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Title:Role of Src phosphorylation of FXR in bile acid regulation
Author(s):Ryerson, Daniel E
Director of Research:Kemper, Jongsook K
Doctoral Committee Chair(s):Kemper, Jongsook K
Doctoral Committee Member(s):Shapiro, David; Raetzman, Lori; Anakk, Sayeepriyadarshini
Department / Program:Molecular & Integrative Physl
Discipline:Molecular & Integrative Physi
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Bile acid
Fibroblast growth factor 19 (FGF19)
Farnesoid X receptor (FXR)
Src
Phosphorylation
Abstract:Bile acids are physiological detergents which aid in the absorption of dietary lipids and lipid soluble vitamins but also function as fed state signaling molecules. Elevated bile acid levels in the liver can lead to cholestatic injury, primary biliary cirrhosis, fibrosis, and liver cancer; therefore, these levels must be tightly regulated. The farnesoid X receptor (FXR) is the primary bile acid nuclear receptor and acts as the master regulator of bile acid homeostasis, preventing liver damage caused by bile acid accumulation. FXR does this by regulating the expression of many target genes in the gut and liver including the intestinal hormone fibroblast growth factor 19 (FGF19) and orphan nuclear receptor small heterodimer partner (SHP). In response to elevated hepatic bile acid levels FXR, acting directly as well as through FGF19 and SHP, inhibits the synthesis of bile acids, downregulates bile acid importers, upregulates bile acid exporters along with genes involved in bile acid conjugation and detoxification. These important roles of FXR are highlighted by the phenotypic effects observed in FXR knockout (FXR-/-) mice. FXR-/- mice display elevated bile acid pool size as well as elevated serum bile acid levels. Additionally, FXR-/- mice show signs of liver damage and develop spontaneous tumors as they age. Understanding how FXR receives signals and translates them into transcriptional responses to mediate these diverse cellular effects will be important for the development of therapeutic agents to treat cholestatic liver disorders. One mechanism through which FXR activity is regulated is signal-induced post-translational modifications. FXR has been shown to undergo multiple types of post-translational modifications including phosphorylation, methylation, acetylation and sumoylation in response to physiological and pathological signals. These modifications affect FXR in many ways including modulating subcellular localization, stability, DNA binding, interaction with transcriptional coregulators and affecting the expression of FXR target genes in a gene selective manner. Mutation of a single amino acid, disrupting one of these post-translational modifications, has been shown to dramatically alter FXR function. Interestingly, some of these post-translational modifications have been shown to be misregulated in models of disease, which highlights the importance of understanding the molecular mechanisms through which FXR is post-translationally modified. In this study a new post-translational modification of FXR was identified which profoundly impacts FXR transcriptional activity. Unbiased mass spectrometry based proteomic analysis showed that tyrosine-67 of FXR is rapidly phosphorylated in liver hepatocytes in response to treatment with either natural bile acids or FGF19. Biochemical analysis paired with bioinformatic tools identified Src as the kinase responsible for this post-translational modification. Feeding mice a diet supplemented with the primary bile acid cholic acid (CA) led to interaction between FXR and Src as well as phosphorylation of FXR. Further studies showed that Src interacts with the DNA binding domain of FXR specifically. In vitro kinase assays utilizing purified Src protein coupled with studies utilizing siRNA knockdown of Src demonstrated that Src is both necessary and sufficient for FXR phosphorylation. Adenoviral reconstitution of wild type and tyrosine-67 phosphorylation deficient mutant (Y67F) FXR in isolated primary mouse hepatocytes (PMH) showed that disruption of this phosphorylation site led to a decrease in FXR/RXR interaction and decreased expression of a subset of FXR target genes involved in bile acid regulation, particularly bile salt export pump (BSEP) and SHP. Disruption of this site in vivo also led to elevated bile acid levels, elevated liver enzyme levels, and increased macrophage infiltration; all signs of liver damage. Additionally, when challenged in models simulating cholestasis, these signs of liver damage are dramatically elevated in mice expressing Y67F-FXR. These in vivo studies demonstrate that disruption of the FXR tyrosine-67 site drastically impairs FXR’s ability to regulate its target genes, maintain bile acid homeostasis, and protect the liver from bile acid induced toxicity. In conclusion, this study identified a previously unknown phosphorylation site of FXR which is mediated by Src. We further showed that this phosphorylation is critical for FXR function, maintenance of bile acid homeostasis, and protecting the liver against bile toxicity; with loss of this phosphorylation site leading to the development of liver damage in vivo. The profound effects FXR tyrosine-67 phosphorylation has on FXR transcriptional activity and metabolic outcomes suggest that this site and the kinase leading to its phosphorylation may prove to be innovative targets for the treatment of hepatobiliary and cholestatic diseases.
Issue Date:2017-04-18
Type:Text
URI:http://hdl.handle.net/2142/97590
Rights Information:Copyright 2017 Daniel Ryerson
Date Available in IDEALS:2017-08-10
Date Deposited:2017-05


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