Distinct binding determinants for ERK2/p38alpha and JNK map kinases mediate catalytic activation and substrate selectivity of map kinase phosphatase-1.

Mitogen-activated protein (MAP) kinase phosphatase 1 (MKP-1/CL100) is an inducible nuclear dual specificity protein phosphatase that can dephosphorylate and inactivate both mitogen- and stress-activated protein kinases in vitro and in vivo. However, the molecular mechanism responsible for the substrate selectivity of MKP-1 is unknown. In addition, it has been suggested that the signal transducers and activators of transcription 1 (STAT1) transcription factor is a physiological non-MAP kinase substrate for MKP-1. We have used the yeast two-hybrid assay to demonstrate that MKP-1 is able to interact selectively with the extracellular signal-regulated kinase 1/2 (ERK1/2), p38alpha, and c-Jun NH(2)-terminal kinase (JNK) MAP kinase isoforms. Furthermore, this binding is accompanied by catalytic activation of recombinant MKP-1 protein in vitro, and these end points show an absolute correlation with MKP-1 substrate selectivity in vivo. In contrast, MKP-1 does not interact with STAT1. Recombinant STAT1 does not cause catalytic activation of MKP-1; nor does MKP-1 block tyrosine phosphorylation of STAT1 in vivo. Both binding and catalytic activation of MKP-1 are abrogated by mutation of a conserved docking site in ERK2, p38alpha, and JNK1 MAP kinases. Within MKP-1, MAP kinase binding is mediated by the amino-terminal noncatalytic domain of the protein. However, mutation of a conserved cluster of positively charged residues within this domain abolishes the binding and activation of MKP-1 by ERK2 and p38alpha but not JNK1, indicating that there are distinct binding determinants for these MAP kinase isoforms. We conclude that the substrate selectivity of MKP-1 is determined by specific protein-protein interactions coupled with catalytic activation of the phosphatase and that these interactions are restricted to members of the MAP kinase family of enzymes.

Biochemical analysis of mouse FKBP60, a novel member of the FKPB family.

We have identified mouse and human FKBP60, a new member of the FKBP gene family. FKBP60 shares strongest homology with FKBP65 and SMAP. FKBP60 contains a hydrophobic signal peptide at the N-terminus, 4 peptidyl-prolyl cis/trans isomerase (PPIase) domains and an endoplasmic reticulum retention motif (HDEL) at the C-terminus. Immunodetection of HA-tagged FKBP60 in NIH-3T3 cells suggests that FKBP60 is segregated to the endoplasmic reticulum. Northern blot analysis shows that FKBP60 is predominantly expressed in heart, skeletal muscle, lung, liver and kidney. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as a substrate, recombinant GST-FKBP60 is shown to accelerate effectively the isomerization of the peptidyl-prolyl bond. This isomerization activity is inhibited by FK506. mFKBP60 binds Ca2+ in vitro, presumably by its C-terminal EF-hand Ca2+ binding motif, and is phosphorylated in vivo. hFKBP60 has been mapped to 7p12 and/or 7p14 by fluorescence in situ hybridization (FISH).

A dominant role for the Raf-MEK pathway in forskolin, 12-O-tetradecanoyl-phorbol acetate, and platelet-derived growth factor-induced CREB (cAMP-responsive element-binding protein) activation, uncoupled from serine 133 phosphorylation in NIH 3T3 cells.

In this study we describe that platelet-derived growth factor (PDGF), 12-O-tetradecanoyl-phorbol-acetate (TPA), and forskolin induced CREB (cAMP-responsive element-binding protein) Ser-133 phosphorylation with comparable magnitude and kinetics in NIH 3T3 cells. While forskolin was the most potent activator of CREB, TPA or PDGF modestly increased CREB activity. The role of protein kinase C, protein kinase A, and the Raf-MEK kinase pathway in the activation and Ser-133 phosphorylation of CREB by these three stimuli was investigated. We found that inhibition of the Raf-MEK kinase pathway efficiently blocks transcriptional activation of CREB by all three stimuli. This dominant involvement of Raf-MEK in CREB transcriptional activation seems to be uncoupled from CREB Ser-133 phosphorylation. We further demonstrate that although inhibition of Raf-MEK represses forskolin-induced CREB activation, forskolin by itself failed to activate ERK1/2 and Elk-1 mediated transcription. These results suggest that a basal level of Raf-MEK activity is necessary for both PDGF- and forskolin-induced CREB activation, independent of CREB Ser-133 phosphorylation.

Activation of protein kinase A by dibutyryl cAMP treatment of NIH 3T3 cells inhibits proliferation but fails to induce Ser-133 phosphorylation and transcriptional activation of CREB.

