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M Schuermann

Publications and source records attributed to M Schuermann.

34 records · Page 2Linked to original sources
PubMed1994

T1 is a c-Fos- and FosB-responsive gene which is induced by growth factors through multiple signal transduction pathways.

Stimulation of quiescent cells with growth factors triggers changes in gene expression through multiple signal transduction pathways. One of these changes in Swiss 3T3 cells is the strong accumulation of T1 mRNA which encodes a secreted glycoprotein of the immunoglobulin superfamily. Proliferating cells continued to express T1 mRNA at a lower level, whereas growth arrest induced either by serum deprivation or by contact inhibition was paralleled by the disappearance of the T1 mRNA. T1 mRNA synthesis in response to serum and platelet-derived growth factor stimulation is mediated through protein kinase C-dependent and protein kinase C-independent pathways. Activation of protein kinase A also led to T1 gene expression. Ongoing protein synthesis is a prerequisite for T1 gene induction by growth factors which defines T1 as a delayed early serum-responsive gene. The ability of the immediate early transcription factors c-Fos and FosB to directly induce the T1 gene was demonstrated in a conditional expression system in the absence of protein synthesis. Furthermore, all known inducers of the T1 gene also lead to c-fos gene activation. Thus we show that the T1 gene is regulated by signals which are transduced through multiple pathways and provide evidence that the Fos proteins play an important role in the integration of these pathways.

3T3 Cells↗
PubMed1993

Transcriptional activation and transformation by chimaeric Fos-estrogen receptor proteins: altered properties as a consequence of gene fusion.

We have generated a series of conditionally active Fos and FosB proteins by fusion with a C-terminal fragment of the human estrogen receptor (ER) which harbours the ligand binding site and the overlapping hormone-inducible transactivation domain TAF-2. The chimaeric Fos-ER proteins showed estrogen-inducible activation of TRE (TPA-responsive element)-directed transcription and hormone-dependent transformation of fibroblasts. These properties of the fusion proteins were independent of the transregulatory and transforming properties of their normal non-fused counterparts c-Fos, v-Fos, FosB-L and FosB-S. Thus c-Fos-ER and FosB-S-ER were strong transforming proteins in the presence of hormone, although c-Fos and FosB-S possess only marginal oncogenic properties. In addition, all fusion proteins showed increased transactivation in the presence of estrogen, again most noticeable in the case of c-Fos-ER and FosB-S-ER. The ER-fusion thus basically eliminated the differences in the transactivating potential seen among the various native Fos proteins. Our data therefore provide evidence: (i) that the hormone binding domain of the human estrogen receptor, apart from delivering hormonal control to a heterologous protein, can have profound effects on the activity of certain transcription factors, particularly on proteins with weak oncogenic and/or transregulatory potential, and (ii) that the transforming potential of c-Fos and FosB-S can be dramatically elevated by increasing their transactivating properties.

3T3 Cells↗
PubMed1992

Molecular and functional characterisation of a fusion protein suited for tumour specific prodrug activation.

A fusion protein consisting of the humanised Fab fragment of the anti CEA MAb BW 431 and the human beta-glucuronidase was expressed in BHK cells. Functional testing revealed that the specificity and avidity of the humanised V region was similar to the original murine MAb BW 431. Furthermore, the enzymatic activity, pH sensitivity and stability of the human beta-glucuronidase in the fusion protein was comparable to the activity of recombinant human beta-glucuronidase. Using anti-idiotype affinity chromatography, two molecules of a molecular weight of 125 kDa or 250 kDa could be visualized under nonreducing conditions in SDS-PAGE. Reducing conditions revealed a 25 kDa light and 100 kDa heavy chain. Due to its suitable biological characteristics this fusion protein might be an appropriate molecule allowing a site specific antibody directed enzyme prodrug therapy (ADEPT) in vivo.

Carcinoembryonic Antigen↗
PubMed1991

Non-leucine residues in the leucine repeats of Fos and Jun contribute to the stability and determine the specificity of dimerization.

