Abstract 1793 The function and cellular localization of the Dishevelled family of proteins is defined by sterol biochemistry

While cholesterol constitutes approximately 40% of membrane lipids and is critical to membrane ordering and signaling, the structural specificity of cholesterol in mediating these functions is not fully elucidated. For example, the impact of naturally occurring sterols, which accumulate in distinct cell types or within rare disorders of the cholesterol synthetic pathway, on cell signaling is unknown. An important role for cholesterol is regulation of protein localization and function through cholesterol-protein binding. We and others have demonstrated that the Dishevelled (DVL) scaffolding family of proteins require binding of DVL's PSD95/Dlg1/ZO1 (PDZ) domain to cholesterol to allow Wnt/β-catenin signaling. Using the family of DVL proteins as a model, we hypothesized that changes in membrane-associated sterol biochemistry would functionally regulate DVL localization and activity. Through analyses of sterol-protein binding and molecular simulations, we demonstrate a differential binding affinity and specificity of DVL proteins for cholesterol versus other naturally occurring sterols. Both protein-sterol pulldowns and surface plasmon resonance (SPR) analyses demonstrated a range of differential binding affinities of cholesterol vs tested sterols towards both full length and the PDZ domain of DVL proteins. Mechanistically, molecular simulations and SPR demonstrate that a synergistic interaction between cholesterol and phosphatidylinositol 4,5-bisphosphate (PIP2) is required to achieve optimal recruitment of DVL proteins to the cytofacial leaflet of the plasma membrane. DVL membrane association was most strongly impaired by sterols which exhibit high polarity and poor support of liquid ordered domains. While proximity labeling and co-immunoprecipitation demonstrated DVL protein-protein interactions were broadly inhibited by changes in sterol biochemistry, disruption of DVL membrane association by sterols unexpectedly induced significant nuclear accumulation of DVL. Increased nuclear DVL localization occurred in a sterol-dependent manner and resulted in increased DVL protein-protein binding to p53 and other Wnt-independent proteins within the nucleus. DVL nuclear translocation was also dependent on their association with the FoxK family of chaperone proteins. Enhanced nuclear DVL in response to sterol changes was observed across all cell types tested, including immortalized cell lines, neural stem cells, pluripotent stem cells, and within disease models of cholesterol synthesis disorders. In summary, our work suggests disrupted sterol homeostasis dynamically regulates DVL signaling in a sterol-specific manner with important impacts on critical signaling pathways regulating proliferation, differentiation, cytoskeletal organization, and tumorigenesis. Defining the structural and biochemical requirements for sterol interactions with cholesterol-binding proteins such as DVL proteins will further elucidate the critical role sterol homeostasis plays during development and within disease. This study was supported by National Institute of General Medical Sciences grants P20GM103620 and P20GM103548, as well as the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) at the National Institutes of Health.