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Biomedical subjects

A Nordheim

Publications and source records attributed to A Nordheim.

At least 55 records · Page 3Linked to original sources
PubMed1994

Involvement of growth factor receptors in the mammalian UVC response.

Irradiation of HeLa cells with short-wavelength ultraviolet light (UVC) induces the modification and activation of the preexisting transcription factors c-Fos-c-Jun (AP-1) and TCF/Elk-1, as well as the protein synthesis independent transcriptional activation of the c-fos and c-jun genes. This response to UVC is mediated via obligatory cytoplasmic signal transduction, involving Ras and Raf, Src, and MAP kinases. The UVC response is inhibited by prior down-modulation of growth factor receptor signaling upon growth factor prestimulation, by suramin (an inhibitor of receptor activation) or by expression of a dominant negative epidermal growth factor (EGF) receptor mutant. These data suggest the involvement of several growth factor receptors in the UVC response. Indeed, UVC induces the suramin-inhibitable immediate tyrosine phosphorylation of the EGF receptor.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1994

Heterogeneity of ternary complex factors in HeLa cell nuclear extracts.

Ternary complex factors (TCFs) interact with the serum response factor and DNA containing the c-fos serum response element to form a ternary complex that mediates induction of c-fos. TCF activities were partially purified from HeLa cell nuclear extracts by DNA-cellulose and anion-exchange chromatography followed by two-dimensional gel electrophoresis. Four different protein spots (p60TCF, p62TCF, p62.5TCF, and p64TCF) show renaturable TCF activity. One, p62TCF, was indistinguishable from the Ets protein Elk-1 in gel shift analyses, while none of the HeLa TCFs resembled the other cloned TCFs, SAP-1a and SAP-1b. In two-dimensional gel analysis, Elk-1, SAP-1a, and SAP-1b displayed different pI and M(r) values and in vitro synthesized Elk-1 comigrated with the p62TCF spot. Both reacted with Elk-1 specific antisera, as did the major proportion of TCF activity present in HeLa crude extracts. We conclude that p62TCF is composed of Elk-1, whereas the identities of the other identified TCFs (p60TCF, p62.5TCF, and p64TCF) are still unknown.

Base Sequence↗
PubMed1994

Ras/MAP kinase-dependent and -independent signaling pathways target distinct ternary complex factors.

Transcriptional activation of the immediate early genes c-fos and egr-1 by extracellular signals appears to be mediated by ternary complex factors (TCFs). In BAC-1 macrophages, growth factor stimulation leads to the retardation of protein-DNA complexes containing distinct TCFs. One TCF is recognized by Elk-1 antisera, whereas the other is immunologically related to SAP-1. The appearance and decay of hyperphosphorylated TCF/Elk-1-containing complexes after stimulation coincide with the activation of mitogen-activated protein kinase (MAPK) and the induction and repression of c-fos and egr-1, whereas modified TCF/SAP-1-containing complexes decay more slowly. Suppression of MAPK activation in macrophages and fibroblasts correlates with the failure to induce TCF/Elk-1 hyperphosphorylation without blocking TCF/SAP-1 modification. Accordingly the modified Elk-1 complex is generated in vitro by activated MAPK, whereas that of SAP-1 is not. Expression of a dominant-negative Ras mutant (RasAsn17) in BAC-1 cells does not affect CSF-1-induced TCF/SAP-1 modification while suppressing TCF/Elk-1 phosphorylation. Neither PKC down-regulation by TPA nor inhibition of Gi proteins by pertussis toxin pretreatment influences CSF-1-induced signaling to TCFs. These data indicate the existence of two separate signaling pathways for the modification of distinct TCFs: one dependent on Ras and MAPK and converging on TCF/Elk-1, and the other targeting TCF/SAP-1 independently of Ras and MAPK.

3T3 Cells↗
PubMed1994

Regulatory squelching.

An important function of transcription factors may be to sequester coactivators or corepressors of transcription. In this manner transcription factors could regulate in trans the activity of promoters to which they do not bind. This may be of widespread significance as a mechanism to control cell cycle-dependent and differentiation-specific transcriptional activity within eukaryotic cells. Therefore squelching in vivo may be important than hitherto appreciated.

