Received for publication October 4, 2005.
Revised December 23, 2005.
Accepted for publication December 23, 2005.

Serine-202 regulates the nuclear translocation of constitutive active/androstane receptor CAR

Abstract

The constitutive active receptor CAR in mouse primary hepatocytes undergoes okadaic acid (OA)-sensitive nuclear translocation following 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 Ser202. Mutation of Ser202 to Asp (S202D) prevented mCAR translocation into the nucleus of TCPOBOP-treated HepG2 cells. Also, 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.

Key words: Protein ser/thr Phosphatases, Structure-activity relationships and modeling, Regulation of gene expression, Cytochrome P450, Genetics, Regulation - transcriptional, Regulation - xenobiotic, Cholesterol metabolism/lipoproteins

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