Various transcription factors, including C/EBP, GCN4 and members of the Fos, Jun and Myc families have been shown to form highly specific complexes via alpha-helical structures referred to as leucine zippers. Experimental evidence has suggested that dimerization involves the formation of hydrophobic bonds between leucine residues in laterally aligned coiled coil structures. However, the specificity of interaction between leucine zipper proteins is not understood. In this study, we show that amino acids, which are located in positions a, e, and g are instrumental in the formation of Fos/Jun heterodimers, presumably by establishing intermolecular electrostatic and hydrophobic interactions. These residues are highly conserved in proteins of the Fos or Jun families but completely different between Fos and Jun, suggesting that these residues determine the specificity of interaction. This conclusion is supported by the observation that the substitution of amino acids in position a or g in Fos with the corresponding Jun amino acids facilitates the association of two Fos leucine repeats. In addition, we show that a conserved histidine residue, located 7 amino acids (i.e., two alpha-helical turns) C-terminally to the 5th leucine in Fos and Jun, is also important for complex formation.

Amino Acid Sequence↗
PubMed1991

Alternative splicing of fosB transcripts results in differentially expressed mRNAs encoding functionally antagonistic proteins.

We show that serum-stimulated fibroblasts transiently express two different forms of fosB mRNA, which are generated by alternative splicing of the transcript from a single gene. In addition to the known long form (fosB-L), encoding a protein of 338 amino acids (FosB-L), a second shorter form (fosB-S) with a deletion of 140 bp was detected. This deletion creates a stop codon 3' to the leucine repeat, giving rise to a protein of 237 amino acids (FosB-S) lacking the carboxyl terminus of FosB-L. Only the long FosB form efficiently induces transformation in mouse and rat fibroblast cell lines and trans-represses the c-fos promoter. Both of these functions are suppressed by coexpressed FosB-S. Upon serum stimulation, maximum expression of the oncogenic fosB-L form precedes the expression of the antagonistic fosB-S form, indicating a new mechanisms regulating the action of members of the Fos family. However, FosB-L and FosB-S do not differ in all trans-regulatory properties: Trans-activation of a 5x TRE-CAT reporter construct in HeLa and NIH-3T3 cells was found with both FosB forms. These observations suggest a correlation between fosB-induced transformation and trans-repression, thus pointing to different mechanisms involved in transformation by fosB and c-fos/v-fos.

Animals↗
PubMed1991

Stage and tissue-specific expression of fosB during mouse development.

The product of the fos-related fosB gene shares many properties with c-Fos such as inducibility by growth factors, complex formation with members of the Jun family and cooperative binding with Jun to the TPA response element (TRE). To investigate whether in contrast to these functional similarities, the two genes might be differentially regulated, we have analysed the expression of fosB during mouse development by in situ hybridization. A spatially restricted accumulation of fosB mRNA in the visceral yolk sac and the nervous system was observed during late gestation. The highest levels of fosB mRNA were found in the cortex and the dorsal columns of the spinal cord. Moreover, stage-specific expression was seen in sensory organs such as retina and vibrissae, where the levels of fosB RNA either increased (retina) or decreased (vibrissae) between days 15 and 18. Our results suggest that fosB may have a specific function in the development of ectoderm-derived tissues. Expression of fosB during prenatal development differs markedly from the known expression pattern of c-fos, pointing to different tissue-specific functions for c-fos and fosB.

Animals↗
PubMed1991

fosB is a transforming gene encoding a transcriptional activator.

The fosB gene encodes a nuclear protein that shows a high degree of homology with c-Fos in several of the known functionally crucial domains, e.g., the leucine zipper and the DNA-binding site, but shows considerable divergence in other regions. Here, we report that FosB, when placed under the control of a constitutive promoter, exhibits clear transforming properties in focus assays using mouse NIH3T3 or rat 208F fibroblasts. The transforming potential of FosB is considerably stronger than that of a corresponding c-fos construct and resembles that of viral fos genes. Using chimeric fos/fosB constructs we show that the C-terminal half of FosB is responsible for these stronger transforming properties, apparently by giving rise to significantly higher levels of protein as compared with the corresponding c-fos sequence. Surprisingly, substitution of the N-terminus of Fos with that of FosB decreases its transforming potential. These differences in the transforming potential are not related to DNA or protein expression, but rather seem to reflect differences in the molecular function(s) encoded in the N-terminal halves of Fos and FosB protein. Both, fosB- and v-fos transformed cells show increased expression of a number of endogenous genes, including c-jun, transin, alpha 1(III) collagen and tissue plasminogen activator. Transactivation by FosB and v-fos of the c-jun and alpha 1(III) collagen gene promoters and of a 3 x TRE-tk chimeric promoter could be shown in transient CAT assays. v-Fos, but not FosB-transformed cells, also show elevated levels of urokinase and plasminogen activator inhibitor mRNAs, pointing to potential differences in the gene regulatory properties of the two Fos family members.

