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Mechanistic Analyses of kinase signaling complexes

激酶信号复合物的机制分析

基本信息

批准号：
10926302
负责人：
Ping Zhang
金额：
$ 149.81万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/10926302
关键词：
Adolescent and Young Adult Affect Ankyrins Armadillo Repeat Automobile Driving BRAF gene Binding Biochemical Biological Biological Models Biological Process Biology Biophysics Catalytic Domain Cell physiology Cells Chimera organism Chimeric Proteins Complex Cryoelectron Microscopy Cyclic AMP-Dependent Protein Kinases Data Disease Family Family Dasypodidae Fibrolamellar Hepatocellular Carcinoma Genetic Goals Guanosine Triphosphate Phosphohydrolases Health Heat-Shock Response Holoenzymes Human Individual Inherited Interdisciplinary Study Knowledge LRRK2 gene Length Leucine-Rich Repeat Link Liver diseases MEKs Malignant Childhood Neoplasm Malignant Neoplasms Malignant neoplasm of liver Mammalian Cell Medicine Modeling Molecular Molecular Chaperones Molecular Conformation Monomeric GTP-Binding Proteins Mutation N-terminal Oncogenic Parkinson Disease Pathologic Pathway interactions Patients Phosphorylation Phosphotransferases Physiological Protein Kinase Proteins Ras Signaling Pathway Recording of previous events Regulation Research Risk Role Signal Transduction Structure System Tertiary Protein Structure Therapeutic Vision WD Repeat Work X-Ray Crystallography bone dimer genetic regulatory protein human disease inhibitor insight interest member monomer novel therapeutic intervention programs protein function raf Kinases transmission process

项目摘要

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.

致癌激酶融合蛋白是一类重要的癌症驱动因子。纤维层状肝细胞癌（FLHCC）是一种罕见的肝癌，主要发生在没有肝脏病史的青少年和年轻人身上。它是由J-PKAca驱动的，J-PKAca是热休克共伴侣DNAJB1的j结构域与PKAca的激酶融合嵌合体，PKAca是PKA的催化亚基，在过去的40年里被用作激酶家族的模型系统。我们确定了嵌合的RIa2:J-PKAca2复合物，这是第一个嵌合的PKA全酶，以及它的野生型对应物RIa2:PKAca2全酶。随后的工作揭示了有关RIa嵌合和野生型全酶构象的机制见解。它们的生物学相关性是通过分析这些结构以及生物化学和生物物理数据得出的。我们将继续研究j域融合对PKA复合物结构和调控的影响。利用我们所获得的结构知识，我们正在进一步开发针对FLHCC的致命儿科癌症驱动因子J-PKAca的抑制剂化合物。此外，我们对J-PKAca的研究可以为探索其他癌症中致癌激酶融合转化的途径提供模型。RAF激酶是Ras信号通路的关键中间体，它们本身是人类癌症的重要驱动因素。阐明调节RAF信号传导的分子机制和确定在人类疾病状态下破坏信号传递的策略是一项重大的科学挑战。我们确定了来自哺乳动物细胞的全长BRAF复合物的低温电镜结构：自抑制的单体BRAF:14-3-3 32、MEK和BRAF:14-3-3 32复合物，以及抑制结合的二聚体braf2:14-3-3 32复合物。这些结果与基于结构的突变数据一起，提供了关于RAS结合如何促进BRAF单体向二聚体转变的见解。我们继续进一步阐明RAF激酶家族个体成员之间的结构和调控差异，以理解这一关键致癌途径的调控的长期观点。我的小组主要关注富含亮氨酸的重复激酶LRRK1和LRRK2的结构和调控。它们是大的多结构域蛋白，含有两个假定的催化结构域，一个GTPase ROCO结构域和一个激酶结构域，此外还有犰狳结构域、锚蛋白结构域、富白细胞结构域和WD40结构域。由于缺乏n端犰狳重复结构域，LRRK1比LRRK2略小。尽管结构域组织相似，但LRRK1和LRRK2具有不同的相互作用组和不同的生理功能。增强激酶活性的LRRK2突变是遗传性帕金森病（PD）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。有趣的是，LRRK1并未被证明与PD或癌症相关，而是在骨生物学中发挥重要作用。揭示LRRK1和LRRK2不同功能状态的分子机制，将极大地增强目前对它们的认识。我们研究的长期目标是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通过揭示全长LRRK1和LRRK2的结构和分子机制，全面了解LRRKs的失活状态及其激活，无论是单独的还是与调控蛋白或底物（如14-3-3蛋白和Rab小GTPases）复合的。总的来说，我们的目标是更好地了解LRRK蛋白在健康和疾病状态下的功能，并以更长远的眼光开发针对这一途径的治疗策略。