Animals↗
PubMed1994

Refined mapping of the human Ets-related gene Elk-1 to Xp11.2-p11.4, distal to the OATL1 region.

The gene for human Elk-1, an Ets-related transcription factor, has previously been localized to a region that lies on the short arm of chromosome X and that is involved in specific chromosomal translocations associated with synovial sarcoma and renal adenocarcinomas. We have used fluorescence in situ hybridization and a panel of tumor-derived somatic cell hybrids to refine the localization of Elk-1, in particular with regard to the rearrangements in these tumors. Elk-1 has been assigned to Xp11.2-p11.4, distal to the OATL1 region.

Animals↗
PubMed1994

Transient activation of RAF-1, MEK, and ERK2 coincides kinetically with ternary complex factor phosphorylation and immediate-early gene promoter activity in vivo.

We have investigated the early in vivo signaling events triggered by serum that lead to activation of the c-fos proto-oncogene in HeLa cells. Both RAF-1 and MEK kinase activities are fully induced within 3 min of serum treatment and quickly decrease thereafter, slightly preceding the activation and inactivation of p42MAPK/ERK2. ERK2 activity correlates tightly with a transient phosphatase-sensitive modification of ternary complex factor (TCF), manifested by the slower electrophoretic mobility of TCF-containing protein-DNA complexes. These induced complexes in turn correlate with the activity of the c-fos, egr-1, and junB promoters. Phorbol ester treatment induces the same events but with slower and prolonged kinetics. Inhibition of serine/threonine phosphatase activities by okadaic acid treatment reverses the repression of the c-fos promoter either after induction or without induction. This corresponds to the presence of the induced complexes and of ERK2 activity, as well as to the activation of a number of other kinases. Inhibition of tyrosine phosphatase activities by sodium vanadate treatment delays but does not block ERK2 inactivation, TCF dephosphorylation, and c-fos repression. The tight linkage in vivo between the activity of MAP kinase, TCF phosphorylation, and immediate-early gene promoter activity is consistent with the notion that a stable ternary complex over the serum response element is a direct target for the MAP kinase signaling cascade. Furthermore, serine/threonine phosphatases are implicated in regulating the kinase cascade, as well as the state of TCF modification and c-fos promoter activity, in vivo.

Calcium-Calmodulin-Dependent Protein Kinases↗
PubMed1994

The N-terminal signal peptide of the murine cyclophilin mCyP-S1 is required in vivo for ER localization.

Cyclophilins are a class of enzymes that are thought to be involved in protein folding by accelerating the isomerization of Xaa-Pro peptide bonds and that mediate the immunosuppressive effect of cyclosporin A. We described previously a murine cyclophilin, mCyP-S1, whose cDNA encoded a putative NH2-terminal signal sequence which was not present in the mature protein. Here we investigate the intracellular localization of mCyP-S1. We show by overexpression of the wild-type and an NH2-terminally truncated derivative that its signal sequence is necessary and functional in vivo for the translocation into the endoplasmic reticulum (ER). Immunocytochemistry and cell fractionations demonstrate the preferential localization of endogenous mCyP-S1 in the ER, or subcompartments thereof. In addition, the results indicate the presence of this cyclophilin in the nucleus.

3T3 Cells↗
PubMed1994

Functional dissection of the transcription factor Elk-1.

The ternary complex factor Elk-1 belongs to the Ets oncoprotein family. We demonstrate that this transcription factor is localized predominantly in the nucleus, for which at least two regions of Elk-1 are required. One of these regions is part of the N-terminal ETS-domain, while the other encompasses amino acids 137-157. In conjunction with the ETS-domain, which mediates autonomous binding of Elk-1 to some DNA target sequences, the conserved B-region is both necessary and sufficient for ternary complex formation with the c-fos serum response element and the serum response factor. However, the B-region must be linked to the ETS-domain by a spacer. Furthermore, the B-region impedes autonomous DNA-binding, possibly by masking the ETS-domain. A point mutation within the ETS-domain, homologous to the ts1.1 point mutation of v-Ets in the E26 virus, affects DNA-binding of Elk-1 in a temperature-dependent manner, which by analogy might be causative for the altered phenotype of ts1.1 E26. Finally we show that amino acids 83-428 contribute to Elk-1 mediated transactivation. In particular, the region 376-404 is indispensable for transactivation, while flanking amino acids on both sides are only required for enhancement of transcriptional efficacy.

