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: 10388723
• 负责人: SUSAN S. TAYLOR
• 金额: $ 17.99万
• 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/10388723
• 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 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.

抽象的。 我的整个职业生涯由尼格姆斯的伞资金资助，一直受到结构将会的原则的指导 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相关的分子的结构 信号从催化（C）亚基的晶体结构开始，该催化（C）亚基是第一个蛋白激酶 要解决的结构。尽管许多功能见解来自监管（R）和C-的结构 亚基和R：C异二聚体，细胞中的PKA信号传导是由全长的R2C2全酶介导的 通常通过激酶锚定蛋白（AKAP）靶向谨慎的位点。 基材。不可能在没有详细的情况下全面理解单元中的PKA信号传导 目标全酶的肖像，这不仅包括R：C域，这些域揭示了很多 对称，催化和变构，以及逃避经典晶体学的动态接头和域。 这些接头嵌入了如此重要的生物学，以驱动组装，针对所有人的组装 激酶。我们最近在解决结构和阐明全长全酶的特征方面的工作显示 如何实现更高水平的复杂性和特异性。它还揭示了出色的结构和 四个PKA全酶的功能性不差，这对于达到特异性至关重要。这 现在的主要挑战是了解灵活的接头如何驱动每个全酶的组装和调节。 为了应对这一挑战，我们正在构建冷冻电子显微镜（冷冻），最终是冷冻电子 层析成像（冷冻）进入我们需要的技术组合以及高分辨率马赛克成像 （HRMI）在组织中。借助这些工具，我们希望创建RIIB和RIA的动态肖像 全酶在其主动状态和无活性状态之间切换时。同时增强我们的 了解疾病，我们将重点关注三种直接由突变PKA亚基引起的疾病。 FLHCC是一种罕见的儿童肝癌，由DNA-JB1的J域与N末端的融合驱动 PKA CA亚基。 Carney复合病（CNC）和杂技病（ACRDYS）是内分泌疾病 由RIA突变引起。我们认为由这些突变体形成的全酶会推动我们的理解 WT蛋白。同时，我们将进行肝脏的HRMI剖面，并将正常肝脏与组织进行比较 FL-HCC表示。 RIA中的ACRDYS和CNC突变体突出了控制的变构网络 激活。对于有针对性的PKA，我们将重点关注两个系统：RIIB全酶和钙调神经酶结合到 AKAP79和RIA与新发现的AKAP图案结合在Cilia特异性GPCR的C末端尾部， GPR161。与我们杰出的合作者团队一起，我们准备取得快速的进步。我们的长期目标 是建立PKA作为原型激酶，以证明如何多价大分子信号传导 复合物是组装和调节的，并因疾病而变得功能失调。