Critical role of a common transcription factor, IRF-1, in the regulation of IFN-beta and IFN-inducible genes.

Interferon regulatory factor 1 (IRF-1) is a protein that binds to cis-elements within the promoter of interferon (IFN)-beta and some IFN-inducible genes. We used a human fibroblast line, GM-637, to generate stable transfectants constitutively expressing IRF-1 mRNA in either the sense or antisense orientation. Upon induction with poly-(I).poly(C) or Newcastle disease virus, cells expressing sense IRF-1 mRNA produced significantly higher levels of IFN-beta mRNA and protein than control cells, whereas cells expressing antisense IRF-1 mRNA produced little or no IFN-beta mRNA and protein. Furthermore, clear differences were seen among the transfectants in the level of expression of two IFN-induced genes (2'-5'-oligoadenylate synthetase and class I HLA). Our data show that IRF-1 is essential for the induced expression of the IFN-beta gene. The results also indicate an important role of IRF-1 in the expression of IFN-inducible genes and suggest a role for IRF-1 in many other cytokine actions.     

Two separate regions essential for nuclear import of the hnRNP D nucleocytoplasmic shuttling sequence

Heterogeneous nuclear ribonucleoprotein (hnRNP) D/AUF1 functions in mRNA genesis in the nucleus and modulates mRNA decay in the cytoplasm. Although it is primarily nuclear, it shuttles between the nucleus and cytoplasm. We studied the nuclear import and export of the last exon-encoding sequence common to all its isoforms by its expression as a green fluorescent protein-fusion protein in HeLa cells and by heterokaryon assay. The C-terminal 19-residue sequence (SGYGKVSRRGGHQNSYKPY) was identified as an hnRNP D nucleocytoplasmic shuttling sequence (DNS). In vitro nuclear transport using permeabilized cells indicated that nuclear import of DNS is mediated by transportin-1 (Trn-1). DNS accumulation in the nucleus was dependent on Trn-1, Ran, and energy in multiple rounds of nuclear transport. Use of DNS with deletions, alanine scanning mutagenesis and point mutations revealed that two separate regions (the N-terminal seven residues and the C-terminal two residues) are crucial for in vivo and in vitro transport as well as for interaction with Trn-1. The N- and C-terminal motifs are conserved in the shuttling sequences of hnRNP A1 and JKTBP.     

Nuclear localization of C-terminal domains of the kinesin-like protein MKLP-1.

The successful execution of mitosis in mammalian cells requires the activities of numerous kinesin-like proteins. The Mitotic Kinesin-Like Protein-1 (MKLP-1) localizes to the spindle equator and is believed to participate in the separation of spindle poles during anaphase B. Injection of antibodies against MKLP-1 into dividing cells results in cell cycle arrest, suggesting that MKLP-1 is essential for mitosis. To further characterize MKLP-1, constructs encoding C-terminal domains of MKLP-1 were expressed as fusions to the green fluorescent protein and localized in HeLa cells. All constructs localized to the nucleus indicating the presence of at least one nuclear localization sequence in the C-terminus of the protein. C-terminal domains of MKLP-1 expressed in insect cells also localized to the nucleus as shown by subcellular fractionation. These proteins remained tightly associated with the nucleus following both detergent and salt extraction, suggesting a tight interaction with a component of the nucleus.     

Characterization of photolyase/blue-light receptor homologs in mouse and human cells.

We isolated and characterized mouse photolyase-like genes, mCRY1 (mPHLL1) and mCRY2 (mPHLL2), which belong to the photolyase family including plant blue-light receptors. The mCRY1 and mCRY2 genes are located on chromosome 10C and 2E, respectively, and are expressed in all mouse organs examined. We raised antibodies specific against each gene product using its C-terminal sequence, which differs completely between the genes. Immunofluorescent staining of cultured mouse cells revealed that mCRY1 is localized in mitochondria whereas mCRY2 was found mainly in the nucleus. The subcellular distribution of CRY proteins was confirmed by immunoblot analysis of fractionated mouse liver cell extracts. Using green fluorescent protein fused peptides we showed that the C-terminal region of the mouse CRY2 protein contains a unique nuclear localization signal, which is absent in the CRY1 protein. The N-terminal region of CRY1 was shown to contain the mitochondrial transport signal. Recombinant as well as native CRY1 proteins from mouse and human cells showed a tight binding activity to DNA Sepharose, while CRY2 protein did not bind to DNA Sepharose at all under the same condition as CRY1. The different cellular localization and DNA binding properties of the mammalian photolyase homologs suggest that despite the similarity in the sequence the two proteins have distinct function(s).     

