Mechanistic Analyses of kinase signaling complexes

致癌激酶融合蛋白代表了一类重要的癌症驱动因素。纤维板层状肝细胞癌是一种罕见的肝癌，主要影响青少年和无肝病病史的年轻人。它是由J-PKAca驱动的，它是热休克辅助伴侣DNAJB1的J-结构域与PKA的催化亚单位PKAca的融合嵌合体，在过去的40年里一直被用作激酶家族的模型系统。我们确定了首次发现嵌合PKA全酶的嵌合RIa2：J-PKAca2复合体和野生型RIa2：PKAca2全酶。随后的工作揭示了关于RIA嵌合和野生型全酶构象的机械论见解。它们的生物学相关性是通过分析这些结构以及生化和生物物理数据得出的。我们继续研究J-结构域融合对PKA复杂结构和调控的影响。利用我们已经获得的结构知识，我们正在进一步开发针对这一致命的儿科癌症驱动因子J-PKAca的抑制化合物。此外，我们对J-PKAca的研究可以为探索致癌激酶在其他癌症中的融合转化途径提供一个模型。RAF激酶是RAS信号通路的关键中间体，它们本身就是人类癌症的重要驱动因素。阐明调节RAF信号的分子机制并确定在人类疾病状态下干扰信号传递的策略是一项重大的科学挑战。我们测定了来自哺乳动物细胞的全长BRAF复合体的冷冻-EM结构：自抑制的单体BRAF：14-3-32：MEK和BRAF：14-3-32复合体，以及抑制剂结合的二聚体BRAF2：14-3-32复合体。这些结果，加上基于结构的突变数据，提供了关于RAS结合如何促进BRAF单体到二聚体转变的见解。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控差异，并从长远的角度理解这一关键致癌途径的调控。我的小组的一个主要焦点是富含亮氨酸的重复蛋白激酶LRRK1和LRRK2的结构和调控。它们是含有两个可能的催化结构域的大的多结构域的蛋白质，其中包括一个GTP酶的ROCO结构域和一个激活域，此外还有犰螂、锚蛋白、富含亮蛋白和WD40的结构域。LRRK1比LRRK2略小，这是因为缺乏N-末端的螳螂重复结构域。尽管LRRK1和LRRK2具有相似的结构域组织，但它们具有不同的相互作用和不同的生理功能。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。带有最常见的LRRK2突变的患者也可能总体上增加患几种癌症的风险。有趣的是，LRRK1并未被证明与帕金森病或癌症有关，而是在骨生物学中起着重要作用。通过揭示LRRK1和LRRK2不同功能状态的分子机制，将极大地提高人们对LRRK1和LRRK2的认识。我们研究的长期目标是更好地了解这些大的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的结构和分子机制来全面了解LRRKs的失活状态和激活，LRRK1和LRRK2单独或与调控蛋白或底物(如14-3-3蛋白和Rab小GTP酶)形成复合体。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，并制定针对这一途径的治疗策略。

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.