Proteomic Analysis of Native Hepatocyte Nuclear Factor-4α (HNF4α) Isoforms, Phosphorylation Status, and Interactive Cofactors

Abstract

Hepatocyte nuclear factor-4α (HNF4α, NR2A1) is a nuclear receptor that has a critical role in hepatocyte differentiation and the maintenance of homeostasis in the adult liver. However, a detailed understanding of native HNF4α in the steady-state remains to be elucidated. Here we report the native HNF4α isoform, phosphorylation status, and complexes in the steady-state, as shown by shotgun proteomics in HepG2 hepatocarcinoma cells. Shotgun proteomic analysis revealed the complexity of native HNF4α, including multiple phosphorylation sites and inter-isoform heterodimerization. The associating complexes identified by label-free semiquantitative proteomic analysis include the following: the DNA-dependent protein kinase catalytic subunit, histone acetyltransferase complexes, mRNA splicing complex, other nuclear receptor coactivator complexes, the chromatin remodeling complex, and the nucleosome remodeling and histone deacetylation complex. Among the associating proteins, GRB10 interacting GYF protein 2 (GIGYF2, PERQ2) is a new candidate cofactor in metabolic regulation. Moreover, an unexpected heterodimerization of HNF4α and hepatocyte nuclear factor-4γ was found. A biochemical and genomewide analysis of transcriptional regulation showed that this heterodimerization activates gene transcription. The genes thus transcribed include the cell death-inducing DEF45-like effector b (CIDEB) gene, which is an important regulator of lipid metabolism in the liver. This suggests that the analysis of the distinctive stoichiometric balance of native HNF4α and its cofactor complexes described here are important for an accurate understanding of transcriptional regulation.

The FBXL10/KDM2B Scaffolding Protein Associates with Novel Polycomb Repressive Complex-1 to Regulate Adipogenesis

“…The samples were reduced in 1.2 mM tris(2-carboxyethyl)phosphine for 15 min at 50°C and alkylated in 3 mM iodoacetamide for 30 min at room temperature, respectively. The samples were digested overnight with 100 ng of trypsin (Promega) at 37°C as described (40). Aliquots of trypsinized samples were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) as we described previously (40).…”

“…The samples were digested overnight with 100 ng of trypsin (Promega) at 37°C as described (40). Aliquots of trypsinized samples were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) as we described previously (40). Protein quantification was done by processing the acquired LC-MS data with the software Progenesis LC-MS (version 2.6; Nonlinear Dynamics, Newcastle, UK).…”

Phosphatidylinositol 3-Phosphatase Myotubularin-related Protein 6 (MTMR6) Is Regulated by Small GTPase Rab1B in the Early Secretory and Autophagic Pathways

“…Eluted samples were separated by SDS-PAGE and analyzed by tandem mass spectrometry. For shotgun proteomics, HeLa cells were harvested with lysis buffer supplemented with 0.1% Nonidet P-40 and analyzed as described (38).…”

Identification of Wilms' Tumor 1-associating Protein Complex and Its Role in Alternative Splicing and the Cell Cycle

“…After centrifugation for 30 min at 12,000 ϫ g at 4°C, the supernatant was frozen with liquid nitrogen and stored at Ϫ80°C as the cell lysate. Immunopurification was performed with 2 g of antibody-cross-linked Dynabeads protein G (Invitrogen), as described previously (24).…”

“…The immunopurification of the native protein complexes was based on a highly sensitive immunoprecipitation system (24). WTAP was immunoprecipitated effectively by each antibody but not with the antiviral protein antibody K7124, which was used as a negative control (Fig.…”