Animals↗
PubMed1991

Mutagenesis of the DNA contact site in Fos protein: compatibility with the scissors grip model and requirement for transformation.

To elucidate the mechanisms involved in the transformation by fos we have initiated a study pertaining to the identification of molecular functions of Fos protein that are crucial for transformation. We have previously reported that the presence of an intact leucine zipper in Fos is an absolute requirement for the induction of transformation, but that the autorepression function of Fos is dispensable. We now show that Fos protein also needs an intact DNA (TRE)-binding site to be able to transform. Amino acid substitutions in this domain of Fos which impair DNA binding also destroy the transforming potential of Fos, suggesting that the interaction of Fos-Jun complexes with TREs may be a crucial part of Fos-induced transformation. This hypothesis is further strengthened by our observation that Fos and Jun can cooperate in the induction of transformation. We show that a Fos protein which contains a Jun leucine zipper and is thus capable of dimerization is still dependent on the presence of exogenous Jun to induce transformation. The critical positions in the Fos DNA-binding site include those which the 'scissors grip' model predicts to be crucial, although the DNA-binding site in Fos seems to extend beyond the basic region into an adjacent cluster of acidic amino acids.

Amino Acid Sequence↗
PubMed1989

Two functionally different regions in Fos are required for the sequence-specific DNA interaction of the Fos/Jun protein complex.

THE products of the cellular and retroviral fos genes associate with other nuclear proteins, among them the transcription factor AP1/Jun (see ref. 3 for a review). The Fos/Jun complex binds to a specific symmetrical DNA recognition sequence (termed TRE), thus stimulating transcription of the respective gene. Here, we show that two distinct regions in Fos are required for the formation of a Fos/Jun/TRE complex. These are the leucine zipper, involved in the association with Jun, and a directly adjacent basic region. Specific amino-acid substitutions in this basic, presumably alpha-helical, region abolish the interaction of Fos/Jun with the TRE but not the association of the two proteins. The functionally crucial amino acids are located in a region of Fos which is structurally similar to the putative DNA-binding sites in Jun and in the yeast transcriptional activator GCN4 (refs 15 and 16).

Amino Acid Sequence↗
PubMed1989

The leucine repeat motif in Fos protein mediates complex formation with Jun/AP-1 and is required for transformation.

Cellular and viral Fos proteins form a tight complex with other nuclear proteins, including the transcription factor and proto-oncogene AP-1/Jun. We have mapped the c-Jun binding site in Fos to a region containing regularly spaced leucine residues recently suggested to interdigitate with a similar structure in Jun. Substitution of single or multiple leucine residues or the alteration of leucine phasing by insertion of additional amino acids reduces or abolishes the binding to Jun, while the substitution of other amino acids has no noticeable effect. These results strongly suggest that the formation of a "leucine zipper" mediates the interaction between Fos and Jun. We also show that the differential binding of the various Fos mutants correlates with their potential to trans-activate AP-1-dependent transcription and to induce morphological transformation.

Animals↗
PubMed1989

The product of a novel growth factor activated gene, fos B, interacts with JUN proteins enhancing their DNA binding activity.

We have identified a gene, fos B, encoding a nuclear protein of 338 amino acids presenting a 70% homology with c-fos, whose expression is activated during G0/G1 transition. Growth factor stimulation of quiescent cells leads to a rapid and transient accumulation of fos B mRNA, with kinetics similar to those of c-fos. The induction of fos B mRNA levels is in part due to a dramatic increase in the transcription of the gene. The half-life of fos B mRNA is in the order of 10-15 min. Both transcriptional activation and mRNA stability are substantially increased in the presence of protein synthesis inhibitors. Immunoprecipitation studies showed that fos B as c-fos protein, forms a complex in vitro with c-jun and jun B proteins in the absence of a target binding sequence. Gel retardation assays demonstrated that fos B protein positively influences the binding of c-jun and jun B proteins to an AP-1 binding consensus sequence, suggesting that fos B protein plays a role in control of gene expression.

