Assembly and regulation of dynamic PKA macromolecular signaling systems

动态PKA大分子信号系统的组装与调控

• 批准号: 8920262
• 负责人: SUSAN S. TAYLOR
• 金额: $ 4.37万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-25 至 2018-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8920262
• 关键词: A kinase anchoring protein; Address; Affinity; Allosteric Regulation; Architecture; Attention; Binding; C-terminal; Calcium/calmodulin-dependent protein kinase; Catalysis; Catalytic Domain; Cell physiology; Cells; Communities; Complex; Coupled; Coupling; Crystallography; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotides; Development; Diffusion; Dimensions; Disease; Docking; Endocrine System Diseases; Event; Gatekeeping; Genes; Goals; Grant; Guanine Nucleotide Exchange Factors; Holoenzymes; Image; Individual; Ions; Kinetics; Length; Link; Macromolecular Complexes; Malignant Neoplasms; Mammalian Cell; Maps; Measurement; Membrane; Memory; Metabolism; Metals; Methods; Modeling; Molecular; Molecular Conformation; Monitor; Mutagenesis; Mutation; N-terminal; Neutrons; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Physiological; Property; Protein Isoforms; Protein Kinase; Proteins; RGS Domain; Recruitment Activity; Recycling; Regulation; Resolution; Roentgen Rays; Role; Sampling; Signal Pathway; Signal Transduction; Signaling Protein; Site; Solutions; Structure; System; Techniques; Trefoil Motif; Work; base; catalyst; cell motility; domain mapping; flexibility; image reconstruction; inorganic phosphate; insight; light microscopy; malignant endocrine gland neoplasm; molecular dynamics; mutant; new technology; particle; phosphoric diester hydrolase; protein kinase R; prototype; public health relevance; tool

DESCRIPTION (provided by applicant): cAMP-dependent protein kinase (PKA), ubiquitous in mammalian cells, regulates a plethora of cell processes including development, differentiation, memory, and metabolism and is associated with many diseases including multiple endocrine disorders and cancer. It serves as a prototype for the protein kinase superfamily and its activation by cAMP is a classic example of allosteric regulation. While our studies of PKA structure and function have given us a molecular understanding of individual PKA catalytic and regulatory subunits, over the past four years we have added a new dimension to our understanding of PKA signaling by solving structures of PKA tetrameric holoenzymes. These first glimpses of the full-length proteins force us to appreciate that PKA functions as a complex and multicomponent signaling system. Protein kinases have evolved to be dynamic and highly regulated molecular switches, not efficient catalysts, and we simply cannot appreciate or understand how this PKA signaling system works and how it is allosterically regulated by cAMP without seeing the full-length holoenzymes. It is these large multi-domain tetrameric complexes that represent the physiological state of PKA signaling in cells, and the quaternary structure of each isoform (RI¿, RI¿, RII¿, and RII¿) is different, as we had predicted based on our low resolution SAXS analyses. Our goals now are to further define the molecular features of these holoenzymes as integrated and finely tuned systems using RI¿ and RII¿ holoenzymes as prototypical isoforms that nucleate dynamic multicomponent macromolecular PKA signaling complexes. Aim I focuses on RI¿ and uses a disease mutation of RI¿ to dissect the pathway for allosteric signaling from one cAMP-binding domain to the other. Crystal structures of the full-length mutant holoenzyme will be validated using solution methods such as SAXS and SANS while mutagenesis will confirm interfaces and provide functional, mechanistic insights. To build higher levels of RI¿ complexity we focus on two newly discovered proteins. P-Rex1, up-regulated in cancers, is a guanine nucleotide exchange factor for Rac1 that binds through its two PDZ domains to the C-terminal PDZ motif of RI¿. To the N-terminal D/D domain we are adding a small newly discovered membrane associated RI-specific AKAP, smAKAP. In Aim II we are building a quantitative model for PKA signaling by RII¿. We specifically define the role of phosphatases, metals (both Mg2+ and Ca2+), and phosphodiesterases. We introduce new concepts by showing the importance of the turnover of a single phosphate in RII¿ and emphasize that the release of cAMP from RII¿ is not diffusion limited. Finally, in Aim III we build higher levels of complexity for the RII¿ "signalosome" by adding a dual-specific AKAP, DAKAP2, to the holoenzyme. DAKAP2 is a multi-domain AKAP that regulates recycling by binding through its PDZ motif to PDZ-K1, which links it to transporters, and through its RGS domains that bind to Rabs. In parallel with crystallography, we will use small angle Xray and neutron scattering as well as single particle image reconstruction to monitor conformational changes and domain organization in solution.

描述（由适用提供）：依赖CAMP的蛋白激酶（PKA），无处不在的哺乳动物细胞中，调节了许多细胞过程，包括发育，分化，记忆和代谢，并且与许多包括多种内分泌疾病和癌症在内的疾病有关。它是蛋白激酶超家族的原型，cAMP激活是变构调节的经典例子。尽管我们对PKA结构和功能的研究使我们对单个PKA催化和调节亚基有了分子的理解，但在过去的四年中，我们通过求解PKA四聚体全酶的结构为我们对PKA信号的理解增加了新的维度。这些全长蛋白的最初瞥见迫使我们欣赏PKA充当复杂且多组分的信号系统。蛋白激酶已经演变为动态和高度调节的分子开关，而不是有效的催化剂，我们根本无法欣赏或理解该PKA信号系统如何工作，以及如何通过CAMP对其进行变构调节，而无需看到全长的全酶。正是这些大型多域四聚体配合物代表细胞中PKA信号的物理状态，以及每个同工型的第四纪结构（RI€）。 ，正如我们基于低分辨率SAXS分析所预测的那样，，RI€，RII¿和RII¿）是不同的。现在，我们的目标是进一步将这些全酶的分子特征定义为使用RI？和RII¿Holoenzys的整合和细调的系统，作为核能动态多组分大分子PKA PKA信号复合物的原型同工型。目的我专注于RI？，并使用RI疾病突变来剖析从一个营地结合域到另一个训练区的变构信号传导的途径。全长突变全酶的晶体结构将使用诸如SAXS和SANS等溶液方法验证，而诱变将确认界面并提供功能性的机械见解。为了建立更高级别的RI层，我们专注于两种新发现的蛋白质。 P-Rex1在癌症中被上调，是Rac1的鸟嘌呤核丁基交换因子，它通过其两个PDZ结构域与RI的C末端PDZ基序结合。在N末端D/D域中，我们正在添加一个小的新发现的膜相关的RI特异性AKAP SMAKAP。在AIM II中，我们正在通过RII构建用于PKA信号的定量模型。我们特别定义了磷酸酶，金属（MG2+和Ca2+）和磷酸二酯酶的作用。我们通过显示RII磷酸单元离职的重要性来介绍新概念，并强调从RII释放CAMP不是扩散的限制。最后，在AIM III中我们建立 通过向全酶添加双重特异性AKAP DAKAP2，RII€“信号体”的复杂性较高。 DAKAP2是一种多域AKAP，通过通过其PDZ基序与PDZ-K1结合来调节回收，该基序与PDZ-K1结合，该基序将其与转运蛋白链接到转运蛋白，并通过其与RABS结合的RGS结构域。与晶体学同时，我们将使用小角度X射线和中子散射以及单个粒子图像重建来监视溶液中的会议变化和域组织。