Study of PTPRF-Mediated Regulation of Wnt Signaling

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.