The cAMP analogue dibutyryl cAMP (dbcAMP) is often used to activate the protein kinase A pathway and to study the expression of cAMP-responsive genes. Here we show that in NIH 3T3 cells dbcAMP is able to activate PKA, but fails to stimulate expression of the cAMP-inducible c-fos gene. Co-expression of A-kinase anchoring protein 75, previously shown to amplify cAMP signalling and to stimulate c-fos expression, could not restore cAMP responsiveness of the c-fos promoter. DbcAMP-induced activation of PKA may result in poor translocation of the catalytic sub-units of PKA to the nucleus, indicated by the lack of both Ser-133 phosphorylation of the cAMP-response element binding factor CREB and stimulation of the transcriptional activity of this factor. DbcAMP treatment, however, inhibited cell proliferation. These results suggest that cAMP-mediated inhibition of proliferation may be independent of translocation of the catalytic sub-units into the nucleus.

Concerted expression of BK virus large T- and small t-antigens strongly enhances oestrogen receptor-mediated transcription.

Previous studies have shown that the human polyomavirus BK (BKV) genome contains an oestrogen response element (ERE). This isolated element binds its cognate receptor in vitro and can mediate 17beta-oestradiol-induced gene expression when linked to a heterologous promoter. The roles of the ERE- and the AP-1-binding sites in oestrogen receptor-directed transcription from the complete BKV promoter/enhancer (Dunlop strain) have been examined and the effects of the general co-activator CBP and large T- and small t-antigens on oestrogen receptor-mediated transcription have been investigated. A constitutive activated oestrogen receptor stimulated BKV promoter activity in HeLa cells. Mutations in either the ERE- or the AP-1-binding sites did not impair oestrogen receptor-induced activation of the BKV Dunlop promoter, while mutations in both binding motifs almost completely abolished oestrogen receptor-induced transcription. Simultaneous expression of large T- and small t-antigens strongly activated oestrogen receptor-mediated transcription. When expressed separately, only large T-antigen moderately stimulated oestrogen receptor-mediated transcription. The stimulatory effect of large T-antigen on the activity of the oestrogen receptor is probably indirect because no physical interaction between the two proteins was detected in a two-hybrid assay. Large T-antigen abrogated the synergistic effect on transcription between this nuclear receptor and the general co-activator CBP. The findings that the BKV early proteins amplify oestrogen receptor-mediated transcription may have important biological implications in individuals with raised oestrogen concentrations.

Human umbilical vein endothelial cells lack expression of the estrogen receptor.

Estrogens may influence the expression of various cytokines, adhesion molecules, von Willebrand factor and prostacyclin produced by endothelial cells. However, reports concerning expression of the estrogen receptor in endothelial cells are controversial. Primary human umbilical vein endothelial cells (HUV-EC), the non continuous human umbilical vein endothelial cell line HUV-EC-C (ATCC CRL 1730) and endothelial cells from 10 frozen umbilical cords were analyzed for the expression of the estrogen receptor. Immunological studies using estrogen receptor specific antibodies failed to detect the expression of the receptor in all human umbilical vein endothelial cells tested. No estrogen receptor transcripts were found in primary HUV-EC or HUV-EC-C by reverse transcriptase-polymerase chain reaction. Weak hybridization signals were detected when the PCR amplicons were hybridized with estrogen receptor cDNA sequences as a probe. In vitro protein-DNA interaction studies revealed no complexes between a fully consensus estrogen response element and HUV-EC-C extracts. Finally, transient transfection studies in HUV-EC-C could not demonstrate 17beta-estradiol-induced transcription of the beta-galactosidase reporter gene linked to a consensus estrogen response element. These observations suggest that human umbilical vein endothelial cells lack the estrogen receptor.

Synergistic increase in c-fos expression by simultaneous activation of the ras/raf/map kinase- and protein kinase A signaling pathways is mediated by the c-fos AP-1 and SRE sites.

Expression of the c-fos proto-oncogene is induced by numerous stimuli some of which are transmitted through the Ras/Raf/MAP kinase or the cAMP-dependent protein kinase (PKA) pathways. The effect of cell-specific interactions between these pathways on c-fos expression was investigated by exposing quiescent NIH3T3 cells to serum, forskolin, or a combination. Co-stimulation with serum and forskolin resulted in a more than additive increase in c-fos transcription. Synergistic increase in c-fos promoter activity was also observed in transient transfection studies after co-stimulation with serum plus forskolin or co-transfection with c-Raf and PKA expression plasmids. Analysis of the cAMP signaling pathway revealed that the synergy was neither due to an increase in PKA activity nor to Ser-133 phosphorylation/activation of CREB. The activation status of the MAP kinases ERK1 and ERK2 in co-treated cells was comparable to that in serum-treated cells. Co-stimulation with forskolin did not alter the phosphorylation state of Elk-1 compared to serum-induced phosphorylation of Elk-1. Deletion of c-fos promoter elements previously shown to be important for regulation of c-fos expression in response to mitogens indicates a role for SRE and FAP-1 elements.

