Structual and function of kinase signaling complexes

RAF激酶家族（a -RAF、B-Raf和C-Raf）是RTK-RAS-RAF-MEK信号级联的核心组成部分，在指导细胞生长、分化和存活中起着重要作用。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细胞质中。与活性gtp结合的Ras直接相互作用破坏Raf激酶的自抑制状态，诱导Raf二聚体的形成和激活。B-Raf和C-Raf在响应Ras激活时比A-Raf更活跃，B-Raf/C-Raf异源二聚体在Ras依赖性信号传导中占主导地位。尽管Raf激酶对控制细胞生长至关重要，但由于激活过程的复杂性，其激活机制尚不完全清楚。本项目的主要目的是阐明Raf活性调控的分子机制。我们的目标是确定B-Raf和C-Raf激酶单体是如何被自抑制的，以及自抑制复合物中的辅助因子是如何促进自抑制和稳定的。我们还旨在定义活性B- raf同型二聚体和B/C异型二聚体的结构。我们将进一步了解Raf的激活过程，并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，我们采用跨学科的方法，结合结构生物学，生物化学和细胞生物学技术。项目2：亮氨酸富重复激酶2 （LRRK2）及其同源物LRRK1的结构-功能分析LRRK2是家族性帕金森病（PD）的主要遗传因子，目前是PD药物设计中最有希望的治疗靶点之一。LRRK2是一个大的多结构域蛋白（2527个残基），包含两个假定的催化结构域：GTPase （ROC-COR）结构域和激酶结构域以及其他结构域。PD病理突变聚集在GTPase和激酶结构域内，并增加激酶活性。LRRK2激酶结构域突变携带者患癌症的风险总体增加，尤其是女性激素相关癌症和乳腺癌。然而，我们对LRRK2的结构知之甚少，对其活性调控的了解就更少了。LRRK2及其单线同源物LRRK1与Raf激酶属于同一家族：酪氨酸激酶样蛋白激酶家族。LRRK1与帕金森病和癌症没有关联。目前对LRRK1的结构和功能知之甚少。由于缺乏结构信息，目前对LRRKs的认识严重受阻，仅确定了LRRK2的ROC和WD40结构域结构。对这些领域的全局关系和这些领域的功能相互作用没有深入的了解。许多重要的机制仍有待阐明，包括结构域如何相互作用和调节，GTPase和激酶活性如何被调节，以及这些活性如何促进LRRK2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化方法来推进对LRRK2和LRRK1结构、构象状态和调控的理解。该项目将产生的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3：激酶融合蛋白DNAJB1-PKACA的结构和功能分析纤维层状肝细胞癌（FLHCC）是一种主要影响青少年和年轻人的肝癌。嵌合的DNAJB1-PKACA蛋白是由19号染色体上热休克蛋白DNAJB1的第一个外显子与蛋白激酶PKA催化亚基PKACA的第一个外显子之间400 kB的缺失产生的，已被认为是FLHCC的驱动因素。抑制DNAJB1-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DNAJB1-PKACA嵌合体的结构和动力学，以及它是如何被调控的，以制定针对这种致命儿科癌症的精准医学策略。野生型PKA在细胞中处于失活状态，是一种由两个PKACA催化亚基和一个调节亚基同二聚体组成的全酶。有四种功能上不冗余的r -亚基亚型。我们的目的是研究dnajb1结构域融合对PKACA全酶形成和抑制的影响。此外，我们进一步努力设计选择性阻断DNAJB1-PKACA活性或RIa2:DNAJB1-PKACA2全酶激活的小分子。

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.