Mutant adenovirus type 9 E4 ORF1 genes define three protein regions required for transformation of CREF cells.

Human adenovirus type 9 (Ad9) elicits exclusively estrogen-dependent mammary tumors in rats, and an essential oncogenic determinant for this virus is Ad9 E4 open reading frame 1 (9ORF1), which encodes a 125-residue cytoplasmic protein with cellular growth-transforming activity in vitro. In this study, we engineered 48 different mutant 9ORF1 genes in an attempt to identify regions of this viral protein essential for transformation of the established rat embryo fibroblast cell line CREF. In initial assays with CREF cells, 17 of the 48 mutant 9ORF1 genes proved to be severely defective for generating transformed foci but only 7 of these defective genes expressed detectable amounts of protein. To further examine the defects of the seven mutant proteins, we selected individual cell pools of stable CREF transformants for the wild-type and mutant 9ORF1 genes. Compared to cell pools expressing the wild-type 9ORF1 protein, most cell pools expressing mutant proteins displayed decreased growth in soft agar, and all generated significantly smaller tumors in syngeneic animals. The altered amino acid residues of the seven mutant 9ORF1 polypeptides clustered within three separate regions referred to as region I (residues 34 to 41), region II (residues 89 to 91), and C-terminal region III (residues 122 to 125). By using indirect immunofluorescence, we also assessed whether the mutant proteins localized properly to the cytoplasm of cells. The region I and region II mutants displayed approximately wild-type subcellular localizations, whereas most region III mutants aberrantly accumulated within the nucleus of cells. In summary, we have identified three 9ORF1 protein regions necessary for cellular transformation and have demonstrated that C-terminal region III sequences significantly influence the proper localization of the 9ORF1 polypeptide in cells.     

The stability and anti-apoptotic function of A1 are controlled by its C terminus

Most Bcl-2 family members can localize to intracellular membranes via hydrophobic sequences within their C-terminal portion. We found that the C terminus of the anti-apoptotic family member A1 did not function as a membrane anchor. Instead, this stretch of the protein rendered A1 highly unstable by mediating its polyubiquitination and rapid proteasomal degradation. Moreover, the domain did not only function independently of its position within the A1 protein but when transferred could even destabilize unrelated proteins like enhanced green fluorescent protein and caspase-3. A1 was, however, much more stable in the presence of the Bcl-2 homology-only protein BimEL, suggesting that direct interaction of A1 with pro-apoptotic members of the Bcl-2 family strongly reduces its rate of turnover. We further show that the C-terminal end of A1 also contributes to the anti-apoptotic capacity of the protein. In conclusion, our data demonstrate that the C terminus serves a dual function by controlling the stability of A1 and by amplifying the capacity of the protein to protect cells against apoptosis.     

Antiproliferative proteins of the BTG/Tob family are degraded by the ubiquitin-proteasome system.

BTG/Tob family proteins, which are characterized by similarities in their N-terminal BTG/Tob homology domains, control cell growth negatively. Among the BTG/Tob family members, BTG2/TIS21/PC3 proteins have been reported to have short lives and to be degraded by the proteasome. However, the mechanisms regulating the stabilities of other BTG/Tob family proteins have not yet been clarified. Here, we report that BTG1, Tob, and Tob2 proteins, as well as BTG2 protein, are degraded by the ubiquitin-proteasome system; the degradation of Tob protein in HeLa cells and the degradation of BTG1, BTG2, Tob and Tob2 proteins transiently expressed in HEK293 cells were inhibited by treatments with proteasome-specific inhibitors. Co-expression of BTG1, BTG2, Tob, or Tob2 protein with ubiquitin in HEK293 cells revealed specific multiubiquitination of each of the four proteins. Although the full-length and N-terminal truncated forms of BTG1, BTG2, Tob, and Tob2 proteins were unstable, the respective C-terminal truncated forms were found to be almost stable, suggesting that the C-terminal regions control the stabilities of BTG1, BTG2, Tob, and Tob2 proteins. In addition, it was found that the respective C-terminal regions confer instability on green fluorescent protein, a normally stable protein. Thus, it can be concluded that the C-terminal regions are necessary and sufficient to control the stabilities of BTG1, BTG2, Tob, and Tob2 proteins.     

