Functional analysis of tyrosine residues in human hepatocyte nuclear factor 4alpha

Hepatocyte nuclear factor 4alpha (HNF4alpha), a member of the nuclear receptor superfamily of ligand-dependent transcription factors, is critical for the development and function of the liver, kidney, pancreas and gastrointestinal tract. HNF4alpha is classically associated with normal cellular differentiation in these tissues, but there is an increasing body of literature suggesting that HNF4alpha may also play a role in cancer. Human HNF4alpha exists as six different transcriptional variants (isoforms) due to alternate promoter usage and splicing. Isoforms HNF4alpha 1-3 are derived from the P1 promoter, whereas isoforms HNF4alpha7-alpha9 are from the P2 promoter which is ∼45 kb upstream of P1. The expression and activity of HNF4alpha is tightly regulated by post-translational modifications, in response to external and internal stimuli. This thesis examines one such modification, namely phosphorylation of tyrosine residues. To elucidate the importance of tyrosine residues in regulating the activity of HNF4alpha, we mutated each of the 13-tyrosine residues to phenylalanine (function blocking) and glutamic acid (phosphomimetic mutant). In Chapter 2, we show that the tyrosine residues in the DNA binding domain (DBD) (Tyr63 and Tyr85) are critical for DNA binding and transcriptional activity of HNF4alpha. In addition, we show that phosphomimetic double mutant in the ligand binding domain (LBD) (Y277, Y279) has an effect on the DNA binding, transactivation, nuclear localization and protein stability of HNF4alpha.;To further validate the results obtained using the tyrosine mutants we identified the tyrosine kinases that phosphorylate human HNF4alpha in the DBD and LBD. In Chapter 3, we found that Src phosphorylates P1-driven HNF4alpha sequentially at Tyr14, Tyr277 and Tyr279. Due to the absence of Tyr14 in the P2-driven HNF4alpha, Src phosphorylates P2-driven HNF4alpha less than the P1 isoforms. In addition, phosphorylation of P1-driven HNF4alpha leads to the cytoplasmic localization and proteasomal degradation. Interestingly, in Chapter 4, we found that Src decreases the transcriptional activity of both P1- and P2-driven HNF4alpha. We show that this transcriptional repression is dependent on the kinase activity and SH3 binding of Src, and A/B and F domain of HNF4alpha have opposing effects on the Src-mediated transcriptional repression. We also show that Abl, a tyrosine kinase closely related to the Src family of kinases and a downstream target of Src, phosphorylates HNF4alpha in the DBD and decreases its transcriptional activity. We propose that Src activation of Abl is responsible for the effect of Src on the function of both P1- and P2-driven HNF4alpha isoforms.;In the concluding Chapter 5, we relate our findings to human liver and colorectal cancer and propose that the isoform-specific phosphorylation by Src could be the basis for the dysregulation of HNF4alpha isoforms during cancer.