Mechanistic Analyses of kinase signaling complexes

癌基因融合蛋白J-PKAca是肝癌发生的主要驱动力。在J-PKAca项目中，我们提出了以下问题：(1)J-结构域融合对PKA全酶结构和功能有何影响？(2)融合癌蛋白能否作为一种特异性靶向治疗肝癌？我们首次确定了J-PKAca在其最重要的生理状态之一的结构，并进一步确定了其野生型的结构。我们的研究结果表明，J-结构域融合对PKA全酶结构和调控的影响，也为研究人员开发肝癌的治疗方法提供了高度的信息。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体的影响。此外，我们还利用结构分析对针对J-PKAca的抑制剂化合物进行了表征。我们的工作一直并将继续为致癌J-PKACa嵌合体的结构和功能提供洞察力，有可能开发针对这种致命的儿科癌症的治疗方法。RAF激酶的突变，尤其是BRAF，是人类癌症的主要诱因。RAF激酶项目的主要目标是揭示RASs介导的RAF激活过程的结构和分子机制。我们想要解决以下长期存在的问题：(1)自抑制的BRAF和CRAF复合体之间的结构和调控差异是什么？(2)RAS与信号前抑制的RAF单体复合体结合是如何启动形成活性RAF二聚体所需的构象变化的？(3)BRAF同源二聚体复合体与BRAF/CRAF异二聚体复合体有何不同？为了解决这些问题，需要重建生理的RAF复合体，这些复合体在不同状态下的多个高分辨结构，以及对这些结构的仔细分析。我们最近关于BRAF络合物的工作导致了多种新的高分辨率的全长生理BRAF络合物在自抑制和活性状态下的低温电子显微镜结构的确定。这些结果为了解RAS激活时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复合体和活性的BRAF/CRAF异源二聚体复合体的分子机制。根据我们综合的结构和生化信息，我们处于一个独特的位置，可以促进对RAF信号在健康和疾病中的机械理解。LRRK2基因突变是遗传性帕金森病的主要遗传因素。带有最常见的LRRK2突变的患者也可能总体上增加患几种癌症的风险。鉴于有关LRRKs的高分辨率结构和机制信息有限，我们建议解决以下基本问题：(1)全长LRRK蛋白的结构是什么，LRRK2是如何被致病突变错误调控的？(2)LRRK2的结构和活性如何受到与上游调控因子的结合的影响？我们的初步低温电子显微镜数据表明，LRRK2和LRRK1都是紧凑的二聚体，这表明了广泛的分子间和分子内相互作用以及潜在的调控关系。我们的工作将全面了解LRRK蛋白的结构和功能，并有可能为LRRK2驱动的疾病治疗的发展创造新的假设。

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.