Phosphorylation of a Conserved Serine in the Deoxyribonucleic Acid Binding Domain of Nuclear Receptors Alters Intracellular Localization

Abstract

Nuclear receptors (NRs) are a superfamily of transcription factors whose genomic functions are known to be activated by lipophilic ligands, but little is known about how to deactivate them or how to turn on their nongenomic functions. One obvious mechanism is to alter the nuclear localization of the receptors. Here, we show that protein kinase C (PKC) phosphorylates a highly conserved serine (Ser) between the two zinc fingers of the DNA binding domain of orphan receptor hepatocyte nuclear factor 4alpha (HNF4alpha). This Ser (S78) is adjacent to several positively charged residues (Arg or Lys), which we show here are involved in nuclear localization of HNF4alpha and are conserved in nearly all other NRs, along with the Ser/threonine (Thr). A phosphomimetic mutant of HNF4alpha (S78D) reduced DNA binding, transactivation ability, and protein stability. It also impaired nuclear localization, an effect that was greatly enhanced in the MODY1 mutant Q268X. Treatment of the hepatocellular carcinoma cell line HepG2 with PKC activator phorbol 12-myristate 13-acetate also resulted in increased cytoplasmic localization of HNF4alpha as well as decreased endogenous HNF4alpha protein levels in a proteasome-dependent fashion. We also show that PKC phosphorylates the DNA binding domain of other NRs (retinoic acid receptor alpha, retinoid X receptor alpha, and thyroid hormone receptor beta) and that phosphomimetic mutants of the same Ser/Thr result in cytoplasmic localization of retinoid X receptor alpha and peroxisome proliferator-activated receptor alpha. Thus, phosphorylation of this conserved Ser between the two zinc fingers may be a common mechanism for regulating the function of NRs.

The ERK1/2-Hepatocyte Nuclear Factor 4α Axis Regulates Human ABCC6 Gene Expression in Hepatocytes

“…It has been reported that HNF4␣ is under the control of various kinases (PKC and PKA) (38,39) and the role of ERK1/2 in the regulation of HNF4␣ has also been reported (40,41). This latter study suggested that the decreased endogenous HNF4␣ protein level due to various stimuli led to down-regulation of the expression of the target gene.…”

p38 MAP Kinase Links CAR Activation and Inactivation in the Nucleus via Phosphorylation at Threonine 38

“…Recently, estrogen receptor alpha (ERa) phosphorylated at the corresponding serine 216 was also found in mouse neutrophils and appears to regulate their infiltration into the uterus (Shindo et al, 2013). Although their phosphorylation in tissues in vivo have not yet been confirmed, studies with phosphomimetic mutants suggest that these residues in hepatocyte nuclear factor 4 alpha (HNF-4a), vitamin D receptor (VDR), peroxisome proliferator-activated receptor alpha, retinoid X receptor alpha (RXRa), and farnesoid X receptor may regulate various functionalities of these nuclear receptors, such as cytoplasmic retention, degradation, and transactivation (Hsieh et al, 1993;Sun et al, 2007;Gineste et al, 2008). Therefore, conserved phosphorylation has provided us with the opportunity to uniformly investigate nuclear receptors but has only been studied as the target of PKC or dephosphorylation by PP2A.…”

Site-specific Androgen Receptor Serine Phosphorylation Linked to Epidermal Growth Factor-dependent Growth of Castration-recurrent Prostate Cancer

“…However, the corresponding residue in the AR subgroup of steroid receptors is alanine and is Ala-586 in human AR. PKC phosphorylation at Ser-78 in the DNA binding domain of hepatocyte nuclear factor-4␣ and other nuclear receptors regulates nuclear localization (60). Hepatocyte nuclear factor-4␣-S78A bound DNA, but the S78D phosphomimetic did not.…”