Amino Acid Sequence↗
PubMed1993

Activation of ternary complex factor Elk-1 by MAP kinases.

Ternary complex factors (TCFs), one of which is Elk-1, have been implicated in mediation of c-fos induction. They have been shown to be phosphorylated by mitogen-activated protein kinases (MAPKs) in vitro. We demonstrate that recombinant Elk-1 is hyperphosphorylated in vivo upon joint overexpression of MAPKs and constitutively activated Raf-1 kinase, the latter serving as an indirect in vivo activator of MAPKs. This phosphorylation is accompanied by a conformational change and results in an elevated transactivation potential of Elk-1. Mutation of mapped in vivo phosphorylation sites, which are potential targets for MAPKs, reduced Elk-1-mediated transcription. Thus, MAPKs are very probably controlling Elk-1 activity by direct phosphorylation in vivo. Furthermore, Elk-1 was shown to stimulate transcription from both the c-fos serum response element and also from an Ets binding site. While binding of TCFs to the c-fos promoter is dependent on the serum response factor, TCFs can autonomously interact with Ets binding sites. This indicates that TCFs may participate in the transcriptional regulation of two different sets of genes.

Animals↗
PubMed1993

C-terminal phosphorylation of the serum-response factor.

The serum-response factor (SRF) is essential for the induction and repression of the protooncogene c-fos. Phosphorylation of SRF has been implicated to be involved in these processes and five phosphorylation sites have already been mapped within the N-terminal region. Here we show that in vivo additional phosphorylation of SRF does occur. This modification is located primarily within amino acids 206-289, which probably contain more than one phosphorylation site. Microsequencing allowed the identification of one phosphorylation site at Ser253, which is a potential target of casein kinase II. Mutational analysis revealed that, in contrast to N-terminal phosphorylation, Ser253 phosphorylation does not affect DNA-binding properties. In addition, phosphorylation at Ser253 does not seem to change transactivation activity of SRF but rather influences its contribution to transcriptional repression. Thus, C-terminal phosphorylation of SRF may modulate c-fos basal repression.

3T3 Cells↗
PubMed1993

c-fos transcriptional activation and repression correlate temporally with the phosphorylation status of TCF.

EGF-induction of human astrocytoma and A431 cells leads to c-fos transcriptional activation and then repression. This could be correlated with changes in the DNA binding characteristics of the c-fos regulatory protein ternary complex factor (TCF) present in nuclear extracts from these cells. Band shifts showed the appearance of induction-related slowly migrating protein-DNA complexes, detected as ternary complexes on the c-fos SRE using a truncated SRF molecule and by direct binding to the Drosophila E74 Ets-protein recognition sequence. By several criteria both types of complexes represented TCF. The appearance of the slow ternary and direct complexes correlated with c-fos transcriptional activation, and their disappearance coincided with the ensuing c-fos shut-off. Blocking c-fos transcriptional repression with the phosphatase inhibitor okadaic acid led to their continued presence. They were sensitive to protein phosphatase 2A but not 1 alpha, and similar slow complexes were formed by partially purified p62TCF phosphorylated by a copurifying kinase activity. Thus the phosphorylation state of TCF correlated strongly with c-fos promoter activity. Since ternary complex formation mediated by full-sized SRF was only slightly affected under comparable conditions, we propose a model for c-fos regulation involving modification of constitutively bound TCF.

Cell Division↗
PubMed1992

Affinity purification of histidine-tagged proteins transiently produced in HeLa cells.