Amino Acid Sequence↗
PubMed1989

A putative int domain for mouse mammary tumor virus on mouse chromosome 7 is a 5' extension of int-2.

We extended the physical map of the mouse int-2 locus by demonstrating that the site of insertion for mouse mammary tumor virus DNA in plaque-type mammary tumors of GR mice is directly linked to int-2. An additional example of proviral integration is described in which a provirus in a presumed enhancer-insertion mode 15 kilobases upstream of the int-2 promoters is capable of activating expression of the gene at levels typical of other virally induced mammary tumors.

Animals↗
PubMed1987

The Drosophila homolog of the mouse mammary oncogene int-1 is identical to the segment polarity gene wingless.

We have isolated the Drosophila melanogaster homolog (Dint-1) of int-1, a conserved cellular oncogene implicated in viral mammary tumorigenesis in mice. The deduced Dint-1 protein sequence contains 468 amino acids and starts with a hydrophobic leader; it is 54% identical to the int-1 sequence, and all 23 cysteine residues are conserved. The putative Drosophila protein has an extra sequence of 85 amino acids, encoded on an additional exon. Dint-1 is expressed throughout development, but transcripts are barely detectable in adult flies. Hybridization in situ to embryos reveals a segmented pattern of expression. We show that Dint-1 and the segment polarity gene wingless are identical and map to the same location. The sequence of the gene suggests that the Dint-1/wingless protein functions in morphogenesis as a signal in cell-cell communication.

Amino Acid Sequence↗
PubMed1987

A rare common integration site of proviruses of the mouse mammary tumor virus in P-type mammary tumors of mouse strain GR.

The mouse mammary tumor virus (MMTV) can induce mammary tumors in mice by proviral activation of the cellular oncogenes int-1 or int-2. Activation of these genes, however, is observed in only a few hormone- and pregnancy-dependent mammary tumors of the mouse strain GR. To study the possible involvement of other oncogenes we cloned three MMTV proviral-host fragments (MT 40, 42, and 53) from different mammary tumors of GR with a single acquired MMTV provirus. From a genomic library of normal mouse DNA we isolated phages with insert DNAs that covered 20-30 kb of the uninterrupted regions. Suitable probes devoid of repetitive DNA sequences were isolated in order to screen other mammary tumors for MMTV proviral integrations in these regions. Only two mammary tumors, MT 40 and 42, showed integration of extra MMTV proviruses within the same region. The integrations occurred only 60 bp apart. The other mammary tumors, however, did not contain MMTV proviral integrations in this region, nor in the MT 53 region. Using mouse-hamster somatic cell hybrid DNA, the MT 40/42 integration region was assigned to mouse chromosome 7, and the second region, MT 53, to chromosome 16. The two regions bear no homology to known cellular oncogenes. We did not observe any mRNA being expressed from these cloned segments either in tumors or in normal mammary glands. These findings indicate that plaque(P)-type mammary tumors in mouse strain GR do not originate from MMTV provirus insertions in a particularly favored integration region, but that there may be a variety of integration sites in these tumors.

Animals↗
PubMedDate not supplied

The p16/Cdkn2a/Ink4a gene is frequently deleted in nitrosourea-induced rat glial tumors.

The present study investigates nitrosourea-induced rat (Rattus norvegicus) glioma cell lines for the functional status of the p16/Cdkn2a/Ink4a gene, which encodes the p16 cdk4 inhibitor and the alternative reading frame protein, p19ARF. We detected homozygous deletions of the p16/Cdkn2a/Ink4a gene locus in 4 of 5 glioma cell lines (C6, F98, RG2, and RGL.3), but not in the 9L gliosarcoma cell line or in a rat primary fibroblast cell line. RT-PCR demonstrated expression of the p16 and p19ARF mRNAs only in 9L cells and in rat fibroblasts. Comparative genomic in situ hybridization showed that the copy number of rat chromosome RNO5 was not altered in any of the glioma cell lines investigated, indicating that the deletions result from a discrete loss in the region of the p16/Cdkn2a/Ink4a locus. This is the first report of p16/Cdkn2a/Ink4a deletions present in nitrosourea-induced rat glioma cell lines. Since this genetic alteration is also commonly observed in human malignant glial tumors, our results validate the use of chemically induced rat glioma cell lines as an experimental model in the development of gene therapy strategies.

Animals↗