Dephosphorylation of threonine 38 is required for nuclear translocation and activation of human xenobiotic receptor CAR (NR1I3).
Upon activation by therapeutics, the nuclear xenobiotic/ constitutive active/androstane receptor (CAR) regulates various liver functions ranging from drug metabolism and excretion to energy metabolism. CAR can also be a risk factor for developing liver diseases such as hepatocellular carcinoma. Here we have characterized the conserved threonine 38 of human CAR as the primary residue that regulates nuclear translocation and activation of CAR. Protein kinase C phosphorylates threonine 38 located on the alpha-helix spanning from residues 29-42 that constitutes a part of the first zinc finger and continues into the region between the zinc fingers. Molecular dynamics study has revealed that this phosphorylation may destabilize this helix, thereby inactivating CAR binding to DNA as well as sequestering it in the cytoplasm. We have found, in fact, that helix-stabilizing mutations reversed the effects of phosphorylation. Immunohistochemical study using an anti-phospho-threonine 38 peptide antibody has, in fact, demonstrated that the classic CAR activator phenobarbital dephosphorylates the corresponding threonine 48 of mouse CAR in the cytoplasm of mouse liver and translocates CAR into the nucleus. These results define CAR as a cell signal-regulated constitutive active nuclear receptor. These results also provide phosphorylation/dephosphorylation of the threonine as the primary drug target for CAR activation.
Mutoh
Shingo
S
Laboratories of Reproductive and Developmental Toxicology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
Osabe
Makoto
M
Inoue
Kaoru
K
Moore
Rick
R
Pedersen
Lee
L
Perera
Lalith
L
Rebolloso
Yvette
Y
Sueyoshi
Tatsuya
T
Negishi
Masahiko
M
D021581:Active Transport, Cell Nucleus / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D000090702:Constitutive Androstane Receptor; D018757:GABA Modulators / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008099:Liver / Q000166:cytology / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010634:Phenobarbital / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology; D013912:Threonine / Q000378:metabolism*; D016335:Zinc Fingers
D000090702:Constitutive Androstane Receptor; D018757:GABA Modulators; C000716327:NR1I3 protein, human; C000716328:Nr1i3 protein, mouse; D018160:Receptors, Cytoplasmic and Nuclear; D011993:Recombinant Fusion Proteins; D013912:Threonine; D010634:Phenobarbital