Protein kinase A-dependent phosphorylation modulates DNA-binding activity of hepatocyte nuclear factor 4.
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 DNA-binding 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 Mutants of HNF4 in which two potentially phosphorylatable serines have been replaced by either neutral or charged amino acids were able to bind DNA in vitro with affinity similar to that of the wild-type protein.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.
Viollet
B
B
Institut Cochin de Génétique Moléculaire, U.129 INSERM, Universite René Descartes, Paris, France.
Kahn
A
A
Raymondjean
M
M
D000595:Amino Acid Sequence; D000818:Animals; D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D019556:COS Cells; D006528:Carcinoma, Hepatocellular; D002457:Cell Extracts; D002467:Cell Nucleus / Q000378:metabolism; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005508:Food Deprivation / Q000502:physiology; D051557:Hepatocyte Nuclear Factor 4; D006801:Humans; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D012694:Serine / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation / Q000502:physiology*; D014407:Tumor Cells, Cultured
D051778:Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; D002457:Cell Extracts; D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; C403128:MLX protein, human; D010750:Phosphoproteins; D014157:Transcription Factors; D012694:Serine; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases