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 α-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.     

Induction of cytochrome P4502B: Role of regulatory elements and nuclear receptors

Cytochrome P450 of the 2B subfamily is easily induced by many xenobiotics. In spite of intensive investigations, the molecular mechanisms of regulation of the CYP2B genes are not clear. The nuclear receptor CAR is shown to play a crucial role in the activation of CYP2B genes by xenobiotics, but many problems of CAR activation in different animal species and humans remain unsolved. This review focuses on signaling pathways involved in the control of CYP2B gene expression in mammals.     

CAR and PXR: the xenobiotic-sensing receptors

The xenobiotic receptors CAR and PXR constitute two important members of the NR1I nuclear receptor family. They function as sensors of toxic byproducts derived from endogenous metabolism and of exogenous chemicals, in order to enhance their elimination. This unique function of CAR and PXR sets them apart from the steroid hormone receptors. In contrast, the steroid receptors, exemplified by the estrogen receptor (ER) and glucocorticoid receptor (GR), are the sensors that tightly monitor and respond to changes in circulating steroid hormone levels to maintain body homeostasis. This divergence of the chemical- and steroid-sensing functions has evolved to ensure the fidelity of the steroid hormone endocrine regulation while allowing development of metabolic elimination pathways for xenobiotics. The development of the xenobiotic receptors CAR and PXR also reflect the increasing complexity of metabolism in higher organisms, which necessitate novel mechanisms for handling and eliminating metabolic by-products and foreign compounds from the body. The purpose of this review is to discuss similarities and differences between the xenobiotic receptors CAR and PXR with the prototypical steroid hormone receptors ER and GR. Interesting differences in structure explain in part the divergence in function and activation mechanisms of CAR/PXR from ER/GR. In addition, the physiological roles of CAR and PXR will be reviewed, with discussion of interactions of CAR and PXR with endocrine signaling pathways.     

Nuclear and cytoplasmic peroxiredoxin-1 differentially regulate NF-kappaB activities

Peroxiredoxins (Prx) are widely distributed and abundant proteins, which control peroxide concentrations and related signaling mechanisms. Prx1 is found in the cytoplasm and nucleus, but little is known about compartmentalized Prx1 function during redox signaling and oxidative stress. We targeted expression vectors to increase Prx1 in nuclei (NLS-Prx1) and cytoplasm (NES-Prx1) in HeLa cells. Results showed that NES-Prx1 inhibited NF-kappaB activation and nuclear translocation. In contrast, increased NLS-Prx1 did not affect NF-kappaB nuclear translocation but increased activity of a NF-kappaB reporter. Both NLS-Prx1 and NES-Prx1 inhibited NF-kappaB p50 oxidation, suggesting that oxidation of the redox-sensitive cysteine in p50's DNA-binding domain is regulated via peroxide metabolism in both compartments. Interestingly, following treatment with H(2)O(2), nuclear thioredoxin-1 (Trx1) redox status was protected by NLS-Prx1, and cytoplasmic Trx1 was protected by NES-Prx1. Compartmental differences from increasing Prx1 show that the redox poise of cytoplasmic and nuclear thiol systems can be dynamically controlled through peroxide elimination. Such spatial resolution and protein-specific redox differences imply that the balance of peroxide generation/metabolism in microcompartments provides an important specific component of redox signaling.     

Examining the mechanism of Erk nuclear translocation using green fluorescent protein

In neuronal cells, the mitogen-activated protein kinase (MAP kinase) cascade is an important mediator of neurotrophin signaling from cell surface receptors to the nucleus, resulting in changes in gene expression. Nuclear localization of Erk is thought to be required for these effects. To examine the mechanism and regulation of Erk nuclear translocation, we have created a green fluorescent protein (GFP)-labeled Erk2 construct, which provides a sensitive means to follow the movement of Erk from the cytoplasm to the nucleus following receptor-mediated MAP kinase activation. Using this system in PC12 cells, we have examined a number of mechanisms that have been implicated in regulating the translocation of Erk. In PC12 cells, NGF and EGF induce a rapid translocation of GFP-Erk that requires Ras and Mek. We have found that prolonged phosphorylation of Erk is not required for the rapid and early influx of Erk into the nucleus following growth factor stimulation. Furthermore, following influx, GFP-Erk rapidly returned to the cytoplasm regardless of its phosphorylation state. The release of Erk from its cytoplasmic activator, Mek, followed by the dimerization of Erk, was sufficient to stimulate nuclear uptake, whereas Erk kinase activity was dispensable. PKA activity has been reported to be required for Erk translocation in PC12 cells. However, PKA activity was also not necessary for the early translocation of Erk into the nucleus by NGF or Ras, but it was able to induce a small influx of Erk that could be measured with GFP-Erk2.     

