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.

抽象的。 我的整个职业生涯都是在 NIGMS 的资助下，遵循以下原则：结构将 提供对功能的理解，最终目标是阐明蛋白质磷酸化的方式 调节生物学。我的具体重点是解决与 PKA 相关的分子结构 信号传导从催化 (C) 亚基的晶体结构开始，这是第一个蛋白激酶 待解决的结构。虽然许多功能见解都来自监管 (R) 和 C 的结构- 亚基和 R:C 异二聚体，细胞中的 PKA 信号传导是由全长 R2C2 全酶介导的 通常通过 A 激酶锚定蛋白 (AKAP) 靶向细胞中专用位点附近的离散位点 基材。如果没有详细的了解，就不可能全面了解细胞中的 PKA 信号传导。 目标全酶的肖像，这不仅包括 R:C 结构域，它揭示了很多关于 对称性、催化作用和变构作用，还有逃避经典晶体学的动态连接子和结构域。 这些连接子中嵌入了如此多重要的生物学特性，驱动着所有这些连接子的组装、靶向和调节。 激酶。我们最近在解决全长全酶的结构和阐明特征方面的工作表明 如何实现更高水平的复杂性和特异性。它还揭示了非凡的结构和 四种 PKA 全酶的功能非冗余性，这对于实现特异性至关重要。这 现在的主要挑战是了解柔性连接体如何驱动每种全酶的组装和调节。 为了应对这一挑战，我们正在构建冷冻电子显微镜 (cryoEM)，并最终构建冷冻电子显微镜 将断层扫描 (cryoET) 纳入我们所需的技术组合以及高分辨率镶嵌成像 （HRMI）在组织中。有了这些工具，我们期望创建 RIIb 和 RIa 的动态肖像 全酶在活性和非活性状态之间切换。为了同时增强我们的 为了了解疾病，我们将重点关注由 PKA 亚基突变直接引起的三种疾病。 FLHCC 是一种罕见的儿童肝癌，由 DNA-JB1 的 J 结构域与 N 末端融合驱动 PKA Ca 亚基。卡尼复合病 (CNC) 和肢端骨质增生 (ACRDYS) 属于内分泌疾病 由RIa突变引起。我们相信这些突变体形成的全酶将推动我们的理解 wt 蛋白质。同时，我们将对肝脏进行 HRMI 分析，并将正常肝脏与以下组织进行比较： 表达FL-HCC。 RIa 中的 ACRDYS 和 CNC 突变体突出了控制的变构网络 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 AKAP79 和 RIa 与纤毛特异性 GPCR C 端尾部新发现的 AKAP 基序结合， 探地雷达161。凭借我们杰出的合作团队，我们准备取得快速进展。我们的长期目标 是建立PKA作为原型激酶来演示多价大分子信号传导如何 复合物被组装和调节，并因疾病而变得功能失调。