Search PubMed⌕ Search

Biomedical subjects

T Curran

Publications and source records attributed to T Curran.

At least 145 records · Page 8Linked to original sources
PubMed1991

Transcriptional regulation by Fos and Jun in vitro: interaction among multiple activator and regulatory domains.

The proteins encoded by the proto-oncogenes c-fos and c-jun (Fos and Jun, respectively) form a heterodimeric complex that regulates transcription by interacting with the DNA-regulatory element known as the activator protein 1 (AP-1) binding site. Fos and Jun are members of a family of related transcription factors that dimerize via a leucine zipper structure and interact with DNA through a bipartite domain formed between regions of each protein that are rich in basic amino acids. Here we have defined other domains in the Fos-Jun heterodimer that contribute to transcriptional function in vitro. Although DNA-binding specificity is mediated by the leucine zipper and basic regions, Jun also contains a proline- and glutamine-rich region that functions as an ancillary DNA-binding domain but does not contribute directly to transcriptional activation. Transcriptional stimulation in vitro was associated with two regions in Fos and a single N-terminal activation domain in Jun. These activator regions were capable of operating independently; however, they appear to function cooperatively in the heterodimeric complex. The activity of these domains was modulated by inhibitory regions in Fos and Jun that repressed transcription in vitro. In the context of the heterodimer, the Jun activation domain was the major contributor to transcriptional stimulation and the inhibitory regions in Fos were the major contributors to transcriptional repression in vitro. Potentially, the inhibitory domains could serve a regulatory function in vivo. Thus, transcriptional regulation by the Fos-Jun heterodimer results from a complex integration of multiple activator and regulatory domains.

Binding Sites↗
PubMed1991

Fos is phosphorylated by p34cdc2, cAMP-dependent protein kinase and protein kinase C at multiple sites clustered within regulatory regions.

The proto-oncogene c-fos encodes a nuclear protein (Fos) that functions in transcriptional regulation in response to extracellular signals. Fos is extensively modified in the nucleus by serine and threonine phosphorylation. It has been suggested that phosphorylation may play an important role in regulating Fos function in normal and transformed cells. As a first step in addressing this issue, we have used purified Fos as a substrate for several serine-threonine protein kinases, including cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and p34cdc2. Each of these kinases phosphorylated Fos at several unique sites. These sites were located within two regions that were previously shown to reduce the transcriptional activity of Fos in vitro. Several of the sites modified in vitro were also shown to be phosphorylated in serum-stimulated fibroblasts. These findings demonstrate that Fos is a target for several protein kinases involved in signal transduction and suggest that phosphorylation could regulate the transcriptional properties of Fos.

Amino Acid Sequence↗
PubMed1991

Inhibition of jun transformation by a mutated fos gene: design of an anti-oncogene.

The protein products of the fos and jun oncogenes (Fos and Jun) function as transcriptional regulators in the form of homo- or heterodimeric complexes that bind to DNA. Dimerization is mediated by a leucine zipper structure that serves to juxtapose alpha-helical regions of each protein, rich in basic amino acids, that form a bipartite DNA-binding domain. Although Fos participates exclusively in heterodimeric complexes, Jun can function either as a homodimer that has a low apparent affinity for DNA or as a more stable heterodimer with Fos that has a higher apparent affinity for DNA. We have used these properties of Fos and Jun to design a mutated fos gene, lacking a functional DNA-binding domain (supfos1), that suppresses the transforming activity of jun in trans. Here we show that chicken embryo fibroblasts transformed by jun revert to a normal phenotype after infection by a retroviral vector encoding supFos1. Furthermore, infection of normal cells with the supfos1 vector renders them resistant to subsequent transformation by jun. Inhibition of jun transformation was associated with the appearance of supFos1-Jun heterodimers and a reduction in the AP-1 DNA-binding activity contributed by Jun homodimers. These findings demonstrate that the function of leucine zipper-containing transcription factors can be investigated by the procedure of intracellular immunization.

Animals↗
PubMed1990

Binding of the Wilms' tumor locus zinc finger protein to the EGR-1 consensus sequence.

