Study of PTPRF-Mediated Regulation of Wnt Signaling

摘要 蛋白质磷酸化是细胞内最重要和最普遍的调节机制之一 信号精确控制磷酸化和去磷酸化之间的平衡对维持生命至关重要。 维持正常的生理功能。虽然蛋白激酶吸引了大量的 考虑到开发靶向治疗抑制剂的前景， 蛋白磷酸酶的相反作用。本研究的总体目标是调查 受体型酪氨酸磷酸酶PTPRF调节Wnt信号传导的分子机制， 正常的肠道内稳态大量的证据表明，Wnt信号传导是细胞凋亡所必需的。 维持正常的肠道干细胞; Wnt信号的失调通常是导致肠道干细胞死亡的第一步。 结直肠癌的发展。在我们研究Wnt信号调节机制的努力中， 我们鉴定了PTPRF，一种受体蛋白酪氨酸磷酸酶，作为Wnt通路中的一种新的正调节因子 其促进β-连环蛋白破坏复合物上游Wnt信号传导的激活。以进一步确定 PTPRF的功能重要性，我们利用Ptprf基因敲除小鼠研究Ptprf缺失是否改变Wnt 信号传导和体内肠干细胞的增殖和分化。我们令人振奋的新数据显示 从Ptprf基因敲除小鼠中分离的肠隐窝形成类器官的能力降低，这与 Wnt靶基因表达降低。此外，我们还发现PTPRF与 Wnt刺激后的LRP 6和小窝蛋白;以及CRISPR/Cas9介导的293 T细胞中PTPRF的敲除 减弱Wnt刺激的TOP-Flash报告基因活性以及LRP 6磷酸化。此外，我们发现， NEDD 4L作为一种新型的E3连接酶，控制PTPRF泛素化和降解。总的来说，中央 推动这一提议假设是PTPRF通过促进Wnt信号传导而正向调节Wnt信号传导， Wnt信号体的形成和PTPRF的表达维持肠干细胞的功能。以下 具体目标是：1）阐明PTPRF介导调控的分子机制 2）确定NEDD 4L在控制PTPRF蛋白稳定性中的作用;和3）确定Wnt信号传导的作用。 PTPRF在体内调节肠干细胞增殖和分化中的功能重要性。 我们的研究结果将填补一个重要的知识空白，蛋白磷酸酶是如何参与 调节复杂的生物过程。为了确保这项研究的成功，我们已经组建了一个强大的 在蛋白磷酸酶、Wnt信号传导和肠上皮细胞方面拥有集体专业知识的调查团队 生物学最终，通过对Wnt信号转导的PTPRF依赖性调节提供新的机制见解， 我们的研究结果将有助于确定以PTPRF为靶点治疗Wnt驱动的疾病的新策略。

ABSTRACT Protein phosphorylation defines one of the most important and pervasive regulatory mechanisms in cell signaling. A precise control of the balance between phosphorylation and dephosphorylation is crucial for living organisms to maintain normal physiological functions. While protein kinases have attracted a significant amount of attention given the promise of developing inhibitors for targeted therapies, significant knowledge gaps exist on the opposing actions of protein phosphatases. The overall objective of this study is to investigate the molecular mechanisms by which PTPRF, a receptor-type tyrosine phosphatase, regulates Wnt signaling and normal intestinal homeostasis. A large body of evidence indicates that Wnt signaling is required for the maintenance of normal intestinal stem cells; and dysregulation of Wnt signaling is often the first step leading to the development of colorectal cancer. In our efforts to investigate the regulatory mechanisms of Wnt signaling, we identified PTPRF, a receptor protein tyrosine phosphatase, as a novel positive regulator in the Wnt pathway that promotes the activation of Wnt signaling upstream of the β-catenin destruction complex. To further determine the functional importance of PTPRF, we utilized Ptprf knockout mice to investigate if Ptprf-loss alters Wnt signaling and the proliferation and differentiation of intestinal stem cells in vivo. Our exciting new data showed that intestinal crypts isolated from Ptprf knockout mice have reduced ability to form organoids, which coincides with decreased expression of Wnt target genes. In addition, we found that PTPRF interacts and co-localizes with LRP6 and caveolin upon Wnt stimulation; and CRISPR/Cas9-mediated knockout of PTPRF in 293T cells attenuates Wnt-stimulated TOP-Flash reporter activity as well as LRP6 phosphorylation. Moreover, we identified NEDD4L as a novel E3 ligase that controls PTPRF ubiquitination and degradation. Collectively, the central hypothesis driving this proposal is that that PTPRF positively regulates Wnt signaling by promoting the formation of Wnt signalosome and the expression of PTPRF sustains intestinal stem cell function. The following specific aims are proposed: 1) to delineate the molecular mechanisms underlying PTPRF-mediated regulation of Wnt signaling; 2) to determine the role of NEDD4L in controlling PTPRF protein stability; and 3) to define the functional importance of PTPRF in regulating the proliferation and differentiation of intestinal stem cells in vivo. Results from our studies will fill an important knowledge gap on how protein phosphatases are involved in regulating complex biological processes. To ensure the success of this study, we have assembled a strong investigative team with collective expertise in protein phosphatases, Wnt signaling and intestinal epithelial cell biology. Ultimately, by providing new mechanistic insights into PTPRF-dependent regulation of Wnt signaling, our findings will help identify new strategies for treating Wnt-driven diseases by using PTPRF as a target.