Integrating regulatory mechanisms of Wnt signaling in development and tissue homeostasis

Evolutionarily conserved signal transduction pathways mediate the ability of cells to respond to their environment and coordinate with each other for proper development and homeostasis of an organism. The Wnt/Wingless (Wg) pathway is required for proliferation, differentiation, stem-cell renewal and homeostasis, and when disrupted leads to disease. Wnt signaling does not control all these processes alone, its activity is extensively regulated by interaction with other signaling pathways and cellular mechanisms. This is mediated predominantly through phospho-regulation of the key pathway components by kinases and phosphatases. Our lab conducted an in vivo RNAi screen designed to identify novel kinase and phosphatase regulators of the Wnt pathway. In my PhD thesis research I further characterized three potential regulators: Downstream of Raf1 (Dsor1), Protein phosphatase 4 (PP4), and myosin phosphatase. Knockdown of Dsor1 reduced Wnt target gene expression and decreased stabilized β-catenin, the key effector protein of the Wnt pathway. Dsor1 and β-catenin had a close physical interaction, and catalytically inactive Dsor1 caused a reduction in active β-catenin, suggesting that Dsor1 counteracts destruction of β-catenin. Additionally, Ras-Dsor1 activity was independent of EGFR, and likely activated by the insulin-like receptor to promote Wnt. This work demonstrates novel crosstalk between Insulin and Wnt signaling via Dsor1. The reduction of PP4 inhibited Wg pathway activity, by reducing Notch-driven wg transcription. PP4 was found to promote Notch signaling within the nucleus of the receiving cell. Furthermore, PP4 regulates proliferation independently of its Notch interaction. This study identified a new role for PP4 in Notch signaling, and subsequently transcriptional regulation of wg. Reduced myosin phosphatase inhibited Wnt signaling by causing increased non-muscle myosin II (NMII) activation and cellular contraction. NMII activation stabilizes cortical F-actin resulting in accumulation of E-cadherin to the adherens junctions (AJ). E-cadherin titrates available β-catenin to the AJs in order to maintain cell-cell adhesion under contraction. The decreased cytoplasmic β-catenin results in insufficient nuclear translocation for full Wnt target gene transcription. This work elucidates that the dynamic activation of actomyosin contractility refines patterning of Wnt target gene expression. These studies identified three novel regulatory mechanisms for controlling Wnt signaling in development and homeostasis.