The Wilms' tumor locus (WTL) at 11p13 contains a gene that encodes a zinc finger-containing protein that has characteristics of a DNA-binding protein. However, binding of this protein to DNA in a sequence-specific manner has not been demonstrated. A synthetic gene was constructed that contained the zinc finger region, and the protein was expressed in Escherichia coli. The recombinant protein was used to identify a specific DNA binding site from a pool of degenerate oligonucleotides. The binding sites obtained were similar to the sequence recognized by the early growth response-1 (EGR-1) gene product, a zinc finger-containing protein that is induced by mitogenic stimuli. A mutation in the zinc finger region of the protein originally identified in a Wilms' tumor patient abolished its DNA-binding activity. These results suggest that the WTL protein may act at the DNA binding site of a growth factor-inducible gene and that loss of DNA-binding activity contributes to the tumorigenic process.

Amino Acid Sequence↗
PubMed1990

Altered protein conformation on DNA binding by Fos and Jun.

The protein products of the c-fos and c-jun proto-oncogenes (Fos and Jun, respectively) form a heterodimeric protein complex that interacts with the activator protein-1 (AP-1) binding site and regulates gene transcription in response to extracellular stimuli. Protein dimerization is mediated primarily by a coiled-coil-like structure termed the leucine-zipper and DNA binding occurs primarily through regions of each protein rich in basic amino acids that contact both strands of the AP-1 site. The precise nature of the protein-DNA interaction is unknown as studies concerned with dimerization and DNA binding by Fos and Jun have relied on indirect methods to investigate protein-protein-DNA interactions. Here we have developed assay systems using fluorescence spectroscopy and circular dichroism to monitor dimerization and DNA binding directly. The results indicate that the interaction of Fos and Jun with DNA results in an altered conformation of the protein dimers and an increased alpha-helical content. These techniques may have general application in studies concerning the interaction of transcriptional regulatory proteins with specific DNA target sequences.

Binding Sites↗
PubMed1990

Redox regulation of fos and jun DNA-binding activity in vitro.

The proto-oncogenes c-fos and c-jun function cooperatively as inducible transcription factors in signal transduction processes. Their protein products, Fos and Jun, form a heterodimeric complex that interacts with the DNA regulatory element known as the activator protein-1 (AP-1) binding site. Dimerization occurs via interaction between leucine zipper domains and serves to bring into proper juxtaposition a region in each protein that is rich in basic amino acids and that forms a DNA-binding domain. DNA binding of the Fos-Jun heterodimer was modulated by reduction-oxidation (redox) of a single conserved cysteine residue in the DNA-binding domains of the two proteins. Furthermore, a nuclear protein was identified that reduced Fos and Jun and stimulated DNA-binding activity in vitro. These results suggest that transcriptional activity mediated by AP-1 binding factors may be regulated by a redox mechanism.

Amino Acid Sequence↗
PubMed1990

Expression and purification of the leucine zipper and DNA-binding domains of Fos and Jun: both Fos and Jun contact DNA directly.

The protein products of the fos and jun protooncogenes interact cooperatively in the form of a heterodimer with the activator protein 1 (AP-1) regulatory element. To characterize the properties of these proteins, we have expressed polypeptides comprised of the dimerization and DNA-binding domains of Fos and Jun in Escherichia coli. The mini-Fos (wbFos) and the mini-Jun (wbJun) proteins were purified to apparent homogeneity by using a nickel affinity chromatography procedure. Purified wbFos and wbJun associated rapidly in vitro and interacted cooperatively with the human metallothionein IIA AP-1-binding site. However, efficient DNA binding of wbJun and wbFos-wbJun complexes required an additional activity present in nuclear extracts. This activity was sensitive to alkylating agents and could be partially mimicked by the presence of reducing and stabilizing agents. DNase I footprinting experiments demonstrated that Jun homodimeric complexes and Fos-Jun heterodimeric complexes interacted with the same site on the human metallothionein IIA gene. Moreover, UV-crosslinking studies demonstrated that Fos and Jun contact DNA directly and that both proteins interacted equivalently with either strand of the AP-1-binding site.

