Serine 202 regulates the nuclear translocation of constitutive active/androstane receptor.
The constitutive active receptor (CAR) in mouse primary hepatocytes undergoes okadaic acid (OA)-sensitive nuclear translocation after activation by xenobiotics such as phenobarbital (PB) and 1,4 bis[2-(3,5-dichloropyridyloxy)]benzene (TCPOBOP). We have now mimicked this TCPOBOP-dependent and OA-sensitive translocation of mouse CAR (mCAR) in HepG2 cells and have demonstrated that protein phosphatase 2A regulates this nuclear translocation. Site-directed mutagenesis analysis of various Ser and Thr residues delineated the translocation activity to Ser-202. Mutation of Ser-202 to Asp (S202D) prevented mCAR translocation into the nucleus of TCPOBOP-treated HepG2 cells. In addition, in the livers of Car-/- mice, the YFP-tagged S202D mutant did not translocate into the nucleus after PB treatment. To examine whether Ser-202 can be phosphorylated, flag-tagged wild-type mCAR or flag-tagged S202A mutant was expressed in HepG2 cells and subjected to Western blot analysis using an antibody specific to a peptide containing phospho-Ser-202. A high molecular weight phosphorylated form of CAR was detected only with the wild-type mCAR. These results are consistent with the conclusion that the dephosphorylation of Ser-202 is a required step that regulates the xenobiotic-dependent nuclear translocation of mCAR.
Hosseinpour
F
F
Pharmacogenetics Section, Laboratory of Reproductive and Developmental Toxicology, NIEHS, NIH, Research Triangle Park, NC 2770, USA.
Moore
R
R
Negishi
M
M
Sueyoshi
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D000090702:Constitutive Androstane Receptor; D051379:Mice; D008969:Molecular Sequence Data; D010634:Phenobarbital / Q000494:pharmacology; D010766:Phosphorylation; D021381:Protein Transport; D011725:Pyridines / Q000494:pharmacology; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*
D000090702:Constitutive Androstane Receptor; D011725:Pyridines; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D012694:Serine; C028474:1,4-bis(2-(3,5-dichloropyridyloxy))benzene; D010634:Phenobarbital