Proteomic analysis of native hepatocyte nuclear factor-4α (HNF4α) isoforms, phosphorylation status, and interactive cofactors.
Proteomic analysis of native hepatocyte nuclear factor-4α (HNF4α) isoforms, phosphorylation status, and interactive cofactors.
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DOI：
10.1074/jbc.m110.154732
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发表时间：
2011-01-07
期刊：
影响因子：
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通讯作者：
Hamakubo T
中科院分区：
文献类型：
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作者：
Daigo K;Kawamura T;Ohta Y;Ohashi R;Katayose S;Tanaka T;Aburatani H;Naito M;Kodama T;Ihara S;Hamakubo T
肝细胞核因子-4 α（HNF 4 α，NR 2A1）是一种核受体，在肝细胞分化和维持成人肝脏内环境稳定中起重要作用。然而，对稳态下天然HNF 4 α的详细了解仍有待阐明。在这里，我们报告了天然HNF 4 α亚型，磷酸化状态，并在稳态复合物，如鸟枪蛋白质组学在HepG2肝癌细胞中所示。鸟枪蛋白质组学分析揭示了天然HNF 4 α的复杂性，包括多个磷酸化位点和异构体间异二聚化。通过无标记半定量蛋白质组学分析鉴定的相关复合物包括：DNA依赖性蛋白激酶催化亚基、组蛋白乙酰转移酶复合物、mRNA剪接复合物、其他核受体辅激活因子复合物、染色质重塑复合物以及核小体重塑和组蛋白脱乙酰化复合物。在这些相关蛋白中，GRB 10相互作用蛋白GYF 2（GIGYF2，PERQ2）是一种新的代谢调节辅助因子。此外，HNF-4 α和肝细胞核因子-4 γ之间存在异源二聚体，对转录调控的生物化学和全基因组分析表明，这种异源二聚体激活了基因转录。如此转录的基因包括细胞死亡诱导DEF 45样效应物B（CIDE B）基因，其是肝脏中脂质代谢的重要调节剂。这表明，分析天然HNF 4 α及其辅因子复合物的独特化学计量平衡对于准确理解转录调控非常重要。
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.