Mechanisms of transcriptional regulation of cellular genes by SV40 large T- and small T-antigens.

During the past decade a number of virus-encoded transcriptional trans-activators that regulate the expression of viral genes have been reported. These trans-activators may also affect the expression or activity of several cellular genes or gene products to create an optimal cellular environment that favors viral replication. Among the better-studied viral trans-activating proteins are the Simian virus 40 large T- and small t-antigens. During the last few years, mechanisms by which these two viral proteins influence cellular gene expression start to emerge. They are grouped provisionally and reflect the methods used to determine the effects of large T-antigen. Large T-antigen may influence cellular gene expression by: i. altering mRNA levels of cellular transcription factors; ii. interacting with and regulating the DNA-binding or transcriptional activity of specific transcription factors; iii. functionally substitution of eukaryotic transcription factors; iv. direct binding to DNA; or v. regulating components of signaling transduction pathways. Small t-ag seems to exert its effect mainly through inhibiting a cellular phosphatase, protein phosphatase 2A, thereby modulating components of signal transduction pathways and preventing dephosphorylation of several transcription factors. However, small t-ag may also control cellular gene expression by regulating mRNA levels of transcription factors or by interacting with other transcription factors.

The human polyomavirus BK T antigen induces gene expression in human cytomegalovirus.

Co-infections or co-habitations of cells by two or more viruses may occur in the human organism. Human cytomegalovirus (HCMV) and the human polyomavirus BK (BKV) have common host cells and may both establish lifelong latency/persistence following primary infection. Both viruses may become reactivated by immunosuppression or other conditions which upset host-virus balance, and they encode gene products with the inherent potential of acting as heterologous transacting factors for expression of cellular or viral genes. It has been shown that HCMV induces gene expression and replication of primate polyomaviruses. We now demonstrate that BKV is able to enhance the expression of HCMV immediate early (IE1 and 2) as well as the early (E) protein pp65 during double infections in semi-permissive cells. By transfection experiments it was established that the phenomenon is due to heterologous transcriptional transactivation of the HCMV major IE promoter (MIEP) by the BKV large T antigen, without contribution from the small t antigen.

Macrophage cytotoxicity against murine meth A sarcoma involves nitric oxide-mediated apoptosis.

We have studied the cytotoxic effect of stimulated macrophages on Meth A tumor cells in vitro. When stimulated with interferon-gamma and soluble beta-1,3-D-glucan, macrophages exerted cytotoxicity towards syngeneic Meth A tumor cells. This cytotoxicity was associated with a high level of nitric oxide production. Both cell death and nitric oxide production were significantly inhibited by the addition of aminoguanidine, a specific inhibitor of inducible nitric oxide synthase (iNOS), to the culture medium. The cytotoxic effect was accompanied by internucleosomal cleavage of DNA as shown by electrophoresis and DNA fragmentation assay.

In vivo expression of a single viral DNA-binding protein generates systemic lupus erythematosus-related autoimmunity to double-stranded DNA and histones.

Although the origin of autoimmune antibodies to double-stranded DNA is not known, the variable-region structures of such antibodies indicate that they are produced in response to antigen-selective stimulation. In accordance with this, results from experiments using artificial complexes of DNA and DNA-binding polypeptides for immunizations have indicated that DNA may induce these antibodies. Hence, the immunogenicity of DNA in vivo may depend upon other structures or processes that may render DNA immunogenic. We report that in vivo expression of a single DNA-binding protein, the polyoma virus T antigen, is sufficient to initiate production of anti-double-stranded DNA and anti-histone antibodies but not a panel of other autoantigens. Expression of a mutant, non-DNA-binding T antigen did result in strong production of antibodies to the T antigen, but only borderline levels of antibodies to DNA and no detectable antibodies to histones. Nonexpressing plasmid DNA containing the complete cDNA sequence for T antigen did not evoke such immune responses, indicating that DNA by itself is not immunogenic in vivo. The results represent a conceptual advance in understanding a potential molecular basis for initiation of autoimmunity in systemic lupus erythematosus.

Subpopulations of non-coding control region variants within a cell culture-passaged stock of BK virus: sequence comparisons and biological characteristics.

