The transcriptional activity of hepatocyte nuclear factor 4 alpha is inhibited via phosphorylation by ERK1/2

Hepatocyte nuclear factor 4 alpha (HNF4 alpha) nuclear receptor is a master regulator of hepatocyte development, nutrient transport and metabolism. HNF4 alpha is regulated both at the transcriptional and post-transcriptional levels by different mechanisms. Several kinases (PKA, PKC, AMPK) were shown to phosphorylate and decrease the activity of HNF4 alpha. Activation of the ERK1/2 signalling pathway, inducing proliferation and survival, inhibits the expression of HNF4a. However, based on our previous results we hypothesized that HNF4 alpha is also regulated at the post-transcriptional level by ERK1/2. Here we show that ERK1/2 is capable of directly phosphorylating HNF4 alpha in vitro at several phosphorylation sites including residues previously shown to be targeted by other kinases, as well. Furthermore, we also demonstrate that phosphorylation of HNF4 alpha leads to a reduced trans-activational capacity of the nuclear receptor in luciferase reporter gene assay. We confirm the functional relevance of these findings by demonstrating with ChIP-qPCR experiments that 30-minute activation of ERK1/2 leads to reduced chromatin binding of HNF4 alpha. Accordingly, we have observed decreasing but not disappearing binding of HNF4 alpha to the target genes. In addition, 24-hour activation of the pathway further decreased HNF4 alpha chromatin binding to specific loci in ChIP-qPCR experiments, which confirms the previous reports on the decreased expression of the HNF4 alpha gene due to ERK1/2 activation. Our data suggest that the ERK1/2 pathway plays an important role in the regulation of HNF4 alpha-dependent hepatic gene expression.