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蛋白作为SCF泛素连接酶的底物识别组分起作用。FBW7 促进造血干细胞（HSC）生物学中涉及的几种蛋白质的降解，包括 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。本项目的总体目标是了解 Fbw 7通路在造血干/祖细胞增殖和分化中的作用，并确定其作用机制 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区室中起作用的三种蛋白质同种型。的 目的一的目的是检测HSC/祖细胞中Fbw7同种型的表达和调节， 成熟的造血元件和在造血分化期间。目标二是研究 通过使用两种互补策略来抑制Fbw7在HSC/祖细胞中的功能 表情第一种方法利用shRNA介导的Fbw7敲低来减少Fbw7在细胞中的表达。 第二种方法需要开发条件无效的Fbw7， 敲除小鼠，其将用于在体内将Fbw7转染到HSC/祖细胞中。我们将使用这两个 研究Fbw7在造血干/祖细胞中功能的策略，并确定Fbw7在造血干/祖细胞中的作用。 Fbw7通路在这些细胞中调节特异性底物如Myc、Notch和细胞周期蛋白E。 目的三是了解调控Fbw7介导的Notch的机制 降解我们将首先使用生物化学方法来识别磷酸化事件， Fbw7的缺口降解。然后，我们将研究这一途径如何在HSC/祖细胞中被调控， 开发识别这些关键磷酸化的抗体。最后，我们将探讨 Fbw7介导的Notch降解在HSC/祖细胞中通过开发条件性"敲入"小鼠 Notch不能被Fbw7降解，因为它不能被磷酸化。重要的是这 模型将特别损害Notch降解，同时保持Fbw7途径的其余部分完整。 总之，本研究将提供Fbw7在HSC/祖细胞中功能的全面研究。