Characterization of the phosphorylation status of the hepatitis B virus X-associated protein 2

The cytosolic Ah receptor (AhR) heterocomplex consists of one molecule of the AhR, a 90-kDa heat shock protein (Hsp90) dimer, and one molecule of the hepatitis B virus X-associated protein 2 (XAP2). Serine residues 43,53,131-2, and 329 on XAP2-FLAG were identified as putative phosphorylation sites using site-directed mutagenesis followed by two-dimensional phosphopeptide mapping analysis. Protein kinase CK2 (CK2) was identified as the 45-kDa kinase from COS 1 cell or liver extracts that was responsible for phosphorylation of serine 43 in the XAP2 peptide 39-57. Loss of phosphorylation at any or all of the serine residues did not significantly affect the ability of XAP2-FLAG to bind to the murine AhR in rabbit reticulocyte lysate or Hsp90 in COS-1 cells. Furthermore, all of these serine mutants were able to sequester murine AhR-YFP into the cytoplasm as well as wild-type XAP2. YFP-XAP2 S53A was unable to enter the nucleus, indicating a potential role of phosphorylation in nuclear translocation of XAP2.     

Subcellular localization of the aryl hydrocarbon receptor is modulated by the immunophilin homolog hepatitis B virus X-associated protein 2

The hepatitis B virus X-associated protein 2 (XAP2) is an immunophilin homolog and core component of the aryl hydrocarbon receptor (AhR). Immunophilins are components of many steroid receptor complexes, serving a largely unknown function. Transiently expressed AhR.YFP (yellow fluorescent protein) localized to the nuclei of COS-1 and NIH-3T3 cells. Co-expression of AhR.YFP with XAP2 restored cytoplasmic localization, which was reversed by 2,3,7, 8-tetrachlorodibenzo-p-dioxin treatment (TCDD). The effect of XAP2 on AhR localization was specific involving a nuclear localization signal-mediated pathway. Examination of the ratio of AhR to XAP2 in the AhR complex revealed that approximately 25% of transiently expressed AhR was associated with XAP2, in contrast with approximately 100% when the AhR and XAP2 were co-expressed. Strikingly, TCDD did not influence these ratios, suggesting that ligand binding initiates nuclear translocation prior to complex dissociation. Analysis of endogenous AhR in Hepa-1 cells revealed that approximately 40% of the AhR complex was associated with XAP2, predicting observed AhR localization to cytoplasm and nuclei. This study reveals a novel functional role for the immunophilin-like component of a soluble receptor complex and provides new insight into the mechanism of AhR-mediated signal transduction, demonstrating the existence of two structurally distinct and possibly functionally unique forms of the AhR.     

Characterization of the AhR-hsp90-XAP2 core complex and the role of the immunophilin-related protein XAP2 in AhR stabilization.

The unliganded aryl hydrocarbon receptor (AhR) exists in the cytoplasm in a tetrameric 9S core complex, consisting of the AhR ligand-binding subunit, a dimer of hsp90, and the hepatitis B virus X-associated protein 2 (XAP2), an immunophilin-related protein sharing homologous regions with FKBP12 and FKBP52. Interactions between the recently identified XAP2 subunit and other members of the unliganded AhR complex and its precise role in the AhR signal transduction pathway are presently unknown. Mapping studies indicate that XAP2 requires the PAS, hsp90, and ligand binding domain(s) of the AhR for binding, and that both proteins directly interact in the absence of hsp90. XAP2 is also able to interact with hsp90 complexes in the absence of the AhR, and C-terminal sequences of XAP2 are required for this interaction. XAP2 binds to the C-terminal end of hsp90, which contains a tetratricopeptide repeat domain acceptor site, whereas the AhR binds to a domain in the middle of hsp90. XAP2 was not found to be associated with the AhR-Arnt heterocomplex either in vitro or in nuclear extracts isolated from Hepa 1 cells treated with TCDD. Transient expression of XAP2 in COS-1 cells resulted in enhanced cytosolic AhR levels, suggesting a role for XAP2 in regulating the rate of AhR turnover.     

