Mapping Allosteric Cooperativity in Protein Kinases

绘制蛋白激酶的变构协同性

• 批准号: 9749974
• 负责人: Gianluigi Veglia
• 金额: $ 62.4万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9749974
• 关键词: Active Sites; Affect; Affinity; Allosteric Regulation; Allosteric Site; Behavior; Binding; Biochemical; Biological; Biological Assay; Biophysics; Cardiomyopathies; Catalysis; Catalytic Domain; Cell physiology; Chimera organism; Chimeric Proteins; Communication; Complex; Cushing Syndrome; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Data; Diabetes Mellitus; Disease; Distal; Enzymes; Eukaryotic Cell; Event; Fibrolamellar Hepatocellular Carcinoma; Free Energy; Gene Proteins; Germany; Goals; Grant; Hepatocyte; Holoenzymes; Hydrophobicity; Innovative Therapy; Interruption; Kinetics; Laboratories; Link; Liver; Lobe; Malignant Neoplasms; Mediating; Metabolic; Methods; Molecular; Molecular Chaperones; Molecular Conformation; Motion; Mutation; NMR Spectroscopy; Oncogenic; Pathologic; Pathology; Pathway interactions; Patients; Pharmaceutical Preparations; Phenotype; Phosphorylation; Phosphotransferases; Play; Protein Inhibition; Protein Kinase; Recurrence; Regulation; Regulatory Pathway; Research Personnel; Resolution; Rheumatoid Arthritis; Roentgen Rays; Role; Scientist; Series; Signal Pathway; Signal Transduction; Somatic Mutation; Structure; Testing; Thermodynamics; Time; Translating; Universities; Vertebral column; adenoma; base; biophysical techniques; design; inhibitor/antagonist; mutant; protein function; protein kinase inhibitor; public health relevance; reconstitution; response; scaffold; selective expression; small molecule inhibitor; tumor progression

 DESCRIPTION (provided by applicant): Protein kinases are allosteric enzymes involved in cell signaling pathways and many pathological diseases (cancer, diabetes, rheumatoid arthritis, cardiomyopathy, and others). In the first grant period, we laid out the basis for understanding allostery in the catalytic subunit of protein kinase A (PKA-C), a ubiquitous kinase that regulates a plethora of vital cellular functions. Using a combination of NMR, X-ray and an array of other bio- physical techniques, we described the intra-molecular allosteric communication of PKA-C, showing how distal mutations affect substrate recognition and product release. Importantly, we defined the structural and dynamic signatures for activated and inhibited states of the kinase. In a recent breakthrough in our laboratory, we were able to reconstitute the full holoenzyme for NMR studies. We are now poised to study regulation and inhibition of PKA-C at the atomic level. In this competitive renewal, we propose to study the allosteric mechanism of regulation of PKA and its dysfunctional states linked to somatic mutations. Specifically, Davide Calebiro at the University of Würzburgh (Germany) discovered a series of PKA-C mutations correlated with adenoma-associated Cushing's syndrome (AA-CS). Also, Sandy Simon's laboratory at The Rockefeller University discovered a chimera of PKA-C that is implicated in the progression of the fibrolamellar hepatocellular carcinoma (FLHCC). To study the effects of these mutations, we formed a consortium with Susan Taylor (UCSD), and will collaborate with the Calebiro and Simon groups to elucidate the changes in the intra- and inter-molecular signaling generated by these disease mutations using a combination of NMR spectroscopy, thermodynamics and kinetic methods. In AIM1, we will probe the role of PKA-C in the assembly of the holoenzyme and how the AA-CS mutants affect the R/C complex. In AIM2, we will analyze how AA-CS mutations affect the regulation of PKA-C by endogenous and exogenous inhibitors. In AIM3, we will study the structure and the dynamics of the chimeric construct of PKA-C (PKA-CDNAJB1) to understand how the fused chaperone affects pseudo-substrate recognition and regulation, degenerating into FLHCC. Since the ground states of these mutants are identical, the different functions must be encoded in the excited states that only NMR spectroscopy can unveil at the atomic resolution. Understanding the changes in the kinase energy landscape caused by these mutations is key to the elucidation of the molecular mechanisms leading to disease. This information will help the design of new strategies to control kinase activity for innovative therapies.

 描述(申请人提供)：蛋白激酶是一种变构酶，与细胞信号通路和许多病理疾病(癌症、糖尿病、类风湿性关节炎、心肌病等)有关。在第一个赠款期间，我们为了解蛋白激酶A催化亚单位(PKA-C)的变构作用奠定了基础，PKA-C是一种普遍存在的激酶，调节着大量重要的细胞功能。结合核磁共振、X射线和一系列其他生物物理技术，我们描述了PKA-C的分子内变构通讯，展示了远端突变如何影响底物识别和产物释放。重要的是，我们定义了激活和抑制状态的激酶的结构和动态特征。在我们实验室最近的一项突破中，我们能够重建用于核磁共振研究的完整全酶。我们现在准备在原子水平上研究PKA-C的调节和抑制。在这一竞争性更新中，我们建议研究PKA的变构调节机制及其与体细胞突变相关的功能失调状态。具体地说，德国维尔茨堡大学的Davide CaleBiro发现了一系列与腺瘤相关库欣综合征(AA-CS)相关的PKA-C突变。此外，桑迪·西蒙在洛克菲勒大学的实验室发现了一个PKA-C嵌合体，该嵌合体与纤维板层状肝细胞癌的进展有关。为了研究这些突变的影响，我们与Susan Taylor(UCSD)组成了一个联盟，并将与CaleBiro和Simon小组合作，使用核磁共振光谱、热力学和动力学方法相结合的方法来阐明这些疾病突变产生的分子内和分子间信号的变化。在AIM1中，我们将探讨PKA-C在全酶组装中的作用以及AA-CS突变体如何影响R/C复合体。在AIM2中，我们将分析AA-CS突变如何影响内源和外源抑制物对PKA-C的调节。在AIM3中，我们将研究PKA-C嵌合结构(PKA-CDNAJB1)的结构和动力学，以了解融合的伴侣如何影响伪底物的识别和调节，从而退化为肝癌。由于这些突变体的基态是相同的，不同的功能必须编码在只有核磁共振光谱才能以原子分辨率揭示的激发态中。了解这些突变引起的激酶能量格局的变化是阐明导致疾病的分子机制的关键。这些信息将有助于设计新的策略来控制创新疗法中的激酶活性。