Cell Proliferation and Differentiation By the Fbw 7 Tumor Suppressor

Fbw 7 肿瘤抑制因子促进细胞增殖和分化

• 批准号: 7226081
• 负责人: Bruce E Clurman
• 金额: $ 46.48万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-12-01 至 2011-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7226081
• 关键词: Antibodies; B-Lymphocytes; Binding; Biochemical; Biological Assay; Cell Proliferation; Cells; Cellular biology; Complex; Cyclin E; Data; Development; Elements; Event; Excision; Exhibits; Genes; Goals; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; In Vitro; Investigation; JUN gene; Knock-out; Knockout Mice; Left; Lentivirus Vector; Maps; Mass Spectrum Analysis; Mediating; Messenger RNA; Modeling; Mus; Mutation; Myelogenous; Pathway interactions; Pattern; Phosphopeptides; Phosphorylation; Phosphorylation Site; Process; Protein Isoforms; Proteins; Proteolysis; RNA Splicing; Regulation; Research; Resistance; Rest; Role; SCF(Fbw7) Ubiquitin Ligase; SKP Cullin F-Box Protein Ligases; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Stem cells; Tissues; Tumor Suppressor Proteins; c-myc Genes; in vivo; mouse model; notch protein; progenitor; protein degradation; protein function; recombinase; self-renewal; small hairpin RNA; stem

The Fbw7 protein functions as the substrate-recognition component of SCF ubiquitin ligases. Fbw7 promotes the degradation of several proteins implicated in hematopoietic stem cell (HSC) biology, including Myc, Notch, cyclin E, and c-Jun. Our preliminary data indicate that Fbw7 is expressed in HSCs/progenitors and regulates hematopoietic differentiation. The overall goal of this project is to understand the role of the Fbw7 pathway in hematopoietic stem/progenitor cell proliferation and differentiation, and to determine the importance of specific Fbw7 substrates in these processes. The Fbw7 gene encodes three protein isoforms that function in different subcellular compartments. The goal of Aim One is to examine the expression and regulation of the Fbw7 isoforms in HSCs/progenitors, mature hematopoietic elements, and during hematopoietic differentiation. The goal of Aim Two is study the functions of Fbw7 in HSCs/progenitors by using two complementary strategies to inactivate Fbw7 expression. The first approach utilizes shRNA-mediated Fbw7 knockdown to reduce Fbw7 expression in HSCs/progenitors in vitro, whereas the second approach entails the development of a conditional-null Fbw7 knockout mouse that will be used to inactivate Fbw7 in HSCs/progenitors in vivo. We will use these two strategies to study Fbw7 functions in hematopoietic stem/progenitor cells, and to determine the role of the Fbw7 pathway in regulating specific substrates such as Myc, Notch, and cyclin E in these cells. The goal of Aim Three is to understand the mechanisms that regulate Fbw7-mediated Notch degradation. We will initially use a biochemical approach to identify the phosphorylation events that signal Notch degradation by Fbw7. We will than study how this pathway is regulated in HSCs/progenitors by developing antibodies that recognize these key phosphorylation(s). Finally, we will examine the importance of Fbw7-mediated Notch degradation in HSCs/progenitors by developing a conditional "knockin" mouse model in which Notch cannot be degraded by Fbw7 because it cannot be phosphorylated. Importantly, this model will specially impair Notch degradation while leaving the rest of the Fbw7 pathway intact. Overall this research will provide a comprehensive investigation of Fbw7 function in HSCs/progenitors.

FBW7蛋白充当SCF泛素连接酶的底物识别成分。 FBW7 促进与造血干细胞（HSC）生物学有关的几种蛋白质的降解，包括 MYC，Notch，Cyclin E和C-Jun。我们的初步数据表明FBW7在HSC/祖细胞中表达 并调节造血分化。该项目的总体目标是了解 造血干/祖细胞增殖和分化的FBW7途径，并确定 在这些过程中特定的FBW7底物的重要性。 FBW7基因编码在不同亚细胞室中起作用的三种蛋白质同工型。这 目标的目标是检查HSC/祖细胞中FBW7同工型的表达和调节， 成熟的造血元素以及造血分化期间。目标二的目标是研究 FBW7在HSC/祖细胞中的功能通过使用两种互补策略灭活FBW7 表达。第一种方法利用shRNA介导的FBW7敲低来降低FBW7的表达 体外HSC/祖细胞，而第二种方法需要开发有条件的无效FBW7 敲除小鼠将用于在体内HSC/祖细胞中灭活FBW7。我们将使用这两个 研究FBW7在造血干/祖细胞中起作用的策略，并确定 在这些细胞中调节特定底物的FBW7途径，例如MYC，Notch和Cyclin e。 目标三的目的是了解调节FBW7介导的Notch的机制 降解。我们最初将使用生化方法来识别信号的磷酸化事件 FBW7降解。我们将研究如何通过研究该途径在HSC/祖细胞中如何调节该途径 开发识别这些关键磷酸化的抗体。最后，我们将研究重要性 通过开发有条件的“敲击”小鼠，HSC/祖细胞中FBW7介导的缺口降解 由于无法磷酸化，因此Notch不能被FBW7降解的模型。重要的是，这个 在离开其余的FBW7途径完整时，模型将特别损害缺口降解。 总体而言，这项研究将对HSC/祖细胞中FBW7功能进行全面研究。