Structual and function of kinase signaling complexes

激酶信号复合物的结构和功能

• 批准号: 10262432
• 负责人: Ping Zhang
• 金额: $ 101.86万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 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/10262432
• 关键词: Adolescent and Young Adult; Affect; Architecture; Biochemical; Biochemistry; Biological; Catalytic Domain; Cells; Cellular biology; Chimera organism; Chimeric Proteins; Chromosome 19; Complex; Cryoelectron Microscopy; Cyclic AMP-Dependent Protein Kinases; Cytosol; Disabled Persons; Disease; Drug Design; Exons; Family; Fibrolamellar Hepatocellular Carcinoma; GTP Binding; Genetic; Goals; Guanosine Triphosphate Phosphohydrolases; Heat shock proteins; Holoenzymes; Homologous Gene; Human; Intramural Research Program; LRRK2 gene; Leucine-Rich Repeat; Link; MEKs; Malignant Childhood Neoplasm; Malignant Neoplasms; Malignant neoplasm of liver; Molecular; Molecular Conformation; Mutation; Oncogenic; Output; Parkinson Disease; Pathologic; Pathology; Phosphotransferases; Play; Process; Protein Family; Protein Isoforms; Protein Kinase; Protein Tyrosine Kinase; Proteins; Proto-Oncogene Proteins c-raf; Ras/Raf; Regulation; Research; Role; Signal Transduction; Structure; Techniques; Tertiary Protein Structure; Therapeutic; WD Repeat; Woman; X-Ray Crystallography; base; cancer risk; cell growth; design; dimer; hormone related cancer; insight; interdisciplinary approach; malignant breast neoplasm; monomer; mutation carrier; novel therapeutic intervention; particle; precision medicine; programs; protein kinase A kinase; raf Kinases; response; small molecule; structural biology; therapeutic target; tumor

Project 1: Structure-function analyses of autoinhibited and active RAF kinases The family of Raf kinases (A-Raf, B-Raf and C-Raf) constitute core components of the RTK-RAS-RAF-MEK signaling cascade, which plays a major role in directing cell growth, differentiation and survival. Raf activity is frequently dysregulated in cancers. In quiescent cells, the wild type Raf kinase exists as an autoinhibited monomer in cytosol. Direct interaction with active GTP-bound Ras disrupts the autoinhibited state of Raf kinase and induces Raf dimer formation and activation. B-Raf and C-Raf are more active than the A-Raf in response to Ras activation and B-Raf/C-Raf heterodimers predominate in Ras-dependent signaling. Although Raf kinases are critical for controlling cell growth, their mechanism of activation is incompletely understood due to the complexity of the activation process. The major goal of this project is to elucidate the molecular mechanisms of Raf activity regulation. We aim to define how B-Raf and C-Raf kinase monomers are autoinhibited and how co-factors in the autoinhibited complexes contribute to the autoinhibition and stabilization. we also aim to define the architectures of active B-Raf homodimers and B/C hetero dimers. We will further our understanding of the Raf activation process as well as study the structural and functional differences of the Raf isoforms as related to this process. To achieve this goal, we use an interdisciplinary approach that combines structural biology, biochemistry and cell biology techniques. Project 2: Structure-function analyses of Leucine Rich Repeat Kinase 2 (LRRK2) and its homologue LRRK1 LRRK2 is the leading genetic contributor to familial Parkinson's disease (PD) and currently one of the most promising therapeutic targets for drug design in PD. LRRK2 is a large multi-domain protein (2527 residues) containing two putative catalytic domains: a GTPase (ROC-COR) domain and a kinase domain as well as other domains. The PD pathological mutations clusters within the GTPase and kinase domains and increase kinase activities. LRRK2 kinase domain mutation carriers have an overall increased risk of cancer, especially for hormone-related cancer and breast cancer in women. However, little is known about the LRRK2 structure and even less is known about the regulation of its activity. LRRK2 and its singlet homologue LRRK1 belong to the same family as Raf kinases: Tyrosine Kinase Like family of protein kinases. LRRK1 is not shown be associated with PD and cancer. Little is known about the structure and function of LRRK1. The present understanding of LRRKs is severely handicapped by the lack of structural information, as only the ROC and WD40 domain structures of LRRK2 have been determined. There is no insight into the global relationship of the domains and the functional interactions of these domains. Many important mechanisms remain to be elucidated, including how the domains interact with and regulate each other, how the GTPase and kinase activities are regulated, and how these activities contribute to the overall functional output of LRRK2. The major goal to reveal the molecular mechanisms of LRRK function and pathology. We aim to advance the understanding of LRRK2 and LRRK1 structures, conformational states and regulation through interdisciplinary structural and biochemical approaches. This project will generate results that allow us to elucidate the structure and function of LRRKs and create new hypotheses for how to intervene with therapeutic strategies. Project 3: Structure and function analyses of kinase fusion protein DNAJB1-PKACA Fibrolamellar hepatocellular carcinoma (FLHCC) is a liver cancer that predominantly affects adolescents and young adults. The chimeric DNAJB1-PKACA protein is generated by a deletion of 400 kB between the first exon of the heat shock protein DNAJB1 and the first exon of the catalytic subunit of Protein Kinase PKA, PKACA, on one copy of chromosome 19 and has been recognized as the driver of FLHCC. Inhibition of the DNAJB1-PKACA chimeric tumor driver offers tremendous potential to treat FLHCC. We study the structure and dynamics of the DNAJB1-PKACA chimera and how it is regulated with the long-term goal of developing precision medicine strategies against this fatal pediatric cancer. In its inactive state in cells, wildtype PKA exists as a holoenzyme composed of two PKACA catalytic subunits and one regulatory subunit homodimer. There are four functionally nonredundant R-subunit isoforms. We aim to investigate the impact of the presence of the DNAJB1-domain fusion on PKACA holoenzyme formation and inhibition. In addition, we further our effort to design small molecules that selective block DNAJB1-PKACA activity or RIa2:DNAJB1-PKACA2 holoenzyme activation.

