Mechanistic Analyses of kinase signaling complexes

激酶信号复合物的机制分析

• 批准号: 10486948
• 负责人: Ping Zhang
• 金额: $ 108.28万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10486948
• 关键词: Address; Affect; BRAF gene; Binding; Biochemical; Biological Process; Biophysics; Chimera organism; Complex; Cryoelectron Microscopy; Cyclic AMP-Dependent Protein Kinases; Data; Development; Disease; Family; Fusion Oncogene Proteins; Genetic; Goals; Health; Holoenzymes; Human; Inherited; LRRK2 gene; Leucine-Rich Repeat; Link; Malignant Childhood Neoplasm; Malignant Neoplasms; Molecular; Molecular Conformation; Mutation; Oncogenic; Pathogenicity; Patients; Phosphotransferases; Physiological; Positioning Attribute; Process; Protein Kinase; Regulation; Research; Research Personnel; Risk; Signal Pathway; Signal Transduction; Structure; Therapeutic; Work; X-Ray Crystallography; base; biological systems; clinically relevant; dimer; inhibitor/antagonist; insight; interest; monomer; novel therapeutic intervention; programs; raf Kinases; reconstitution

The oncogenic kinase fusion J-PKAca is the primary driver for FLHCC. In the J-PKAca project, we ask the following questions: (1) What is the impact of the J-domain fusion on PKA holoenzyme structures and function? (2) Can the fusion oncoprotein be specifically targeted as a therapeutic approach for FLHCC? We were the first to determine the structure of J-PKAca in one of its most important physiological states, where it is associated in a holoenzyme complex, and further determined the structure of its wild-type counterpart. Our findings have demonstrated the J-domain fusion impact on PKA holoenzyme structure and regulation and are also highly informative to researchers for developing FLHCC therapeutics. We are continuing our studies on the impact of J-domain fusion on PKA holoenzymes and larger signaling complexes. Furthermore, we are also characterizing inhibitor compounds directed against J-PKAca using structural analysis. Our work has been and will continue to provide insights into the structure and function of the oncogenic J-PKACa chimera, with a potential to develop therapeutics against this fatal pediatric cancer. Mutations in the RAF kinases especially BRAF are a major contributor to human cancers. The major goal of the RAF kinase project is to reveal the structural and molecular mechanisms of the RASmediated RAF activation process. We want to address the following longstanding questions: (1) What are the structural and regulatory differences between autoinhibited BRAF and CRAF complexes? (2) How does RAS binding to the presignaling autoinhibited RAF monomer complex initiate the conformational changes required to form active RAF dimers? (3) Do BRAF homodimer complexes differ from BRAF/CRAF heterodimer complexes? To address these questions, reconstitution of physiological RAF complexes, multiple highresolution structures of these complexes in various states, and careful analysis of these structures are required. Our recent work on the BRAF complexes led to the determination of multiple new highresolution cryoEM structures of fulllength physiological BRAF complexes in both autoinhibited and active states. These results provide molecular basis for understanding the structural and biochemical changes of BRAF that occur upon RAS activation. We continue to decipher the molecular mechanism of the presignaling autoinhibited CRAF complex and the active BRAF/CRAF heterodimer complex. We are in a unique position, based on our combined structural and biochemical information, to advance the mechanistic understanding of RAF signaling in health and disease. Mutations in LRRK2 are a major genetic contributor to inherited PD. Patients with the most common LRRK2 mutation can also have an overall increased risk of several cancers. Given the limited highresolution structural and mechanistic information regarding the LRRKs, we propose to address the following essential questions: (1) What are the structures of fulllength LRRK proteins and how is LRRK2 misregulated by pathogenic mutations? (2) How is the structure and activity of LRRK2 affected by binding to upstream regulators? Our preliminary cryoEM data showed that both LRRK2 and LRRK1 are compact dimers, suggesting extensive inter and intramolecular interaction and potential regulatory relationships. Our work will provide a comprehensive understanding of the structure and function of LRRK proteins and has the potential to create new hypotheses for the development of LRRK2 driven disease therapeutics.

致癌激酶融合J-PKAca是FLHCC的主要驱动因素。在J-PKAca项目中，我们提出了以下问题：（1）J结构域融合对PKA全酶的结构和功能有什么影响？(2)融合癌蛋白能否作为FLHCC的特异性靶向治疗方法？我们是第一个确定J-PKAca在其最重要的生理状态之一的结构的人，其中它与全酶复合物相关，并进一步确定了其野生型对应物的结构。我们的研究结果已经证明了J-结构域融合对PKA全酶结构和调节的影响，并且对研究人员开发FLHCC治疗方法也具有高度的信息性。我们正在继续研究J结构域融合对PKA全酶和更大的信号复合物的影响。此外，我们还使用结构分析来表征针对J-PKAca的抑制剂化合物。我们的工作已经并将继续深入了解致癌J-PKACa嵌合体的结构和功能，有可能开发针对这种致命的儿科癌症的治疗方法。RAF激酶尤其是BRAF中的突变是人类癌症的主要贡献者。RAF激酶项目的主要目标是揭示RAS介导的RAF激活过程的结构和分子机制。我们希望解决以下长期存在的问题：（1）自抑制BRAF和CRAF复合物之间的结构和调节差异是什么？(2)RAS如何与前信号自身抑制RAF单体复合物结合，引发形成活性RAF二聚体所需的构象变化？(3)BRAF同源二聚体复合物与BRAF/CRAF异源二聚体复合物不同吗？为了解决这些问题，生理RAF复合物的重建，这些复合物在各种状态下的多个高分辨率结构，并仔细分析这些结构是必需的。我们最近对BRAF复合物的研究确定了多种新的高分辨率cryoEM结构的全长生理BRAF复合物在自抑制和活性状态。这些结果为理解RAS激活后BRAF的结构和生化变化提供了分子基础。我们继续破译前信号自身抑制的CRAF复合物和活性BRAF/CRAF异二聚体复合物的分子机制。基于我们结合的结构和生物化学信息，我们处于独特的位置，以促进对健康和疾病中RAF信号传导的机制理解。LRRK 2突变是遗传性PD的主要遗传因素。具有最常见的LRRK 2突变的患者也可能具有几种癌症的总体风险增加。鉴于有限的高分辨率的结构和机制的信息，关于LRRK，我们建议解决以下基本问题：（1）全长LRRK蛋白的结构是什么，LRRK 2是如何被致病性突变失调？(2)LRRK 2的结构和活性如何受到与上游调节因子结合的影响？我们的初步cryoEM数据表明LRRK 2和LRRK 1都是紧凑的二聚体，这表明广泛的分子间和分子内相互作用以及潜在的调节关系。我们的工作将提供对LRRK蛋白的结构和功能的全面了解，并有可能为LRRK 2驱动的疾病治疗方法的发展创造新的假设。