Regulation of Ras-Dependent Signal Transduction Pathways

Ras 依赖性信号转导途径的调节

• 批准号: 9556265
• 负责人: Deborah Morrison
• 金额: $ 83.27万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9556265
• 关键词: Alleles; Area; Attenuated; Binding; Binding Proteins; Biochemical; Cell membrane; Cell physiology; Cell surface; Cellular Stress; Colorectal; Colorectal Cancer; Cytosol; Defect; Dimerization; Disease; Event; Family; Feedback; Germ-Line Mutation; Goals; Growth Factor; Human; Impairment; KSR gene; LEOPARD Syndrome; Link; Lung; Lung Adenocarcinoma; MAPK8 gene; Malignant Neoplasms; Malignant neoplasm of ovary; Malignant neoplasm of pancreas; Mediating; Mission; Mitotic; Mutation; Noonan Syndrome; Normal Cell; Oncogenic; Ovarian; Oxidative Stress; Paclitaxel; Papillary thyroid carcinoma; Pathway interactions; Patients; Pharmaceutical Preparations; Phospholipids; Phosphorylation; Phosphotransferases; Physiological; Play; Process; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins B-raf; RAS inhibition; Ras Signaling Pathway; Ras/Raf; Receptor Protein-Tyrosine Kinases; Recruitment Activity; Regulation; Research; Role; Roter; SAM Domain; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Site; Somatic Mutation; Stress; Structure; Therapeutic; Thyroid carcinoma; Treatment Efficacy; Treatment Factor; Work; anticancer research; cardiofaciocutaneous syndrome; design; dimer; human disease; inhibitor/antagonist; insight; melanoma; member; mutant; prevent; raf Kinases; ras Guanine Nucleotide Exchange Factors; ras Proteins; response; scaffold; senescence; therapeutic target; tumor; tumorigenesis

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.

当正常细胞功能的控制因关键信号转导途径的缺陷而出现错误时，癌症通常会发生。 RasGTPase 调节的信号通路就是这样一种通路，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/苏氨酸激酶家族的成员是 Ras 通路中的关键中间体，用于将信号从激活的 Ras 传递到下游蛋白激酶 MEK 和 ERK。哺乳动物有 3 种 Raf 蛋白：A-Raf、B-Raf 和 C-Raf（也称为 Raf-1）。正如对参与细胞信号转导的蛋白质所预期的那样，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（例如受体酪氨酸激酶和 Ras）的突变或扩增，经常导致在含有这些等位基因的肿瘤中通过 Raf/MEK/ERK 级联产生组成型信号传导。此外，Raf 蛋白本身的突变也可以充当疾病驱动因素。 C-Raf 中的种系突变是 Noonan 和 LEOPARD 综合征的病因，而 B-Raf 突变见于 Noonan、LEOPARD 和心面皮肤 (CFC) 综合征，其中 75% 的 CFC 患者发生 B-Raf 突变。此外，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了 B-Raf 的体细胞突变。我们的研究阐明了调节正常和突变 Raf 信号传导的几种重要机制。我们的研究表明，KSR1 支架在调节质膜响应生长因子处理而发出的 Raf 信号强度和持续时间方面发挥着关键作用。此外，我们的工作还提供了有关 KSR1 支架如何响应生长因子信号从胞质溶胶招募到质膜的见解。通过结构/功能分析，我们的研究表明，KSR1 的保守 CA1 区域形成了一个延伸的无菌 α 基序结构域，在生长因子信号传导下，该结构域暴露并结合质膜中的磷脂。因此，KSR1 结合的 MEK 定位于细胞表面，可被激活的 Raf 激酶磷酸化。最后，我们的研究表明，KSR1 的表达水平可以改变 ATP 竞争性 Raf 抑制剂对致癌 Ras 至 ERK 信号传导的影响。具体来说，KSR1 与 C-Raf 竞争抑制剂诱导的与 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（例如，抑制性反馈位点的磷酸化和介导 14-3 的残基的磷酸化）来改变。 -3 结合）和蛋白质相互作用（例如 14-3-3 结合和 Raf 二聚化）。此外，我们发现 Raf 二聚化对于由人类疾病相关的具有中度、低度或受损激酶活性的 Raf 突变体诱导的信号上调至关重要，或者在该通路由激活的 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有可能通过 Ras 通路信号传导的升高来改变人类疾病状态的进展和治疗。此外，我们的研究提供了第一个“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 信号传导。总而言之，这些发现对于通过 Ras 通路信号传导升高来治疗人类疾病状态具有重要意义，并将 Raf 二聚体界面确定为治疗靶点。最后，我们最近的研究发现了一种先前未知的抑制 Ras/Raf/MEK/ERK 信号传导的机制，该信号传导是由应激激活的 JNK 级联介导的。我们发现关键的 Ras 通路组成部分，包括 RasGEF Sos1 和 Rafs，在响应 JNK 激活的多个 S/TP 位点上被磷酸化，并且这些位点的过度磷酸化使 Rafs 和 Sos1 对上游信号无反应。这种磷酸化调节回路与癌症治疗药物（例如 Rigosertib 和紫杉醇/紫杉醇）有关，它们通过有丝分裂和氧化应激激活 JNK，并与 JNK 级联的生理调节因子以及作为信号检查点的功能参与，以抑制 Ras 通路信号传导。细胞应激条件。