Lessons Learned from PKA: Assembly of Dynamic Macromolecular Switches

抽象的。 我的整个职业生涯都是在NIGMS的保护伞下资助的，一直遵循这样的原则：结构将 提供对功能的理解，最终目标是阐明蛋白质磷酸化是如何 调节着生物学。我的重点一直是解决与PKA相关的分子结构 信号从催化(C)亚单位的晶体结构开始，这是第一个蛋白激酶 结构有待解决。虽然许多功能见解来自监管(R)和C-C的结构- 亚基和R：C异源二聚体，细胞中的PKA信号是由全长R2C2全酶介导的，这些全酶是 靶向，通常是通过A激酶锚定蛋白(AKAP)，定位到细胞中接近专用的谨慎位置 底物。如果没有详细的研究，就不可能全面了解细胞中的PKA信号。 目标全酶的肖像，这不仅包括R：C结构域，它揭示了如此多的 对称性、催化和变构，但也有避开经典结晶学的动态连接物和结构域。 如此多重要的生物学被嵌入在这些连接子中，它们驱动着所有的组装、靶向和调节 激活剂。我们最近在解决全长全酶的结构和阐明其特征方面的工作表明 如何实现更高级别的复杂性和专用性。它还揭示了引人注目的结构和 四个PKA全酶的功能非冗余性，这对于实现特异性是至关重要的。这个 现在的主要挑战是了解灵活的连接子如何驱动每个全酶的组装和调节。 为了迎接这一挑战，我们正在建造低温电子显微镜，并最终建造低温电子。 在我们所需的技术组合以及高分辨率马赛克成像中加入断层扫描(冷冻成像) (HRMI)。有了这些工具，我们希望创建RIIb和RIA的动态肖像 全酶在活性和非活性状态之间切换。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。 摘要肝细胞癌是一种少见的儿童期肝癌，由DNA-JB1的J结构域与N末端融合所致。 PKA钙亚基的表达。Carney综合征和肢端营养不良均为内分泌疾病 由RIA基因突变引起。我们相信，由这些突变体形成的全酶将推动我们对 重量蛋白的含量。与此同时，我们将对肝脏进行HRMI分析，并将正常肝脏与下列组织进行比较 表达FL-肝细胞癌。RIA中的ACRDYS和NC突变体突出了控制 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到 AKAP79和RIA与新发现的纤毛特异GPCR的C端尾部的AKAP基序结合， GPR161。有了我们出色的合作团队，我们将取得快速进展。我们的长期目标 是建立PKA作为展示多价大分子信号转导的原型激酶 复合体被组装和调节，并因疾病而变得功能失调。

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.