Mechanistic Analyses of kinase signaling complexes

致癌激酶融合蛋白代表一类重要的癌症驱动因子。 纤维板层型肝细胞癌（FLHCC）是一种罕见的肝癌， 影响没有肝病史的青少年和年轻人。它是由 J-PKAca，其是热休克共伴侣DNAJB 1的J结构域的激酶融合嵌合体 与PKA的催化亚基PKAca，其已被用作激酶的模型系统 家庭在过去的40年。我们确定了嵌合RIa 2：J-PKR 2复合物，这是第一个 嵌合PKA全酶及其野生型对应物RIa 2：PKA 2全酶。后续 这项工作揭示了RIa嵌合体和野生型 全酶构象它们的生物学相关性来自于对这些 结构连同生物化学和生物物理数据。我们继续研究 PKA复合物J结构域融合的结构与调控。利用结构知识， 我们正在进一步开发针对这种致命的抑制剂化合物， FLHCC的儿科癌症驱动因子J-PKAca。此外，我们对J-PKAca的研究可以提供 一个探索其他癌症中致癌激酶融合转化途径的模型。 RAF激酶是Ras信号通路中的关键中间体，它们本身是 人类癌症的主要驱动因素。阐明调节RAF的分子机制 在人类疾病状态下， 一个重大的科学挑战我们确定了全长BRAF复合物的冷冻电镜结构， 来源于哺乳动物细胞：自身抑制，单体BRAF：14-3-32：MEK和BRAF：14-3-32 复合物，和结合于受体的二聚BRAF 2：14-3-32复合物。这些结果，连同 基于结构的突变数据，提供了关于RAS结合如何促进 BRAF单体向二聚体的转变。我们继续进一步阐明结构和 RAF激酶家族成员之间的调节差异， 了解这一关键致癌途径的调节。我们组的一个主要焦点是 是富含亮氨酸的重复序列激酶LRRK 1和LRRK 2的结构和调节。他们是 含有两个推定的催化结构域（一个GTTRO ROCO结构域）的大的多结构域蛋白质 和激酶结构域，以及犰狳、锚蛋白、富含亮氨酸和WD 40结构域。LRRK 1是 由于缺乏N-末端Armadillo重复结构域，LRRK 2略小于LRRK 2。尽管 类似的结构域组织，LRRK 1和LRRK 2具有不同的相互作用组， 生理功能。增强激酶活性的LRRK 2突变是一种主要的遗传学机制， 遗传性帕金森病（PD）的贡献者。最常见的LRRK 2患者 基因突变也会增加患几种癌症的风险。有趣的是，LRRK 1 未显示与PD或癌症相关，但在骨骼中具有重要作用 生物学通过揭示LRRK 1和LRRK 2，将大大增强目前对LRRK 1和LRRK 2的理解。 不同功能状态的分子机制。我们学习的长期目标 是为了更好地了解这些大型多结构域激酶如何影响人类健康。 我们正在进行的研究旨在全面了解非活动状态 通过揭示LRRKs的结构和分子机制， 全长LRRK 1和LRRK 2，无论是单独的还是与调节蛋白或底物复合的， 如14-3-3蛋白和Rab小GTP酶。总的来说，我们的目标是获得更好的 了解LRRK蛋白如何在健康和疾病状态下发挥作用， 发展针对这一途径的治疗策略的愿景。

Oncogenic kinase fusion proteins represent an important class of cancer drivers. Fibrolamellar hepatocellular carcinoma (FLHCC) is a rare liver cancer that predominantly affects adolescent and young adults with no history of liver diseases. It is driven by J-PKAca, which is a kinase fusion chimera of the J-domain of heat shock co-chaperone DNAJB1 with PKAca, the catalytic subunit of PKA, which has been used as a model system for the kinase family for the last 40 years. We determined the chimeric RIa2:J-PKAca2 complex, the first for chimeric PKA holoenzymes, and its wild-type counterpart RIa2:PKAca2 holoenzyme. Subsequent work has revealed mechanistic insights with respect to the RIa chimeric and wild-type holoenzyme conformations. Their biological relevance has been derived from analysis of these structures together with biochemical and biophysical data. We continue to study the impact of J-domain fusion of PKA complex structures and regulation. Using the structural knowledge we have gained, we are further developing inhibitor compounds directed against this fatal pediatric cancer driver J-PKAca for FLHCC. Additionally, our studies of J-PKAca could provide a model for exploring the pathways of oncogenic kinase fusion transformation in other cancers. The RAF kinases are key intermediates in the Ras signaling pathway, and they themselves are prominent drivers of human cancer. Elucidating the molecular mechanisms that regulate RAF signaling and identifying strategies to disrupt signal transmission in human disease states is a major scientific challenge. We determined cryo-EM structures of full-length BRAF complexes derived from mammalian cells: autoinhibited, monomeric BRAF:14-3-32:MEK and BRAF:14-3-32 complexes, and an inhibitor-bound, dimeric BRAF2:14-3-32 complex. These results, together with structure based mutational data, provide insights regarding how RAS binding facilitates the BRAF monomer to dimer transition. We continue to further elucidate the structural and regulatory differences between individual members of the RAF kinase family, with a long-term view of understanding the regulation of this key oncogenic pathway. A main focus in my group is the structure and regulation of the leucine-rich repeat kinase LRRK1 and LRRK2.. They are large multi-domain proteins containing two putative catalytic domains, a GTPase ROCO domain and a kinase domain, in addition to armadillo, ankyrin, leucin rich and WD40 domains. LRRK1 is slightly smaller than LRRK2 due to the lack of an N-terminal armadillo repeat domain. Despite similar domain organizations, LRRK1 and LRRK2 have distinct interactomes and distinct physiological functions. Mutations in LRRK2 that enhance kinase activity are a major genetic contributor to inherited Parkinson's disease (PD). Patients with the most common LRRK2 mutation can also have an overall increased risk of several cancers. Interestingly, LRRK1 has not been shown to associate with PD or cancer, but instead has an important role in bone biology. The current understanding of LRRK1 and LRRK2 will be greatly enhanced by revealing molecular mechanisms of their different functional states. The long-term goal of our studies is to gain a better understanding of how these large multi-domain kinases affect human health. Our ongoing studies are aimed at obtaining a comprehensive understanding of the inactive state of the LRRKs and their activation by revealing the structures and molecular mechanisms of full-length LRRK1 and LRRK2, both alone and in complex with regulatory proteins or substrates, such as the 14-3-3 proteins and the Rab small GTPases. Broadly, our goal is to gain a better understanding of how the LRRK proteins function in health and disease states, with an extended vision of developing therapeutic strategies to target this pathway.