Sef is a spatial regulator for Ras/MAP kinase signaling

Spatiotemporal control of the Ras/ERK MAP kinase signaling pathway is among the key mechanisms for regulating a wide variety of cellular processes. In this study, we report that human Sef (hSef), a recently identified inhibitor whose action mechanism has not been fully defined, acts as a molecular switch for ERK signaling by specifically blocking ERK nuclear translocation without inhibiting its activity in the cytoplasm. Thus, hSef binds to activated forms of MEK, inhibits the dissociation of the MEK-ERK complex, and blocks nuclear translocation of activated ERK. Consequently, hSef inhibits phosphorylation and activation of the nuclear ERK substrate Elk-1, while it does not affect phosphorylation of the cytoplasmic ERK substrate RSK2. Downregulation of endogenous hSef by hSef siRNA enhances the stimulus-induced ERK nuclear translocation and the activity of Elk-1. These results thus demonstrate that hSef acts as a spatial regulator for ERK signaling by targeting ERK to the cytoplasm.     

Overexpression of the Rho-guanine nucleotide exchange factor ECT2 inhibits nuclear translocation of nuclear receptor CAR in the mouse liver

Various drugs such as phenobarbital (PB) trigger translocation of constitutive active/adrostane receptor (CAR) from the cytoplasm into the nucleus of mouse liver cells without directly binding to the receptor. We have now characterized the guanine nucleotide exchange factor epithelial cell-transforming gene 2 (ECT2) as a PB-inducible factor as well as a cellular signal that represses PB-triggered nuclear translocation of CAR. When CFP-tagged ECT2 was co-expressed with YFP-tagged CAR in the liver of Car(-/-) mice, ECT2 repressed CAR nuclear translocation. Coexpression of various deletion mutants delineated this repressive activity to the tandem Dbl homology/pleckstrin homology domains of ECT2 and to their cytosolic expression. CAR directly bound to the PH domain. Thus, ECT2 may comprise a part of the PB response signal regulating the intracellular trafficking of CAR.     

Constitutive androstane receptor (CAR) is a xenosensor and target for therapy

Constitutive androstane receptor (CAR, NR1I3), which is under consideration in this review, is a member of the superfamily of nuclear receptors. However, certain features distinguish CAR from the variety of nuclear receptors. First, this receptor has structural features that allow it to display constitutive activity in the absence of a ligand and to interact in a species-specific manner with a huge number of ligands diverse in chemical structure and origin. Second, recently many researchers are focused on CAR because the significance is increasingly shown of its influence on a variety of physiological functions, such as gluconeogenesis, metabolism of xenobiotics, fatty acids, bilirubin, and bile acids, hormonal regulation, etc. In addition to the fundamental scientific interest, the study of CAR is of practical importance because changes in CAR activity can lead to disorders in physiological processes, which finally can result in changes in pathological states. However, despite intensive studies, many mechanisms are still unclear, which makes it difficult to understand the role of CAR in the overall picture of molecular regulation of physiological processes. This review analyzes the features and diversity of the functions of CAR.     

TNFalpha induces rapid activation and nuclear translocation of telomerase in human lymphocytes

Maintenance of telomeres regulates chromosomal stability and cellular mitosis through a checkpoint mechanism. Continuous cell proliferation requires telomerase to maintain chromosomal stability and to counteract the cellular mitotic clock. Importantly, nuclear expression of telomerase activity is required for elongation of telomere sequences. In this study, we show that tumor necrosis factor alpha (TNFalpha) induces telomerase activity in the cytoplasm of peripheral blood lymphocytes (PBL) at 60 min, followed by translocation of activated telomerase to the nucleus at 120 min. Conversely, the phosphoinositol 3-kinase (PI3K) inhibitor wortmannin blocks TNFalpha-induced activation of telomerase, whereas the specific NF-kappaB translocation inhibitor SN-50 blocks TNFalpha-induced nuclear translocation of activated telomerase. These studies suggest that activation and nuclear translocation of telomerase are regulated by PI3K/Akt/NF-kappaB signaling pathways in PBL.     

Nucleo-Cytoplasmic Trafficking of TRIM8, a Novel Oncogene,Is Involved in Positive Regulation of TNF Induced NF-κB Pathway

TNF induced nuclear factor kappa B (NF-κB) is one of the central signaling pathways that plays a critical role in carcinogenesis and inflammatory diseases. Post-translational modification through ubiquitin plays important role in the regulation of this pathway. In the current study, we investigated the role of TRIM8, member of RING family ubiquitin ligase in regulation of NF-κB pathway. We observed that TRIM8 positively regulates TNF induced NF-κB pathway. Different domains of TRIM8 showed discrete functions at the different steps in regulation of TNF induced NF-κB pathway. Ubiquitin ligase activity of TRIM8 is essential for regulation of NF-κB activation in both cytoplasm as well as nucleus. TRIM8 negates PIAS3 mediated negative repression of NF-κB at p65 by inducing translocation of PIAS3 from nucleus to cytoplasm as well as its turnover. TNF induces translocation of TRIM8 from nucleus to cytoplasm, which positively regulates NF-κB. The cytoplasmic translocation of TRIM8 is essential for TNF induced NF-κB but not for p65 mediated NF-κB regulation. TRIM8 also enhanced the clonogenic and migration ability of cells by modulating NF-κB. The further study will help to understand the role of TRIM8 in inflammation and cancer.