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: 10376936
• 负责人: SUSAN S. TAYLOR
• 金额: $ 2.68万
• 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 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10376936
• 关键词: A kinase anchoring protein; Acrodysostosis; Allosteric Regulation; Amino Acid Motifs; Atrial myxoma with lentigines; Biology; Calcineurin; Catalysis; Catalytic Domain; Cells; Childhood Liver Cancer; Cilia; Complex; Cryo-electron tomography; Cryoelectron Microscopy; Crystallization; Crystallography; Cyclic AMP-Dependent Protein Kinases; DNA; Disease; Endocrine System Diseases; Funding; G-Protein-Coupled Receptors; Goals; Hand; Holoenzymes; Image; Length; Liver; Mediating; Molecular; Mosaicism; Mutation; National Institute of General Medical Sciences; Phosphorylation; Phosphotransferases; Portraits; Protein Kinase; Proteins; Regulation; Resolution; Second Messenger Systems; Signal Transduction; Site; Specificity; Structure; System; Tail; Techniques; Tissues; Work; career; flexibility; insight; macromolecular assembly; mutant; prototype; tool

ABSTRACT. My entire career, funded under the umbrella of NIGMS, has been guided by the principle that structure will provide an understanding of function with the ultimate goal being to elucidate how protein phosphorylation regulates biology. My specific focus has been to solve structures of molecules that are associated with PKA signaling beginning with the crystal structure of the catalytic (C) subunit, which was the first protein kinase structure to be solved. While many functional insights have come from structures of the regulatory (R) and C- subunits and from R:C heterodimers, PKA signaling in cells is mediated by full-length R2C2 holoenzymes that are targeted, typically through A Kinase Anchoring Proteins (AKAPs), to discreet sites in the cell near dedicated substrates. It is not possible to comprehensively understand PKA signaling in cells without having a detailed portrait of the targeted holoenzymes, and this includes not only the R:C domains which reveal so much about symmetry, catalysis and allostery but also the dynamic linkers and domains that evade classic crystallography. So much important biology is embedded in these linkers that drive the assembly, targeting and regulation of all kinases. Our recent work in solving structures and elucidating features of the full-length holoenzymes shows how higher levels of complexity and specificity are achieved. It also revealed the remarkable structural and functional non-redundancy of the four PKA holoenzymes, which is so essential for achieving specificity. The major challenge now is to understand how flexible linkers drive the assembly and regulation of each holoenzyme. To meet this challenge, we are building cryo electron microscopy (cryoEM) and eventually cryo electron tomography (cryoET) into our portfolio of techniques that we need as well as high-resolution mosaic imaging (HRMI) in tissues. With these tools in hand, we expect to create a dynamic portrait of the RIIb and RIa holoenzymes as they toggle between their active and inactive states. To simultaneously enhance our understanding of disease we will focus on three diseases that are caused directly by mutant PKA subunits. FLHCC is a rare childhood liver cancer that is driven by the fusion of the J domain of DNA-JB1 to the N-terminus of the PKA Ca subunit. Carney Complex Disease (CNC) and Acrodysostosis (ACRDYS) are endocrine disorders caused by mutations in RIa. We believe that holoenzymes formed with these mutants will drive our understanding of the wt proteins. In parallel we will do an HRMI profile of the liver and compare normal liver to tissues where FL-HCC is expressed. The ACRDYS and CNC mutants in RIa highlight the allosteric network that controls activation. For targeted PKA we will focus on two systems: the RIIb holoenzyme and calcineurin bound to AKAP79 and RIa bound to the newly discovered AKAP motif in the C-terminal tail of the cilia-specific GPCR, GPR161. With our exceptional team of collaborators, we are poised to make rapid progress. Our longterm goal is to establish PKA as the prototypical kinase for demonstrating how polyvalent macromolecular signaling complexes are assembled and regulated and become dysfunctional as a consequence of disease.

摘要。 我的整个职业生涯，在NIGMS的保护伞下资助，一直遵循这样一个原则，即结构将 提供功能的理解，最终目标是阐明蛋白质磷酸化如何 调节生物学。我的特别重点是解决与PKA相关的分子结构 信号传导开始于催化（C）亚基的晶体结构，这是第一个蛋白激酶 结构要解决。虽然许多功能性的见解来自于调节（R）和C- 从R2 C2亚基和R：C异二聚体，PKA信号在细胞中是由全长R2 C2全酶介导的， 靶向，通常通过A激酶介导蛋白（AKAP），在细胞中的离散位点附近的专用 印刷受体.如果没有详细的信息，就不可能全面了解细胞中的PKA信号传导。 靶向全酶的肖像，这不仅包括R：C结构域，它揭示了很多关于 对称性、催化和变构性，以及逃避经典晶体学的动态连接体和结构域。 如此重要的生物学是嵌入在这些连接器中的，这些连接器驱动着所有生物的组装、靶向和调节。 激酶。我们最近的工作在解决结构和阐明功能的全长全酶表明， 如何实现更高水平的复杂性和特异性。它还揭示了显着的结构和 四种PKA全酶的功能非冗余，这对于实现特异性至关重要。的 现在的主要挑战是了解柔性连接体如何驱动每个全酶的组装和调节。 为了迎接这一挑战，我们正在建立低温电子显微镜（cryoEM），并最终建立低温电子显微镜。 断层扫描（cryoET）到我们的技术组合，我们需要以及高分辨率镶嵌成像 （HRMI）在组织中。有了这些工具，我们希望创建一个动态的RIIb和RIa的肖像 全酶在活性和非活性状态之间切换。同时提高我们的 为了更好地了解疾病，我们将重点关注由突变PKA亚基直接引起的三种疾病。 FLHCC是一种罕见的儿童肝癌，由DNA-JB 1的J结构域与N末端融合驱动。 PKA Ca亚基的表达。Carney综合征（CNC）和肢端骨发育不全（ACRDYS）是内分泌疾病 由RIa突变引起的。我们相信，这些突变体形成的全酶将推动我们了解 的WT蛋白质。与此同时，我们将做一个肝脏的HRMI曲线，并将正常肝脏与 FL-HCC表达。RIa中的ACRDYS和CNC突变体突出了控制RIa的变构网络。 activation.对于靶向PKA，我们将集中在两个系统：RIIb全酶和钙调神经磷酸酶结合， AKAP 79和RIa与纤毛特异性GPCR的C-末端尾部中新发现的AKAP基序结合， GPR161。凭借我们出色的合作者团队，我们准备取得快速进展。我们的长期目标 是建立PKA作为原型激酶，以证明多价大分子信号传导 复合物被组装和调节，并由于疾病而变得功能失调。