The nuclear xenobiotic receptor CAR (NR1I3) is the key transcription factor that regulates drug metabolism and excretion in the liver (1). Inactive CAR is sequestered as in the cytoplasm of hepatocytes. Various xenobiotics, typified by the classic CAR activator phenobarbital, indirectly activate CAR and translocate it into the nucleus. While the complete molecular mechanism remains elusive, de-phosphorylation has been thought to regulate this indirect CAR activation. This is evidenced by the protein phosphatase 2A inhibitor okadaic acid repressing activation of CAR in mouse primary hepatocytes (2). Recently we have characterized the conserved threonine residue (Thr38 and 48 in human and mouse CARs, respectively) within the DNA binding domain as the target of this de-phosphorylation responsible for CAR activation and nuclear translocation in mouse primary hepatocytes (3). Growth factors have been implicated in the repression of CAR via the activated extracellular signal-regulated kinase ERK1/2 (4). Here, we have determined that growth factors repress de-phosphorylation of threonine 38 and CAR activation. FLAG-tagged CAR T38A and CAR T38D mutants were expressed in Huh-7 cells and co-precipitated by an anti-phospho-ERK1/2 antibody. Phospho-ERK1/2 co-precipitated only the phosphorylation-mimicking CART38D mutant via the XRS peptide localized near the C-terminus of CAR molecules. Correlating with this interaction which occurred in growth factor-dependent manner; the results obtained from our newly developed ELISA revealed that threonine 38 is de-phosphorylated in Huh-7 cells after treatment with U0126. These results indicate that growth factors signal ERK1/2 to directly interact with the phosphorylated form of CAR and inhibit de-phosphorylation, thereby repressing CAR activation. This growth factor regulation was also observed in mouse primary hepatocytes.
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- Mutoh et al., Dephosphorylation of threonine 38 is required for nuclear translocation and activation of human xenobiotic receptor CAR (NR1I3). J. Biol. Chem. 284, 34785-34792, 2009.
- Koike et al., Extracellular signal-regulated kinase is an endogenous signal retaining the nuclear constitutive active/androstane receptor (CAR) in the cytoplasm of mouse primary hepatocytes. Mol. Pharmacol. 71,1217-1221, 2007.