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通路的缺陷而出错时，癌症就会发生。由RasGTPase调控的信号通路就是其中之一，它的功能是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通路的关键中间体，将活化的Ras信号传递给下游蛋白激酶MEK和ERK。哺乳动物有三种Raf蛋白，A-Raf， B-Raf和C-Raf（也称为Raf-1）。正如我们所期望的那样，对于参与细胞信号传导的蛋白质，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上游调控因子（如受体酪氨酸激酶和Ras）的突变或扩增，通常会在含有这些等位基因的肿瘤中通过Raf/MEK/ERK级联导致组成性信号传导。此外，Raf蛋白本身的突变可以作为疾病驱动因素发挥作用。C-Raf的种系突变是Noonan和LEOPARD综合征的病因，而B-Raf突变见于Noonan、LEOPARD和心皮肤（CFC）综合征，其中75%的CFC患者发生B-Raf突变。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都观察到B-Raf的体细胞突变。在过去的财政年度，我们的研究已经阐明了几个重要的机制，有助于正常和突变Raf信号的调节。我们的研究表明，KSR1支架在调节响应生长因子处理的质膜发出的Raf信号的强度和持续时间方面起着关键作用。此外，我们发现KSR表达水平可以改变Raf抑制剂对致癌Ras到ERK信号传导的影响。具体而言，KSR1与C-Raf竞争抑制剂诱导的与B-Raf的结合，从而减弱了这些药物对ERK信号传导的矛盾激活作用。我们的研究还表明，B-Raf激酶的致癌潜能可以通过特定的磷酸化事件（例如，抑制反馈位点的磷酸化和介导14-3-3结合的残基的磷酸化）和蛋白质相互作用（例如，14-3-3结合和Raf二聚化）而改变。此外，我们发现Raf二聚化对于人类疾病相关的Raf突变体诱导的信号上调至关重要，这些突变体具有中度、低或激酶活性受损，或者在该途径由活化的RTK或Ras蛋白诱导的情况下。我们的工作进一步揭示了调节Raf二聚化的体细胞突变具有改变Ras通路信号升高的人类疾病状态的进展和治疗的潜力。最后，我们的工作提供了第一个“原理证明”，即抑制Raf二聚化可以在需要二聚化的条件下抑制Raf信号。综上所述，这些发现对于治疗Ras通路信号升高的人类疾病状态和确定Raf二聚体界面作为治疗靶点具有重要意义。

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, serving to relay signals from activated Ras to the downstream protein kinases, MEK and ERK. 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 are causative for Noonan and LEOPARD syndromes, whereas B-Raf mutations are found in Noonan, LEOPARD, and cardiofaciocutaneous (CFC) 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. During this past fiscal year, our research has elucidated several important mechanisms contributing to the regulation of both normal and mutant Raf signaling. Our studies have revealed that the KSR1 scaffold plays a critical role in modulating the intensity and duration of Raf signaling emanating from the plasma membrane in response to growth factor treatment. In addition, we have found that the KSR expression levels can alter the effects of Raf inhibitors on oncogenic Ras to ERK signaling. Specifically, KSR1 competes with C-Raf for inhibitor-induced binding to B-Raf and in doing so attenuates the paradoxical activating effect of these drugs on ERK signaling. Our studies have also shown that the oncogenic potential of the B-Raf kinase can be altered by specific phosphorylation events (e. g., phosphorylation on inhibitory feedback sites and the phosphorylation of residues that mediate 14-3-3 binding) and protein interactions (e.g., 14-3-3 binding and Raf dimerization). Moreover, we have found that Raf dimerization is critical for upregulated signaling induced by human disease-associated Raf mutants with moderate, low or impaired kinase activity, or in cases where the pathway is induced by activated RTK or Ras proteins. Our work has further revealed that somatic mutations which modulate Raf dimerization have the potential to alter the progression and treatment of human disease states with elevated Ras pathway signaling. Finally, our work has provided the first 'proof of principle' that inhibiting Raf dimerization can suppress Raf signaling under conditions where dimerization is required. Taken together, these findings have important implications for the treatment of human disease states with elevated Ras pathway signaling and identify the Raf dimer interface as a therapeutic target.