Phosphorylation of a conserved serine in the deoxyribonucleic acid binding domain of nuclear receptors alters intracellular localization.
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.
Sun
Kai
K
Environmental Toxicology Graduate Program, University of California, Riverside, California 92521, USA.
Montana
Vedrana
V
Chellappa
Karthikeyani
K
Brelivet
Yann
Y
Moras
Dino
D
Maeda
Yutaka
Y
Parpura
Vladimir
V
Paschal
Bryce M
BM
Sladek
Frances M
FM
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000737:chemistry / Q000378:metabolism; D017124:Conserved Sequence; D003593:Cytoplasm / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D015536:Down-Regulation; D051557:Hepatocyte Nuclear Factor 4 / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D015533:Transcriptional Activation
D051557:Hepatocyte Nuclear Factor 4; D018160:Receptors, Cytoplasmic and Nuclear; D012694:Serine; D004247:DNA; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate