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: 10535033
• 负责人: SUSAN S. TAYLOR
• 金额: $ 8.04万
• 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/10535033
• 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.

抽象的。我的整个职业生涯都是在NIGMS的保护伞下资助的，遵循的原则是 结构将提供对功能的理解，最终目的是阐明蛋白质磷酸是如何- 磷酸化调节生物学。我的重点一直是解决相关分子的结构 PKA信号从催化(C)亚单位的晶体结构开始，这是第一个蛋白质 激酶结构有待解决。虽然许多功能洞察来自监管(R)的结构 和C亚基以及来自R：C异源二聚体，细胞内的PKA信号是由全长R2C2全长全长... MES通常通过A激酶锚定蛋白(AKAP)靶向于细胞中 专用基板。如果细胞中没有PKA信号，就不可能全面了解PKA信号 目标全酶的详细描述，这不仅包括揭示So的R：C结构域 更多关于对称性、催化和变构，但也有避开经典的动态连接物和结构域 结晶学。许多重要的生物学都嵌入在这些连接子中，它们驱动组装、靶向和 调节所有的激活酶。我们最近在解决全息结构和阐明全息特征方面所做的工作- 酶展示了如何实现更高水平的复杂性和专一性。它还揭示了不同寻常的 四种PKA全酶的结构和功能无冗余，这对于实现 专一性。现在的主要挑战是了解灵活的连接子如何驱动组装和调节 每一种全酶。为了迎接这一挑战，我们正在建造低温电子显微镜(CryoEM)，最终 将低温电子断层扫描(CryoET)整合到我们需要的技术组合中，以及高分辨率 组织的马赛克成像(HRMI)。有了这些工具，我们希望创建一幅RIIb的动态肖像 和RIA全酶，因为它们在活性和非活性状态之间切换。同时提升我们的 对于疾病的理解，我们将重点关注由突变的PKA亚单位直接引起的三种疾病。FL- 肝细胞癌是一种罕见的儿童肝癌，由DNA-JB1的J结构域与N末端融合引起 PKA钙亚基的表达。Carney综合征和肢端营养不良是内分泌疾病。 由RIA基因突变引起的序列。我们相信，由这些突变体形成的全酶将推动我们的 对wt蛋白的了解。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 它控制着激活。对于靶向PKA，我们将重点研究两个系统：RIIb全酶和钙调神经磷酸酶 结合AKAP79和RIA与新发现的纤毛特异的C端尾部的AKAP基序结合 GPCR、GPR161。在我们出色的合作团队的帮助下，我们将取得快速进展。我们的长- 学期目标是建立PKA作为展示多价大分子如何 信号复合体被组装和调节，并因疾病而变得功能失调。