Mechanistic Analyses of kinase signaling complexes

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全酶结构和功能的影响是什么？(2)融合癌蛋白能否作为FLHCC的特异性靶向治疗方法？我们首先确定了J-PKAca在其最重要的生理状态之一的结构，在这种状态下，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。我们的研究结果证明了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FLHCC治疗方法提供了大量信息。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物的影响。此外，我们还利用结构分析表征了针对J-PKAca的抑制剂化合物。我们的工作已经并将继续提供对致癌J-PKACa嵌合体的结构和功能的见解，具有开发针对这种致命儿科癌症的治疗方法的潜力。RAF激酶尤其是BRAF的突变是人类癌症的主要诱因。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控差异是什么？(2) RAS与预信号自抑制RAF单体复合物的结合如何引发形成活性RAF二聚体所需的构象变化？(3) BRAF同型二聚体复合物与BRAF/CRAF异源二聚体复合物有何不同？为了解决这些问题，需要重建生理RAF复合物，在不同状态下这些复合物的多个高分辨率结构，并仔细分析这些结构。我们最近对BRAF复合物的研究导致了在自抑制和活性状态下全长生理BRAF复合物的多个新的高分辨率冷冻电镜结构的确定。这些结果为了解RAS激活后BRAF的结构和生化变化提供了分子基础。我们继续破译预信号自抑制的CRAF复合物和活跃的BRAF/CRAF异源二聚体复合物的分子机制。基于我们结合的结构和生化信息，我们处于一个独特的位置，以推进对健康和疾病中RAF信号传导的机制理解。LRRK2突变是遗传性帕金森病的主要遗传因素。最常见的LRRK2突变患者患几种癌症的总体风险也会增加。鉴于目前关于LRRK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如何被致病性突变错误调节？(2) LRRK2与上游调控因子结合对其结构和活性有何影响？我们的初步低温数据显示，LRRK2和LRRK1都是紧凑的二聚体，表明分子间和分子内广泛的相互作用和潜在的调控关系。我们的工作将提供对LRRK蛋白结构和功能的全面了解，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.