Regulation of Ras-Dependent Signal Transduction Pathways

当正常细胞功能的控制由于关键信号转导途径的缺陷而出错时，通常会发生癌症。由RasGTPase调控的信号通路就是其中之一，它调节重要的细胞过程，包括增殖、分化、存活和衰老。Raf丝氨酸/苏氨酸激酶家族成员是Ras途径中的关键中间体，直接与激活的Ras结合，在ERK级联中起起始作用，包括Raf、MEK和ERK蛋白激酶。哺乳动物中有三种Raf蛋白，A-Raf、B-Raf和C-Raf(也称为Raf-1)。正如可能预期的那样，在细胞信号转导中起核心作用的蛋白质，Raf激酶可以直接促进致癌转化和其他人类疾病状态。例如，Raf上游调节因子的突变或扩增，如受体酪氨酸激酶和RAS，经常导致携带这些等位基因的肿瘤中通过Raf/MEK/ERK级联反应的结构性信号。此外，Raf蛋白本身的突变也可以作为疾病的驱动因素。C-Raf的种系突变可能是Noonan和豹子Rasony综合征的病因，而B-Raf突变在Noonan、豹子和心面部皮肤(CFC)Rasology综合征中被发现，其中B-Raf突变发生在75%的CFC患者中。此外，在70%的恶性黑色素瘤以及许多结直肠癌、卵巢癌、肺癌和甲状腺乳头状癌中观察到B-Raf的体细胞突变。多年来，我们研究团队的一个主要目标是阐明调节Raf催化活性的机制。这些研究已经确定了关键的磷酸化事件和有助于Raf激活/失活循环的蛋白质相互作用。此外，我们的工作证明了Raf二聚化在正常和疾病相关信号中的重要性，并确定了Raf二聚体界面是一个治疗靶点。最近进行的研究发现了一种新的磷酸调节电路，可以在细胞应激条件下抑制Raf的激活和Ras信号。该通路作为应激激活的信号检查点发挥作用，并可被癌症治疗药物如rigosertib、紫杉醇和长春新碱激活，通过氧化和有丝分裂应激激活JNK/MAPK级联反应。在此回顾期间，我们开发了生物发光共振能量转移(BRET)技术来研究活细胞条件下Raf的调节相互作用。利用该系统对KIT受体酪氨酸激酶和B-Raf V600E之间的信号串扰进行了评估，结果发现KIT信号激活WT-B-Raf可以干扰B-Raf V600E驱动的黑色素瘤的形成。我们还与分子靶标实验室合作，使用BRET技术进行高通量筛选，以确定可以干扰或阻止活细胞中的Raf二聚化或Ras/Raf相互作用的天然产物化合物。作为这项工作的结果，从海洋水母Macrohynchia Philippina中分离到了新的大菲酮型吡咯亚氨基喹，其中两个新鉴定的化合物被发现能够干扰Raf二聚化和ERK级联信号。这些发现表明，大分子化合物的化学支架可以为在人类疾病状态下靶向ERK级联反应提供小分子治疗线索。

Cancer often arises when the control of normal cell function goes awry due to defects in critical signal transduction pathways. The signaling pathway regulated by the RasGTPase is one such pathway, and it functions to modulate vital cellular processes, including proliferation, differentiation, survival, and senescence. Members of the Raf serine/threonine kinase family are key intermediates in the Ras pathway, binding directly to activated Ras and serving as the initiating kinases in the ERK cascade, comprised of the Raf, MEK and ERK protein kinases. There are three mammalian Raf proteins, A-Raf, B-Raf, and C-Raf (also known as Raf-1). As might be expected for proteins so centrally involved in cell signaling, the Raf kinases can directly contribute to oncogenic transformation and other human disease states. For example, mutation or amplification of upstream regulators of Raf, such as receptor tyrosine kinases and Ras, frequently results in constitutive signaling through the Raf/MEK/ERK cascade in tumors harboring these alleles. In addition, mutations in the Raf proteins themselves can function as disease drivers. Germline-mutations in C-Raf can be are causative for Noonan and LEOPARD RASopathy syndromes, whereas B-Raf mutations are found in Noonan, LEOPARD, and cardiofaciocutaneous (CFC) RASopathy syndromes, with B-Raf mutations occurring in 75% of CFC patients. Moreover, somatic mutations in B-Raf are observed in 70% of malignant melanomas as well as in many colorectal, ovarian, lung and papillary thyroid carcinomas. Over the years, a major goal of our research team has been to elucidate the mechanisms that regulate Raf catalytic activity. These studies have led to the identification of critical phosphorylation events and protein interactions that contribute to the Raf activation/inactivation cycle. In addition, our work has demonstrated the importance of Raf dimerization in normal and disease-associated signaling and has identified the Raf dimer interface as a therapeutic target. Studies conducted more recently have led to the discovery of a new phospho-regulatory circuit that can suppress Raf activation and Ras signaling under conditions of cellular stress. This circuit functions as a stress-activated signaling checkpoint and can be engaged by cancer therapeutics such as rigosertib, taxol, and vincristine, that activate the JNK/MAPK cascade through oxidative and mitotic stress. During this review period, we developed bioluminescence resonance energy transfer (BRET) technologies to study Raf regulatory interactions under live cell conditions. Using this system, signaling cross-talk between the KIT receptor tyrosine kinase and B-Raf V600E was evaluated, resulting in the discovery that activation of WT-B-Raf by KIT signaling could interfere with melanoma formation driven by B-Raf V600E. In collaboration with the Molecular Targets Laboratory, we have also used the BRET technology to conduct a high-throughput screen to identify natural product compounds that can disrupt or prevent Raf dimerization or the Ras/Raf interaction in live cells. As a result of this effort, new macrophilone-type pyrroloiminoquines were isolated from the marine hydroid Macrorhynchia philippina, and two of the newly identified compounds were found to disrupt Raf dimerization and ERK cascade signaling. These findings indicate that the chemical scaffold of the macrophilones could provide small-molecule therapeutic leads for targeting the ERK cascade in human disease states.