Proteomic analysis of native hepatocyte nuclear factor-4α (HNF4α) isoforms, phosphorylation status, and interactive cofactors.
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.
Research Center for Advanced Science and Technology, University of Tokyo, Tokyo 153-8904, Japan.
Kenji
K
Daigo
Takeshi
T
Kawamura
Yoshihiro
Y
Ohta
Riuko
R
Ohashi
Satoshi
S
Katayose
Toshiya
T
Tanaka
Hiroyuki
H
Aburatani
Makoto
M
Naito
Tatsuhiko
T
Kodama
Sigeo
S
Ihara
Takao
T
Hamakubo
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D030562:Databases, Protein; D005110:Extracellular Space / Q000378:metabolism; D056945:Hep G2 Cells; D051557:Hepatocyte Nuclear Factor 4 / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space / Q000378:metabolism; D008280:Magnetics; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D040901:Proteomics / Q000379:methods*; D015398:Signal Transduction; D015533:Transcriptional Activation
D051557:Hepatocyte Nuclear Factor 4