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: 10078616
• 负责人: SUSAN S. TAYLOR
• 金额: $ 65.99万
• 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/10078616
• 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 phos- phorylation 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 holoenzy- mes 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 holo- enzymes 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. FL- HCC 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 dis- orders 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 long- term 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，一直遵循以下原则， 结构将提供功能的理解，最终目标是阐明蛋白质磷酸化是如何进行的。 磷酸化调节生物学。我特别关注的是解决分子的结构 PKA信号始于催化（C）亚基的晶体结构，这是第一个蛋白质 待解的激酶结构。虽然许多功能性的见解来自于监管（R）的结构， 和C亚基以及R：C异二聚体，细胞中PKA信号传导由全长R2 C2全酶介导。 通常通过A激酶介导蛋白（AKAP）靶向细胞中的离散位点， 专用基板。如果没有一个完整的细胞模型，就不可能全面了解细胞中PKA信号传导。 靶向全酶的详细画像，这不仅包括R：C结构域， 很多关于对称性，催化和变构，但也有动态链接器和域，逃避经典 结晶学如此重要的生物学嵌入在这些连接器中，这些连接器驱动组装，靶向和 调节所有激酶。我们最近在解决结构和阐明特征的全长全息， 酶显示了如何实现更高水平的复杂性和特异性。它还揭示了 四种PKA全酶的结构和功能非冗余，这对于实现 的特异性现在的主要挑战是了解柔性连接体如何驱动组装和调节 每一种全酶为了应对这一挑战，我们正在建立低温电子显微镜（cryoEM），并最终 冷冻电子断层扫描（cryoET）到我们的技术组合，我们需要以及高分辨率 镶嵌成像（HRMI）。有了这些工具，我们希望创建一个动态的RIIb画像 和RIa全酶，因为它们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。FL- HCC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复合体病（CNC）和肢端骨发育不全（ACRDYS）是内分泌疾病， 由RIa突变引起的顺序。我们相信，这些突变体形成的全酶将驱动我们的 对WT蛋白质的理解。与此同时，我们将对肝脏进行HRMI分析，并将正常肝脏与 表达FL-HCC的组织。RIa中的ACRDYS和CNC突变体突出了变构网络 控制着激活对于靶向PKA，我们将重点关注两个系统：RIIb全酶和钙调神经磷酸酶 结合AKAP 79和RIa结合新发现的AKAP基序在C-末端尾部的纤毛特异性 GPCR，GPR161。凭借我们出色的合作者团队，我们准备取得快速进展。我们长久以来- 长期目标是建立PKA作为原型激酶，以证明多价大分子如何 信号传导复合物被组装和调节，并由于疾病而变得功能障碍。