Borna disease virus nucleoprotein requires both nuclear localization and export activities for viral nucleocytoplasmic shuttling

Nuclear transport of viral nucleic acids is crucial to the life cycle of many viruses. Borna disease virus (BDV) belongs to the order Mononegavirales and replicates its RNA genome in the nucleus. Previous studies have suggested that BDV nucleoprotein (N) and phosphoprotein (P) have important functions in the nuclear import of the viral ribonucleoprotein (RNP) complexes via their nuclear targeting activity. Here, we showed that BDV N has cytoplasmic localization activity, which is mediated by a nuclear export signal (NES) within the sequence. Our analysis using deletion and substitution mutants of N revealed that NES of BDV N consists of a canonical leucine-rich motif and that the nuclear export activity of the protein is mediated through the chromosome region maintenance protein-dependent pathway. Interspecies heterokaryon assay indicated that BDV N shuttles between the nucleus and cytoplasm as a nucleocytoplasmic shuttling protein. Furthermore, interestingly, the NES region overlaps a binding site to the BDV P protein, and nuclear export of a 38-kDa form of BDV N is prevented by coexpression of P. These results suggested that BDV N has two contrary activities, nuclear localization and export activity, and plays a critical role in the nucleocytoplasmic transport of BDV RNP by interaction with other viral proteins.     

The role of chaperone proteins in the aryl hydrocarbon receptor core complex

The aryl hydrocarbon receptor (AhR) exists in the absence of a ligand as a tetrameric complex composed of a 95-105 kDa ligand binding subunit, a dimer of hsp90, and the immunophilin-like X-associated protein 2 (XAP2). XAP2 has a highly conserved carboxy terminal tetratricopeptide repeat domain that is required for both hsp90 and AhR binding. Hsp 90 appears to be involved in the initial folding of newly synthesized AhR, stabilization of ligand binding conformation of the receptor, and inhibition of constitutive dimerization with ARNT. XAP2 is capable of stabilizing the AhR, as well as enhancing cytoplasmic localization of the receptor. XAP2 binds to both the AhR and hsp90 in the receptor complex, and is capable of independently binding to both hsp90 and the AhR. However, the exact functional role for XAP2 in the AhR complex remains to be fully established.     

Nucleocytoplasmic shuttling of p53 is essential for MDM2-mediated cytoplasmic degradation but not ubiquitination

As a shuttling protein, p53 is constantly transported through the nuclear pore complex. p53 nucleocytoplasmic transport is carried out by a bipartite nuclear localization signal (NLS) located at its C-terminal domain and two nuclear export signals (NES) located in its N- and C-terminal regions, respectively. The role of nucleocytoplasmic shuttling in p53 ubiquitination and degradation has been a subject of debate. Here we show that the two basic amino acid groups in the p53 bipartite NLS function collaboratively to import p53. Mutations disrupting individual amino acids in the NLS, although causing accumulation of p53 in the cytoplasm to various degrees, reduce but do not eliminate the NLS activity, and these mutants remain sensitive to MDM2 degradation. However, disrupting both parts of the bipartite NLS completely blocks p53 from entering the nucleus and causes p53 to become resistant to MDM2-mediated degradation. Similarly, mutations disrupting four conserved hydrophobic amino acids in the p53 C-terminal NES block p53 export and prohibit it from MDM2 degradation. We also show that colocalization of a nonshuttling p53 with MDM2 either in the nucleus or in the cytoplasm is sufficient for MDM2-induced p53 polyubiquitination but not degradation. Our data provide new insight into the mechanism and regulation of p53 nucleocytoplasmic shuttling and degradation.     

Mouse Disabled1 (DAB1) is a nucleocytoplasmic shuttling protein

Disabled1 (DAB1) is an intracellular mediator of the Reelin-signaling pathway and essential for correct neuronal positioning during brain development. So far, DAB1 has been considered a cytoplasmic protein. Here, we show that DAB1 is subject to nucleocytoplasmic shuttling. In its steady state, DAB1 is mainly located in the cytoplasm. However, treatment with leptomycine B, a specific inhibitor of the CRM1 (chromosomal region maintenance 1)-RanGTP-dependent nuclear export, resulted in nuclear accumulation of DAB1. By using deletion or substitutional mutants of DAB1 fused with enhanced green fluorescent protein, we have mapped a bipartite nuclear localization signal and two CRM1-dependent nuclear export signals. These targeting signals were functional in both Neuro2a cells and primary cerebral cortical neurons. Using purified recombinant proteins, we have shown that CRM1 binds to DAB1 directly in a RanGTP-dependent manner. We also show that tyrosine phosphorylation of DAB1, which is indispensable for the layer formation of the brain, by Fyn tyrosine kinase or Reelin stimulation did not affect the subcellular localization of DAB1 in vitro. These results suggest that DAB1 is a nucleocytoplasmic shuttling protein and raise the possibility that DAB1 plays a role in the nucleus as well as in the cytoplasm.