In the circular DNA genome of the human polyomavirus BK an approximately 400 bp non-coding control region (NCCR) separates the early and late genes. The NCCR contains the origin of replication as well as the promoter/enhancer with a mosaic of cis-acting elements involved in the regulation of both early and late transcription. The NCCR has been shown to be very heterogeneous between different BK virus (BKV) strains. This may affect the host cell permissivity and oncogenic potential of a given BKV strain. Our previous studies with BKT-1B, a continuous cell line established from a BKV (Gardner) -induced hamster fibrosarcoma, revealed that the BKV DNA is integrated in the host genome in multiple copies. The sequence of the integrated BKV NCCR was substantially different from (and even contained sequences not found in) that of the BKV (Gardner) strain supposedly used to establish the BKT-1B cell line. PCR amplification, cloning and subsequent sequencing revealed that the original BKV (Gardner) stock contained at least seven different subpopulations of viral genomes. None of them had a control region 'anatomy' which was identical to either the BKV (Gardner) strain, the variant found integrated in BKT-1B cells or any previously published NCCR. In order to study the biological significance of these new BKV NCCR variants we developed a simple cassette model allowing the NCCRs of the new variants to be cloned in an identical genomic background of BKV protein-coding sequences and performed transfection studies with the recombinant genomes in non-permissive rodent cells and in semi-permissive monkey cells. The results demonstrated that the NCCR variants conferred striking differences, in both transforming capacity and host cell permissivity, to the recombinant BKV genomes. Sequence comparisons suggested genetic explanations for these differences, as well as evolutionary relationships between BKV NCCRs.

Noncoding control region of naturally occurring BK virus variants: sequence comparison and functional analysis.

The human polyomavirus BK (BKV) has a proven oncogenic potential, but its contribution to tumorigenesis under natural conditions remains undetermined. As for other primate polyomaviruses, the approximately 5.2 kbp double-stranded circular genome of BKV has three functional regions: the coding regions for the two early (T, t antigens) and four late (agno, capsid proteins; VP1-3) genes separated by a noncoding control region (NCCR). The NCCR contains the origin of replication as well as a promoter/enhancer with a mosaic of cis-acting elements involved in the regulation of both early and late transcription. Since the original isolation of BKV in 1971, a number of other strains have been identified. Most strains reveal a strong sequence conservation in the protein coding regions of the genome, while the NCCR exhibits considerable variation between different BKV isolates. This variation is due to deletions, duplications, and rearrangements of a basic set of sequence blocks. Comparative studies have proven that the anatomy of the NCCR may determine the transcriptional activities governed by the promoter/enhancer, the host cell tropism and permissivity, as well as the oncogenic potential of a given BKV strain. In most cases, however, the NCCR sequence of new isolates was determined after the virus had been passaged several times in more or less arbitrarily chosen cell cultures, a process known to predispose for NCCR rearrangements. Following the development of the polymerase chain reaction (PCR), it has become feasible to obtain naturally occurring BKV NCCRs, and their sequences, in samples taken directly from infected human individuals. Hence, the biological significance of BKV NCCR variation may be studied without prior propagation of the virus in cell culture. Such variation has general interest, because the BKV NCCRs represent typical mammalian promoter/enhancers, with a large number of binding motifs for cellular transacting factors, which can be conveniently handled for experimental purposes. This communication reviews the naturally occurring BKV NCCR variants, isolated and sequenced directly from human samples, that have been reported so far. The sequences of the different NCCRs are compared and analyzed for the presence of proven and putative cellular transcription factor binding sites. Differences in biological properties between BKV variants are discussed in light of their aberrant NCCR anatomies and the potentially modifying influence of transacting factors.

Detection of BK virus DNA in nasopharyngeal aspirates from children with respiratory infections but not in saliva from immunodeficient and immunocompetent adult patients.

Our understanding of important stages in the pathogenesis of the human polyomavirus BK virus (BKV) and JC virus (JCV) infections is limited. In this context, nasopharyngeal aspirates from 201 children with respiratory diseases and saliva from 60 human immunodeficiency virus type 1-infected adults and 10 healthy adult controls were collected and analyzed for the presence of BKV and JCV DNA by PCR. Neither BKV nor JCV DNA was detected in the saliva specimens. We demonstrated BKV DNA, but no infectious BKV, in 2 of 201 nasopharyngeal aspirates. Each sample contained one unique rearranged noncoding control region variant of BKV. The results indicate that (i) BKV and JCV are not regularly associated with respiratory infections in children requiring hospitalization, (ii) nasopharyngeal cells are not an important site for primary replication of human polyomavirus BKV and JCV, and (iii) the salivary glands and oropharyngeal cells seem not to be involved in BKV and JCV persistence. We propose that for the polyomaviruses BKV and JCV the alimentary tract should be considered as a portal of entrance to the human organism.