项目一：Raf激酶家族（A-Raf、B-Raf和C-Raf）构成RTK-RAS-RAF-MEK信号级联的核心组分，其在指导细胞生长、分化和存活中起主要作用。Raf活性在癌症中经常失调。在静止细胞中，野生型Raf激酶作为自抑制单体存在于胞质溶胶中。与活性GTP结合的Ras的直接相互作用破坏Raf激酶的自抑制状态并诱导Raf二聚体形成和活化。B-Raf和C-Raf比A-Raf对Ras活化的反应更活跃，并且B-Raf/C-Raf异二聚体在Ras依赖性信号传导中占主导地位。尽管Raf激酶对于控制细胞生长是至关重要的，但由于激活过程的复杂性，它们的激活机制尚未完全理解。本项目的主要目标是阐明Raf活性调节的分子机制。我们的目的是确定如何B-Raf和C-Raf激酶单体的自抑制和如何在自抑制复合物的辅助因子有助于自抑制和稳定。我们还旨在定义活性B-Raf同二聚体和B/C异二聚体的结构。我们将进一步了解Raf激活过程，以及研究与此过程相关的Raf亚型的结构和功能差异。为了实现这一目标，我们使用了一种跨学科的方法，结合了结构生物学，生物化学和细胞生物学技术。项目二：富含亮氨酸重复序列激酶2（LRRK 2）及其同源物LRRK 1的结构-功能分析LRRK 2是家族性帕金森病（PD）的主要遗传因素，并且是目前PD药物设计中最有希望的治疗靶点之一。LRRK 2是一个大的多结构域蛋白（2527个残基），含有两个推定的催化结构域：一个GTdR（ROC-COR）结构域和一个激酶结构域以及其他结构域。PD病理性突变聚集在GT3和激酶结构域内并增加激酶活性。LRRK 2激酶结构域突变携带者患癌症的风险总体增加，特别是女性乳腺癌和乳腺癌。然而，人们对LRRK 2的结构知之甚少，对其活性的调控更是知之甚少。LRRK 2及其单态同源物LRRK 1与Raf激酶属于同一家族：酪氨酸激酶样蛋白激酶家族。LRRK 1未显示与PD和癌症相关。关于LRRK 1的结构和功能知之甚少。目前对LRRK的理解受到缺乏结构信息的严重阻碍，因为仅确定了LRRK 2的ROC和WD 40结构域结构。没有深入了解这些域的全局关系和这些域的功能相互作用。许多重要的机制仍有待阐明，包括这些结构域如何相互作用和相互调节，如何调节GTPase和激酶活性，以及这些活动如何有助于LRRK 2的整体功能输出。主要目的是揭示LRRK功能和病理的分子机制。我们的目标是通过跨学科的结构和生物化学方法来促进对LRRK 2和LRRK 1结构，构象状态和调控的理解。该项目将产生结果，使我们能够阐明LRRKs的结构和功能，并为如何干预治疗策略创造新的假设。项目三：激酶融合蛋白DNAJB 1-PKACA的结构和功能分析纤维板层型肝细胞癌（FLHCC）是一种主要影响青少年和年轻人的肝癌。嵌合DNAJB 1-PKACA蛋白是通过在19号染色体的一个拷贝上的热休克蛋白DNAJB 1的第一外显子和蛋白激酶PKA的催化亚基PKACA的第一外显子之间缺失400 kB而产生的，并且已经被认为是FLHCC的驱动因子。抑制DNAJB 1-PKACA嵌合肿瘤驱动因子为治疗FLHCC提供了巨大的潜力。我们研究了DNAJB 1-PKACA嵌合体的结构和动力学，以及如何通过开发针对这种致命儿科癌症的精准医学策略的长期目标来调节它。野生型PKA在细胞中处于失活状态时，以全酶形式存在，由两个PKACA催化亚基和一个调节亚基同源二聚体组成。有四种功能上非冗余的R亚基同种型。我们的目的是调查的DNAJB 1结构域融合PKACA全酶的形成和抑制的存在下的影响。此外，我们进一步努力设计选择性阻断DNAJB 1-PKACA活性或RIa 2：DNAJB 1-PKACA 2全酶激活的小分子。