Quantitative Analysis of Cytokine-Induced Hepatocyte Nuclear Factor-4α Phosphorylation by Mass Spectrometry

Hepatocyte nuclear factor-4α (HNF-4α), a liver-enriched transcription factor, is essential for liver development and function. HNF-4α regulates a large number of liver-specific genes, many of which are modulated by injury. While HNF-4α function is regulated by phosphorylation, only a limited number of phosphorylation sites in HNF-4α have been identified, and the roles of HNF-4α phosphorylation after injury are unexplored. To address these issues, we have carried out an extensive quantitative mass spectrometry (MS)-based analysis of HNF-4α serine and threonine phosphorylation in response to cytokine stimulation. Studies were performed in HNF-4α-enriched HepG2 cells treated with cytokines for 3 h or left untreated, followed by chemical derivatization of the phosphoserine and phosphothreonine residues using stable isotopic variants of dithiothreitol (DTT) and MS analysis. This has allowed the identification and relative quantification of 12 serine/threonine phosphorylation sites in HNF-4α. Eight of these phosphorylation sites and their sensitivity to cytokine stimulation have not been previously reported. We found that cytokine treatment leads to an increase ofHNF-4α phosphorylation in several phosphopeptides. The phosphorylation of HNF-4α mediated by protein kinase A (PKA) significantly reduces HNF-4α binding activity, which mimics the repressive effect of cytokines on HNF-4α binding, and the inhibition of PKA activity by PKA inhibitor can partially recover the reduced HNF-4α binding activity induced by cytokines. These results suggest that the mechanism that alters HNF-4α binding after cytokine stimulation involves modulation of specific HNF-4α phosphorylation dependent, in part, on a PKA signaling pathway. Hepatocyte nuclear factor-4α (HNF-4α) is a liver-enriched transcription factor, giving it the potential to regulate hundreds or even thousands of targets. The importance of HNF-4α to liver differentiation and function has been demonstrated by cell-type-specific gene ablation in mutant mice. When HNF-4α was deleted in fetal development, hepatocyte precursors lost their epithelial phenotype.1 When the gene was deleted in the adult liver, defects in metabolism were most prominent.2 HNF-4α is a member of the ligand-dependent nuclear receptor family of transcription factors, making it an interesting target for drug development. The native ligand forHNF-4α remains controversial, and multiple molecules have been proposed, ranging from palmitoyl-coenzyme A to linoleic acid.3,4 The agonists and antagonists have been investigated as a means to manipulate HNF-4α function as a potential therapeutic approach.5 HNF-4α has been shown to contribute to human disease. A dominant mutations in the HNF4A gene cause both maturity onset diabetes of the young6 and neonatal macrosomia and hyperinsulinemic hypoglycemia.7 Previous work by our group has demonstrated that HNF-4α plays a role in the liver’s response to systemic injury and the development of the acute phase response.8–10 To fully understand how HNF-4α accomplishes these important physiologic functions in the liver, knowledge of the mechanisms regulating HNF-4α function itself is required. Protein phosphorylation is a common post-translational modification among transcription factors and has been shown to play a function role in nuclear localization, DNA binding, and transactivation.11,12 Previous studies have demonstrated thatHNF-4α protein can be phosphorylated by multiprotein kinases at multiple sites. Protein kinase A (PKA) phosphorylates HNF-4α at serine (Ser, S) 133 and 134 in the DNA binding domain (DBD) and impairs its DNA binding activity.13 AMP-activated protein kinase phosphorylates HNF-4α at S304 in the ligand binding domain (LBD) and impairs its dimerization and DNA binding activity.14 p38 kinase phosphorylates S158 in the LBD of HNF-4α in response to inflammatory redox, causing an increase in DNA binding and transactivation.15 It has been reported by our group that the activation of Janus kinase-2 pathway is involved in HNF-4α phosphorylation that may account for the rapid decrease in HNF-4α DNA binding and transactivation after injury.9 Despite the acceptance that phosphorylation is a critical modification involved in many cellular events, and the recent advances in mass spectrometry (MS) technology to identify phosphoproteins, the role of phosphorylation and phosphoproteins in physiological processes cannot be considered a static process. Protein phosphorylation is dynamic and specific depending on both the site and state of phosphorylation. However, qualitative and quantitative analyses and understanding of protein phosphorylation remain a challenge for a number of reasons.16 First, phosphopeptides are usually detected with low efficiency or not at all by MS probably due to their acidic character. Second, the phosphorylation may not be stoichiometric at a given site in a protein; i.e., the phosphopeptide of interest may be of low abundance compared to the unmodified peptide. Third, phosphorylated residues may interfere with the enzymatic digestion of the protein resulting in peptide fragments that are either too large or too small for effective analysis by MS. Fourth, phosphopeptides do not efficiently fragment during MS/MS CID (collision-induced dissociation) for sequence identification. Therefore, only a few phosphorylation sites in HNF-4α have been mapped, and quantitative analyses of HNF-4α phosphorylation as well as the significance of its phosphorylation in response to an inflammatory stimulus have not been well characterized. In the current study, we have utilized the overexpressed FLAG-tagged HNF-4α model to generate sufficient quantities of HNF-4α to allow its reliable detection, followed by chemical derivatization of phosphoserine and phosphothreonine residues with either dithiothreitol (DTT) or deuterated [D6]DTT form for quantification.16–19 This enables the cytokine sensitive phosphorylation sites in HNF-4α to be identified, and the extent of phosphorylation to be quantitatively analyzed by liquid chromatography–electrospray ionization–tandem mass spectrometry (LC-ESI-MS/MS) in a cytokine-stimulated HepG2 cell culture model. Our results show that HNF-4α can be phosphorylated at various serine and threonine sites in a peptide-dependent manner in response to cytokine treatment. The impact of in vitro phosphorylation of HNF-4α by several protein kinases on HNF-4α binding activity was also defined. Utilizing novel means facilitate us, for the first time, to map qualitatively and quantitatively HNF-4α’s phosphorylation profile in a cytokine-induced inflammatory response model of injury. It also extends current knowledge about the quantitative changes in HNF-4α phosphorylation during inflammatory response and provides important insight into the mechanism regulating HNF-4α function and the hepatic response to systemic injury.