Regulation of Ras-Dependent Signal Transduction Pathways

Ras 依赖性信号转导途径的调节

• 批准号: 8937711
• 负责人: Deborah Morrison
• 金额: $ 60.81万
• 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/8937711
• 关键词: Alleles; Area; Attenuated; Binding; Binding Proteins; Biochemical; Cell membrane; Cell physiology; Cell surface; Colorectal; Colorectal Cancer; Cytosol; Defect; Dimerization; Disease; Event; Family; Feedback; Germ-Line Mutation; Goals; Growth Factor; Human; LEOPARD Syndrome; Link; Lung; Lung Adenocarcinoma; MEKs; Malignant Neoplasms; Malignant neoplasm of ovary; Malignant neoplasm of pancreas; Mediating; Mission; Mutation; Noonan Syndrome; Normal Cell; Oncogenic; Ovarian; Papillary thyroid carcinoma; Pathway interactions; Patients; Pharmaceutical Preparations; Phospholipids; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Process; Protein Kinase; Protein-Serine-Threonine Kinases; Proteins; Proto-Oncogene Proteins B-raf; Ras Signaling Pathway; Receptor Protein-Tyrosine Kinases; Recruitment Activity; Regulation; Research; Role; SAM Domain; Signal Pathway; Signal Transduction; Signal Transduction Pathway; Somatic Mutation; Structure; Therapeutic; Thyroid carcinoma; Work; anticancer research; cardiofaciocutaneous syndrome; design; dimer; human disease; inhibitor/antagonist; insight; melanoma; member; mutant; prevent; raf Kinases; 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. 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.

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，负责将信号从激活的RAS传递到下游的蛋白激酶MEK和ERK。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的种系突变是Noonan和豹子综合征的病因，而B-Raf突变在Noonan、豹子和心面部皮肤(CFC)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。我们的研究阐明了几个重要的机制，有助于调节正常和突变的Raf信号。我们的研究表明，KSR1支架在调节Raf信号的强度和持续时间方面发挥了关键作用，这些信号来自质膜，以响应生长因子的处理。此外，我们的工作提供了关于KSR1支架如何在生长因子信号作用下从胞浆招募到质膜的见解。通过结构/功能分析，我们的研究表明，KSR1的保守的CA1区形成了一个扩展的无菌α基序结构域，该结构域在生长因子信号转导下暴露出来，并与质膜中的磷脂结合。结果，KSR1结合的MEK被定位于细胞表面，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，KSR1的表达水平可以改变ATP竞争性Raf抑制剂对ERK信号转导致癌RAS的影响。具体地说，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信号的矛盾激活效应。我们的研究还表明，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)来改变。此外，我们还发现，Raf二聚化对于具有中等、低或受损的激酶活性的人类疾病相关Raf突变体诱导的上调信号至关重要，或者在该途径由激活的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改变RAS途径信号升高的人类疾病状态的进展和治疗。最后，我们的工作首次证明了在需要二聚化的条件下，抑制Raf二聚化可以抑制Raf信号转导。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并确定Raf二聚体界面作为治疗靶点具有重要意义。