In order to produce eukaryotic proteins in a functional state, it is often necessary to use eukaryotic instead of prokaryotic expression systems. We have designed vectors which can be employed to express either N- or C-terminally histidine-tagged proteins in transiently transfected eukaryotic cells. The histidine tag allows the rapid enrichment of these proteins by metal chelate affinity chromatography in a native and functional state. Yields of up to 5 micrograms protein/5 x 10(7) cells were achieved.

Base Sequence↗
PubMed1992

Elk-1 protein domains required for direct and SRF-assisted DNA-binding.

The Ets-related Elk-1 protein can bind to purine-rich DNA target sites in a sequence specific fashion and, in addition, can form a ternary complex with the c-fos serum response element (SRE) and the serum response factor (SRF). We demonstrate that Elk-1 can readily interchange between its different interaction partners. The amino terminal ETS-domain of Elk-1 was shown to be necessary and sufficient for direct DNA-binding activity. For ternary complex formation with the SRE and SRF, both the Elk-1 ETS-domain as well as flanking sequences up to amino acid 169 were required. Removal of sequences between the ETS-domain and amino acids 137-169 did not abolish ternary complex formation. This suggests the Elk-1 region spanning amino acids 137-169 to contain a protein-protein interaction domain. Furthermore, we have shown that a single amino acid exchange introduced into the ETS-domain can drastically alter the direct DNA-binding affinity of Elk-1 without severely affecting SRF-assisted binding to the SRE. Thus, Elk-1 requires different propensities of the ETS-domain to exert its different modes of DNA sequence recognition.

Amino Acid Sequence↗
PubMed1992

Identification of multiple SRF N-terminal phosphorylation sites affecting DNA binding properties.

Human serum response factor (SRF) bearing a histidine tag was expressed using vaccinia virus. The recombinant protein was purified and shown to be phosphorylated mainly in its N-terminal part. The corresponding phosphorylation sites were mapped by microsequencing and also appear to be phosphorylated in endogenous serum response factor. Four phosphorylation sites are located on serines within amino acids 77-85, while another phosphorylation site has been identified at Ser103. Mutations that considerably reduced or abolished phosphorylation at amino acids 77-85 caused a decrease in binding to the c-fos serum response element accompanied by markedly reduced association and dissociation rates. In contrast, replacing Ser103 by alanine decreased DNA binding activity without drastically affecting the on/off rates. The combination of abolishing phosphorylation at amino acids 77-85 and 103 displayed greatly reduced on/off rates of DNA binding, but the reduction of DNA binding activity was partially alleviated. None of these mutations affect either the ability to interact with p62TCF or stimulation of transcription in vitro. These findings imply possible roles for SRF phosphorylation in the regulation of c-fos transcription.

Amino Acid Sequence↗
PubMed1992

Progress towards a molecular understanding of cyclosporin A-mediated immunosuppression.

Immunosuppressive drugs like cyclosporin A (CsA), FK506 and rapamycin exert their immunosuppressive potential primarily by interfering with the activation and proliferation of T cells. These substances bind to intracellular receptor proteins, called immunophilins. Immunophilins are ubiquitous and abundant proteins, found in all prokaryotic and eukaryotic cells investigated, as well as in many subcellular compartments. Immunophilins display an inherent enzymatic activity, the prolyl-peptidyl cis-trans isomerase (PPIase). The eukaryotic PPIases are inhibited upon the binding of immunosuppressants. In addition, the complex of immunophilins and CsA or FK506 acquires a new activity, namely the binding and inhibition of the cytoplasmic Ca(2+)-binding phosphatase calcineurin. This inhibition is postulated to prevent the proper assembly and nuclear positioning of the transcription factor NF-AT (nuclear factor of activated T cells). The correct DNA binding of NF-AT to regulatory elements of the interleukin 2 (IL-2) promoter normally contributes to the transcriptional activation of this gene. Thus, immunosuppressive drugs prevent T-cell activation by interfering with Ca(2+)-dependent signal transduction pathways which regulate gene activities.

Amino Acid Sequence↗