Serine/threonine phosphorylation of orphan receptor hepatocyte nuclear factor 4.
Arch Biochem Biophys
; 340(1): 1-9, 1997 Apr 01.
Article
em En
| MEDLINE
| ID: mdl-9126270
ABSTRACT
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.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fosfoproteínas
/
Fosfosserina
/
Fosfotreonina
/
Fatores de Transcrição
/
Proteínas de Ligação a DNA
Limite:
Animals
Idioma:
En
Revista:
Arch Biochem Biophys
Ano de publicação:
1997
Tipo de documento:
Article
País de afiliação:
Estados Unidos