是 鉴定神经元特异性Cβ4亚型和与Shh信号相关的特定突变体。 另一个可能相关的新发现是，有一个类似于PKI的功能性序列嵌入在 Smoothed的C-末端尾巴，与Shh信号相关的GPCR。最后一项发现是RI 亚基经历液体：有助于细胞内cAMP缓冲的液体相分离打开了另一个新的 细胞中非规范PKA信号的前沿。我们发现RIβ也形成生物分子凝聚体， 与RIα不同，我们现在正在鉴定一种RIβ突变体RIβ(R335W)，它与 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 生化特征和结构溶液，以及在人体组织中的高分辨率成像 我们有望将C-β和RI-β的定位和表达变化与致病突变联系起来。在……里面 同时，我们将建立在我们的冷冻-EM结构的全长RIIβ全酶上，我们希望在那里捕获一些 促进高度变构和异构体特异性cAMP介导的激活的结构域动力学 每种全酶的。在我们出色的合作团队的帮助下，我们将取得快速进展。

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.