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.

致癌激酶融合蛋白代表着重要的癌症驱动因素。纤维素肝细胞癌（FLHCC）是一种罕见的肝癌，主要影响没有肝病病史的青少年和年轻人。它是由J-PKACA驱动的，J-PKACA是热电冲击共伴侣DNAJB1的激酶融合嵌合体与PKACA（PKA的催化亚基），在过去40年中，PKA的催化亚基一直用作激酶家族的模型系统。我们确定了Chimeric RIA2：J-PKACA2复合物，第一个用于嵌合PKA Holoenzymes及其野生型RIA2：PKACA2 Holoenzyme。随后的工作揭示了关于RIA嵌合和野生型全酶构象的机理见解。它们的生物学相关性来自对这些结构以及生化和生物物理数据的分析。我们继续研究PKA复合结构和调节的J域融合的影响。利用我们获得的结构知识，我们正在进一步开发针对这种致命的小儿癌驱动器J-PKACA的抑制剂化合物J-PKACA。此外，我们对J-PKACA的研究可以提供一个模型，用于探索其他癌症中致癌激酶融合转化的途径。 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杰出驱动因素。阐明调节RAF信号传导并确定破坏人类疾病状态信号传播的策略的分子机制是一项主要的科学挑战。我们确定了源自哺乳动物细胞的全长BRAF复合物的冷冻EM结构：自身抑制，单体BRAF：14-3-32：MEK和BRAF：14-3-32复合物，以及抑制剂结合的Dimeric BRAF2：14-3-32复合物。这些结果以及基于结构的突变数据提供了有关RAS结合如何促进BRAF单体与二聚体过渡的见解。我们继续进一步阐明RAF激酶家族各个成员之间的结构和调节差异，并长期观察了对这一关键致癌途径的调节。我小组的主要重点是富含亮氨酸的重复激酶LRRK1和LRRK2的结构和调节。它们是大型多域蛋白质，其中包含两个推定的催化域，一个GTPase Roco结构域和一个激酶结构域和一个激酶域，除了Armadillo，ankadillo，Ankyrin，ankyrin，liucin，Leucin，Rish and rice and and and。由于缺乏N末端腋窝重复域，LRRK1比LRRK2小略小。尽管有类似的领域组织，但LRRK1和LRRK2具有独特的相互作用体和不同的生理功能。 LRRK2中增强激酶活性的突变是遗传帕金森氏病（PD）的主要遗传学因素。具有最常见的LRRK2突变的患者也可能会增加多种癌症的风险。有趣的是，LRRK1尚未证明与PD或癌症相关，而是在骨骼生物学中具有重要作用。通过揭示其不同功能状态的分子机制，对LRRK1和LRRK2的当前理解将大大增强。我们研究的长期目标是更好地了解这些大型多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单独的LRRK1和LRRK2的结构和分子机制来获得对LRRK的不活跃状态及其激活的全面理解，这些结构和分子机制单独使用，并与调节蛋白或底物（例如14-3-3-3蛋白）以及RAB小型Gtpase（例如14-3-3-3蛋白）相关。从广义上讲，我们的目标是更好地了解LRRK蛋白在健康和疾病状态中如何发挥作用，并具有开发针对该途径的治疗策略的广泛愿景。