Cell Proliferation and Differentiation By the Fbw 7 Tumor Suppressor

Fbw7蛋白是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在造血干细胞/祖细胞中表达 并调节造血细胞分化。本项目的总体目标是了解 Fbw7通路在造血干/祖细胞增殖分化中的作用 在这些过程中，特定的Fbw7底物的重要性。 Fbw7基因编码三种不同的蛋白质亚型，在不同的亚细胞中发挥作用。这个 目标一的目的是检测Fbw7亚型在HSCs/祖细胞中的表达和调控， 成熟的造血元素，在造血细胞分化过程中。目标二的目标是研究 利用两种互补策略灭活Fbw7在造血干细胞/祖细胞中的作用 表情。第一种方法利用shRNA介导的Fbw7基因敲除来降低Fbw7的表达 体外培养造血干细胞/祖细胞，而第二种方法需要发展条件空的Fbw7 将用于灭活体内HSCs/祖细胞中Fbw7的基因敲除小鼠。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途径调节这些细胞中的特定底物，如Myc、Notch和Cyclin E。 目标三的目标是了解调节Fbw7介导的Notch的机制 退化。我们将首先使用生化方法来识别信号的磷酸化事件 缺口退化由Fbw7。然后我们将研究这一途径是如何在HSCs/前体细胞中调节的 开发识别这些关键磷酸化的抗体(S)。最后，我们将研究其重要性 通过建立条件性“敲击”小鼠研究Fbw7介导的肝星状细胞/祖细胞Notch降解 Notch不能被Fbw7降解的模型，因为它不能被磷酸化。重要的是，这 模型将特别削弱Notch的降解，同时保持Fbw7途径的其余部分完好无损。 总体而言，本研究将对Fbw7在造血干细胞/祖细胞中的功能进行全面的研究。

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.