Received for publication October 26, 2007.
Revised January 17, 2008.
Accepted for publication January 17, 2008.

PPP1R16A, the membrane subunit of protein phosphatase 1, signals nuclear translocation of the nuclear receptor CAR

Abstract

CAR, a member of the nuclear steroid/thyroid hormone receptor family, activates transcription of numerous hepatic genes upon exposure to therapeutic drugs and environmental pollutants. Sequestered in the cytoplasm, this receptor signals xenobiotic exposure, such as phenobarbital (PB), by translocating into the nucleus. Unlike other hormone receptors, translocation can be triggered indirectly without binding to xenobiotics. We have now identified a membrane-associated subunit of protein phosphatase 1(PPP1R16A, or abbreviated as R16A) as a novel CAR binding protein. When CAR and R16A are co-expressed in mouse liver, CAR translocates into the nucleus. Close association of R16A and CAR molecule on liver membrane was shown by FRET analysis using expressed YFP-CAR and CFP-R16A fusion proteins. R16A can form dimer through its middle region where protein kinase A phosphorylation sites are recently identified. Translocation of CAR by R16A correlates with the ability of R16A to form an inter-molecular interaction via the middle region. Moreover, this interaction is enhanced by PB treatment in mouse liver. R16A specifically interacted with PP1 in HepG2 cells despite the highly conserved structure of PP1 family molecules. PP1 activity was inhibited by R16A in vitro and co-expression of PP1 in liver can prevent YFP-CAR translocation into mouse liver. Taken together, R16A at the membrane may mediate the PB signal to initiate CAR nuclear translocation, through a mechanism including its dimerization and inhibition of PP1 activity, providing a novel model for the translocation of nuclear receptors in which direct interaction of ligands and the receptors may not be crucial.

Key words: Fluorescence techniques, Regulation of gene expression, Yeast 2-hybrid, Regulation - transcriptional, Regulation - xenobiotic