Serine/threonine phosphorylation of orphan receptor hepatocyte nuclear factor 4.
We showed previously that hepatocyte nuclear factor 4 (HNF-4) defines a new subclass, Group IV, of nuclear receptors. In order to determine whether members of this subclass are phosphorylated, HNF-4 was overexpressed to high levels in insect cells using a baculovirus expression system. The baculovirus-expressed HNF-4 (HNF4.BV) was characterized and compared to HNF-4 overexpressed in transiently transfected mammalian (COS-7) cells (HNF4.COS). The results indicate that both HNF4.BV and HNF4.COS are phosphorylated although HNF4.BV was hypophosphorylated relative to HNF4.COS. Phosphoamino acid analysis showed that HNF-4 is phosphorylated mainly on serine and to a lesser extent on threonine residues. Phosphopeptide mapping revealed 13 phosphopeptides for HNF4.COS, only 9 of which were present in the HNF4.BV sample. DNA-binding studies also showed that HNF4.BV binds DNA with a lower specificity and affinity, as measured by the equilibrium dissociation constant (Kd), than does HNF4.COS. Partial proteolytic digestion experiments also revealed that HNF4.BV and HNF4.COS adopt somewhat different three-dimensional conformations. Since glycosylation of HNF4.BV was ruled out by a number of methods and since HNF-4 expressed in bacteria exhibited an even lower DNA-binding affinity than HNF4.BV, we propose that serine/theronine phosphorylation may play a role in the DNA-binding activity of HNF-4 and, therefore, possibly of other Group IV receptors as well.
Jiang
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G
Environmental Toxicology Graduate Program, University of California, Riverside 92521, USA.
Nepomuceno
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L
Yang
Q
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Sladek
F M
FM
D000818:Animals; D019556:COS Cells; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006031:Glycosylation; D051557:Hepatocyte Nuclear Factor 4; D009687:Nuclear Proteins / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D010769:Phosphothreonine / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins; D018411:Spodoptera; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*
D004268:DNA-Binding Proteins; D051557:Hepatocyte Nuclear Factor 4; D009687:Nuclear Proteins; D010748:Phosphopeptides; D010750:Phosphoproteins; D011994:Recombinant Proteins; D014157:Transcription Factors; D010769:Phosphothreonine; D010768:Phosphoserine