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，癌症通常会出现。由RasGT 3调节的信号传导途径是这样的途径之一，并且其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/苏氨酸激酶家族的成员是Ras途径中的关键中间体，用于将信号从活化的Ras传递到下游蛋白激酶MEK和ERK。存在三种哺乳动物Raf蛋白，A-Raf、B-Raf和C-Raf（也称为Raf-1）。正如可以预期的那样，Raf激酶可以直接导致致癌转化和其他人类疾病状态。例如，Raf的上游调节因子（如受体酪氨酸激酶和Ras）的突变或扩增经常导致通过携带这些等位基因的肿瘤中的Raf/MEK/ERK级联的组成型信号传导。此外，Raf蛋白本身的突变可以作为疾病驱动因素。C-Raf的种系突变是努南综合征和LEOPARD综合征的病因，而B-Raf突变见于努南综合征、LEOPARD综合征和心面皮肤（CFC）综合征，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、肺癌和乳头状甲状腺癌中观察到B-Raf的体细胞突变。我们的研究已经阐明了几个重要的机制，有助于正常和突变体Raf信号的调节。我们的研究表明，KSR 1支架在调节响应于生长因子处理而从质膜发出的Raf信号的强度和持续时间中起着关键作用。此外，我们的工作提供了关于KSR 1支架如何响应生长因子信号从细胞质募集到质膜的见解。通过结构/功能分析，我们的研究表明，KSR 1的保守的CA 1区域形成了一个扩展的无菌-α-基序结构域，其在生长因子信号传导时暴露并结合质膜中发现的磷脂。因此，KSR 1结合的MEK定位于细胞表面，在那里它可以被激活的Raf激酶磷酸化。最后，我们的研究表明，KSR 1的表达水平可以改变ATP竞争性Raf抑制剂对致癌Ras对ERK信号转导的影响。具体而言，KSR 1与C-Raf竞争与B-Raf的结合，从而减弱这些药物对ERK信号传导的矛盾激活作用。关于Raf蛋白本身的调节，我们的研究表明，B-Raf激酶的致癌潜力可以通过特定的磷酸化事件（如磷酸化）改变。例如，在一个实施例中，抑制性反馈位点上的磷酸化和介导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）。此外，我们已经发现Raf二聚化对于由具有中等、低或受损激酶活性的人类疾病相关Raf突变体诱导的上调信号传导，或在其中途径由活化的RTK或Ras蛋白诱导的情况下是关键的。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具有升高的Ras通路信号传导的人类疾病状态的进展和治疗的潜力。此外，我们的再研究提供了第一个“原理证明”，即在需要二聚化的条件下，抑制Raf二聚化可以抑制Raf信号传导。总之，这些发现对于治疗具有升高的Ras通路信号传导的人类疾病状态具有重要意义，并将Raf二聚体界面鉴定为治疗靶点。最后，我们最近的研究发现了一个以前未知的抑制Ras/Raf/MEK/ERK信号传导的机制，这是由应激激活的JNK级联介导的。我们已经发现，Ras途径的关键成分，包括RasGEFSos 1和Rafs，在多个S/TP位点上被磷酸化，与JNK激活有关，并且这些位点的过度磷酸化使得Rafs和Sos 1对上游信号无反应。这种磷酸调节回路被癌症治疗剂（例如Rigosertib和Paclitazone/Taxol）以及JNK级联的生理调节剂所参与，所述癌症治疗剂通过有丝分裂和氧化应激激活JNK，并且所述磷酸调节回路在细胞应激条件期间作为信号传导检查点发挥功能以抑制Ras途径信号传导。

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. 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, our work has provided insight regarding how the KSR1 scaffold is recruited from the cytosol to the plasma membrane in response to growth factor signals. Through structure/function analysis, our studies showed that the conserved CA1 region of KSR1 forms an extended sterile-alpha-motif domain, which upon growth factor signaling, becomes exposed and binds phospholipids found in the plasma membrane. As a result, KSR1-bound MEK is localized to the cell surface where it can be phosphorylated by activated Raf kinases. Finally, our studies have revealed that the expression levels of KSR1 can alter the effects of ATP-competitive 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. In regard to the regulation of the Raf proteins themselves, our studies have 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. In addition, our reasearch 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. Finally, our recent studies have uncovered a previously unknown rote for the inhibition of the Ras/Raf/MEK/ERK signaling that is mediated by the stress-activated JNK cascade. We have found that key Ras pathway compenents, including the RasGEF Sos1 and the Rafs, are phosphorylated on multiple S/TP sites in resonse to JNK activation and that the hyperphosphorylation of these sites renders the Rafs and Sos1 unresponsive to upstream signals. This phospho-regulatory circuit is engaged by cancer therapeutics, such as Rigosertib and Paclitaxel/Taxol, that activate JNK through mitotic and oxidative stress as well as by physiological regulators of the JNK cascade and my function as a signaling checkpoint to suppress Ras pathway signaling during conditions of cellular stress.