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 overexp ressed to high levels in insect cells using a baculovirus expression s ystem, 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 H NF4.COS are phosphorylated although HNF4.BV was hypophosphorylated rel ative to HNF4.COS. Phosphoamino acid analysis showed that HNF-4 is pho sphorylated mainly on serine and to a lesser extent on threonine resid ues, Phosphopeptide mapping revealed 13 phosphopeptides for HNF4.COS, only 9 of which were present in the HNF4.BV sample, DNA-binding studie s also showed that HNF4.BV binds DNA with a lower specificity and affi nity, 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 numbe r of methods and since HNF-4 expressed in bacteria exhibited an even l ower DNA-binding affinity than HNF4.BV, we propose that serine/theroni ne phosphorylation may play a role in the DNA-binding activity of HNF- 4 and, therefore, possibly of other Group IV receptors as well. (C) 19 97 Academic Press.