Functional characterization of the phosphorelay protein Mpr1p from Schizosaccharomyces pombe

Histidine-containing phosphotransfer (HPt) proteins play an essential role in multistep histidine-aspartate phosphorelay signal transduction systems in prokaryotes and eukaryotes. The putative HPt protein in Schizosaccharomyces pombe, Mpr1p (also known as Spy1p), is a 295 amino acid protein that appears to be composed of more than one functional domain. The amino acid sequence of the N-terminal region of Mpr1p lacks homology to other known proteins, whereas the C-terminal domain is predicted to have structural similarity to the Ypd1p HPt protein from Saccharomyces cerevisiae. This study provides both in vitro and in vivo evidence that the C-terminal domain of Mpr1p indeed functions as an HPt protein in shuttling phosphoryl groups from one response regulator domain to another. Furthermore, we find that various deletions of the N-terminal region diminish both the phosphotransfer activity of Mpr1p and its affinity for response regulator domains, suggesting a possible role for the N-terminal domain in HPt-response regulator domain interactions.     

Docking-dependent ubiquitination of the interferon regulatory factor-1 tumor suppressor protein by the ubiquitin ligase CHIP

Characteristically for a regulatory protein, the IRF-1 tumor suppressor turns over rapidly with a half-life of between 20-40 min. This allows IRF-1 to reach new steady state protein levels swiftly in response to changing environmental conditions. Whereas CHIP (C terminus of Hsc70-interacting protein), appears to chaperone IRF-1 in unstressed cells, formation of a stable IRF-1·CHIP complex is seen under specific stress conditions. Complex formation, in heat- or heavy metal-treated cells, is accompanied by a decrease in IRF-1 steady state levels and an increase in IRF-1 ubiquitination. CHIP binds directly to an intrinsically disordered domain in the central region of IRF-1 (residues 106-140), and this site is sufficient to form a stable complex with CHIP in cells and to compete in trans with full-length IRF-1, leading to a reduction in its ubiquitination. The study reveals a complex relationship between CHIP and IRF-1 and highlights the role that direct binding or "docking" of CHIP to its substrate(s) can play in its mechanism of action as an E3 ligase.     

Herpes simplex virus tegument protein VP22 contains overlapping domains for cytoplasmic localization, microtubule interaction, and chromatin binding

We have previously shown that the 301-amino-acid herpes simplex virus tegument protein VP22 exhibits a range of subcellular localization patterns when expressed in isolation from other virus proteins. By using live-cell analysis of cells expressing green fluorescent protein (GFP)-tagged VP22 we have shown that when VP22 is first expressed in the cell it localizes to the cytoplasm, where, when present at high enough concentrations, it can assemble onto microtubules, causing them to bundle and become highly stabilized. In addition we have shown that when a cell expressing VP22 enters mitosis, the cytoplasmic population of VP22 translocates to the nucleus, where it efficiently binds mitotic chromatin. Here we have investigated the specific regions of the VP22 open reading frame required for these properties. Using GFP-VP22 as our starting molecule, we have constructed a range of N- and C-terminal truncations and analyzed their localization patterns in live cells. We show that the C-terminal 242 residues of VP22 are sufficient to induce microtubule bundling. Within this subregion, the C-terminal 89 residues contain a signal for cytoplasmic localization of the protein, while a larger region comprising the C-terminal 128 residues of the VP22 protein is required for mitotic chromatin binding. Furthermore, a central 100-residue domain of VP22 maintains the ability to bind microtubules without inducing bundling, suggesting that additional regions flanking this microtubule binding domain may be required to alter the microtubule network. Hence, the signals involved in dictating the complex localization patterns of VP22 are present in overlapping regions of the protein.