Study of PTPRF-Mediated Regulation of Wnt Signaling

PTPRF 介导的 Wnt 信号转导调节研究

• 批准号: 10677248
• 负责人: Tianyan Gao
• 金额: $ 34.71万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-06-01 至 2027-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10677248
• 关键词: 3-Dimensional; Attention; Attenuated; Automobile Driving; Bioinformatics; Biological Process; CRISPR/Cas technology; Caveolins; Cell Differentiation process; Cell Proliferation; Cell membrane; Cells; Cellular biology; Colon; Colon Carcinoma; Colorectal Cancer; Complex; Data; Development; Disease; Endocytosis; Ensure; Epithelium; Equilibrium; Genes; Goals; Human; Human body; In Vitro; Internal Ribosome Entry Site; Intestines; Investigation; Knock-out; Knockout Mice; Knowledge; LGR5 gene; Maintenance; Mediating; Modeling; Molecular; Mus; Mutation; Oncogenic; Organism; Organoids; PTPRR gene; Pathway interactions; Phosphoric Monoester Hydrolases; Phosphorylation; Physiological; Physiology; Play; Process; Proliferating; Protein Dephosphorylation; Protein Kinase; Protein Tyrosine Phosphatase; Protein phosphatase; Proteins; Regulation; Reporter; Role; Sampling; Signal Transduction; System; Technology; Testing; Tissues; Ubiquitination; Villus; WNT Signaling Pathway; beta catenin; cancer cell; colon cancer patients; colon tumorigenesis; in vivo; inhibitor; insight; intestinal crypt; intestinal epithelium; intestinal homeostasis; knock-down; novel; novel therapeutic intervention; receptor; self-renewal; single-cell RNA sequencing; stem cell fate; stem cell function; stem cell proliferation; stem cells; success; targeted treatment; tumor growth; ubiquitin-protein ligase

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.

抽象的 蛋白质磷酸化定义了细胞中最重要，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于生活至关重要 维持正常生理功能的生物体。而蛋白质激酶吸引了大量 鉴于有望开发针对靶向疗法的抑制剂的承诺，存在重大的知识差距 关于蛋白质磷酸酶的相反作用。这项研究的总体目的是调查 PTPRF（一种受体型酪氨酸磷酸酶）调节Wnt信号传导和 正常的肠稳态。大量证据表明，Wnt信号是需要的 维持正常肠干细胞； Wnt信号的失调通常是导致的第一步 结直肠癌的发展。在我们研究Wnt信号的调节机制时， 我们确定了一种受体蛋白酪氨酸磷酸酶PTPRF，是Wnt途径中的新型阳性调节剂 这促进了β-catenin破坏复合物上游的Wnt信号的激活。进一步确定 PTPRF的功能重要性，我们利用PTPRF敲除小鼠研究PTPRF-loss是否改变了Wnt 信号传导以及体内肠道干细胞的增殖和分化。我们令人兴奋的新数据 从PTPRF基因敲除小鼠中分离出的肠道加密植物具有降低形成器官的能力，这重合 Wnt靶基因的表达降低。此外，我们发现PTPRF与 Wnt刺激后LRP6和小窝蛋白； 293T细胞中PTPRF的CRISPR/CAS9介导的敲除 减弱了WNT刺激的顶闪式报道器活性以及LRP6磷酸化。而且，我们确定了 NEDD4L是一种控制PTPRF泛素化和降解的新型E3连接酶。集体，中央 推动该建议的假设是，PTPRF通过促进pTPRF积极调节Wnt信号传导 Wnt信号体的形成和PTPRF自杀肠干细胞功能的表达。下列 提出了具体目的：1）描述PTPRF介导的调节的分子机制 Wnt信号传导； 2）确定NEDD4L在控制PTPRF蛋白稳定性中的作用； 3）定义 PTPRF在确定体内肠道干细胞的增殖和分化方面的功能重要性。 我们研究的结果将填补有关蛋白质磷酸酶如何参与的重要知识差距 调节复杂的生物过程。为了确保这项研究的成功，我们组装了一个强大的 具有蛋白质磷酸酶，WNT信号传导和肠上皮细胞的集体专业知识的调查团队 生物学。最终，通过为wnt信号的PTPRF依赖性调节提供新的机械见解， 我们的发现将有助于通过使用PTPRF作为目标来确定治疗WNT驱动疾病的新策略。