Gq. Jiang et al., SERINE THREONINE PHOSPHORYLATION OF ORPHAN RECEPTOR HEPATOCYTE NUCLEAR FACTOR-4/, Archives of biochemistry and biophysics, 340(1), 1997, pp. 1-9
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.