Protein Kinase A-Dependent Phosphorylation Modulates DNA-Binding Activity of Hepatocyte Nuclear Factor 4

Abstract

Hepatocyte nuclear factor 4 (HNF4), a liver-enriched transcription factor of the nuclear receptor superfamily, is critical for development and liver-specific gene expression. Here, we demonstrate that its DNAbinding activity is modulated posttranslationally by phosphorylation in vivo, ex vivo, and in vitro. In vivo, HNF4 DNA-binding activity is reduced by fasting and by inducers of intracellular cyclic AMP (cAMP) accumulation. A consensus protein kinase A (PKA) phosphorylation site located within the A box of its DNA-binding domain has been identified, and its role in phosphorylation-dependent inhibition of HNF4 DNA-binding activity has been investigated. Mutants of HNF4 in which two potentially phosphorylatable serines have been replaced by either neutral or charged amino acids were able to bind DNA in vitro with affinity similar to that of the wild-type protein. However, phosphorylation by PKA strongly repressed the binding affinity of the wild-type factor but not that of HNF4 mutants. Accordingly, in transfection assays, expression vectors for the mutated HNF4 proteins activated transcription more efficiently than that for the wild-type protein when cotransfected with the PKA catalytic subunit expression vector. Therefore, HNF4 is a direct target of PKA which might be involved in the transcriptional inhibition of liver genes by cAMP inducers.The liver-enriched transcription factor hepatocyte nuclear factor 4 (HNF4) (54) is involved in close association with other factors in hormonal and dietary control of liver and intestine genes. In addition, HNF4 has a potential role as a developmental regulator that has been conserved during evolution from invertebrates to vertebrates. HNF4 expression is restricted to the liver, kidney, and intestine (60) and, in Drosophila melanogaster, to the malpighian tubules (61). HNF4 exists as various isoforms. At least five different cDNAs are generated by differential splicing from a single gene, the HNF4␣ gene, in the amino-and carboxy-terminal regions; in all isoforms the DNA-binding domain remains unchanged (6,14,20,33,53,54). Recently, novel human and Xenopus HNF4 isoforms (HNF4␥ and HNF4␤, respectively) have been identified and shown to be derived from two distinct and differentially expressed genes (14,24). HNF4 is expressed very early during embryo development and has been found to be a crucial positive-acting factor for the expression of the HNF1 gene (55), placing HNF4 at the top of a transactivator hierarchy in hepatic cells (23,36). Disruption of the HNF4␣ gene led to early embryonic death due to malfunction of the yolk sac (7).HNF4 is a member of the nuclear receptor superfamily that binds to DNA at direct repeats separated by one nucleotide (DR1). This transactivator preexists as very stable homodimers in solution, and no HNF4-specific ligand has been identified so far (27). Therefore, HNF4 is classified as an orphan nuclear receptor, characterized by two zinc finger DNA-binding motifs, a large conserved hydrophobic domain containing the dimerization, and a putative ...

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Protein Kinase A Suppresses Sterol Regulatory Element-binding Protein-1C Expression via Phosphorylation of Liver X Receptor in the Liver

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