Amino Acid Sequence↗
PubMed1990

Induction of c-fos mRNA expression by afterdischarge in the hippocampus of naive and kindled rats.

Periodic induction of focal electrical seizure [afterdischarge (AD)] is an absolute prerequisite for the development of kindling, an animal model of complex partial epilepsy. Once established, it is a permanent condition. The mechanism(s) that translate ADs, which last tens of seconds, into life-long alterations in the CNS is unclear. Cellular immediate-early genes have been implicated in the conversion of short-term stimuli to long-term alterations in cellular phenotypes by regulating target gene expression. We have investigated the contribution of one such early gene, c-fos, to this process. The relationship between ADs and expression of c-fos gene in the rat hippocampus, a key structure in kindling development, was studied by analysis of mRNA levels. The low constitutive expression of c-fos mRNA in the hippocampus was not altered by kindling. There was an "all-or-none" relationship between induction of c-fos and the duration of AD. The threshold for induction was approximately 30 s of AD. Above-threshold ADs induced c-fos in both naive and kindled animals to the same extent and with identical temporal profiles. Although the expression of c-fos is unchanged with kindling, c-fos may nonetheless contribute to many long-term changes of kindling, both adaptive and epileptogenic.

Animals↗
PubMed1990

Fos and jun cooperate in transcriptional regulation via heterologous activation domains.

The products of c-fos and c-jun (Fos and Jun) function in gene regulation by interacting with the AP-1 binding site. Here we have examined the contribution of Fos and Jun toward transcriptional activity by using Fos and Jun polypeptides purified from Escherichia coli. Fos contained a transcriptional activation domain as well as a region which exerted a negative influence on transcriptional activity in vitro. Moreover, distinct activation domains in both Fos and Jun functioned cooperatively in transcriptional stimulation. Thus, regulation of gene expression by Fos and Jun results from an integration of several functional domains in a bimolecular complex.

Binding Sites↗
PubMed1990

Transcriptional activation and repression by Fos are independent functions: the C terminus represses immediate-early gene expression via CArG elements.

The Fos-Jun complex has been shown to activate transcription through the regulatory element known as the AP-1 binding site. We show that Fos down regulates several immediate-early genes (c-fos, Egr-1, and Egr-2) after mitogenic stimulation. Specifically, we demonstrate that the target for this repression is a sequence of the form CC(A/T)6GG, also known as a CArG box. Whereas Fos bound to the AP-1 site through a domain rich in basic amino acids and associated with Jun via a leucine zipper interaction, mutant Fos proteins lacking these structures were still capable of causing repression. Furthermore, Jun neither enhanced nor inhibited down regulation by Fos. Critical residues required for repression are located within the C-terminal 27 amino acids of c-Fos, since v-Fos and C-terminal truncations of c-Fos did not down regulate. In addition, transfer of 180 c-Fos C-terminal amino acids to Jun conferred upon it the ability to repress. Finally, Fra-1, a Fos-related protein which has striking similarity to Fos in its C-terminal 40 amino acids, also down regulated Egr-1 expression. Thus, Fos is a transcriptional regulator that can activate or repress gene expression by way of two separate functional domains that act on distinct regulatory elements.

Animals↗
PubMed1990

Analysis of dimerization and DNA binding functions in Fos and Jun by domain-swapping: involvement of residues outside the leucine zipper/basic region.

The products of two cellular proto-oncogenes c-fos and c-jun form a heterodimeric complex that contribute to the DNA-binding activity referred to as AP-1 (activator protein-1). Two domains have been proposed to be required for heterodimer formation and protein-DNA complex formation. The leucine zipper domain mediated the interaction between the two proteins and a highly basic region immediately N-terminal to the leucine zipper forms a DNA binding domain. To assess the role of these two domains in dimerization and DNA binding and to determine what contribution, if any, is made by residues outside of these regions, we carried out an extensive domain swap analysis. Restriction sites created in the fos and jun cDNAs flanking the basic region and leucine zipper allowed these domains to be swapped between the two proteins either singly or in various combinations with adjacent domains. The chimeric proteins were assayed for their ability to dimerize with each other and to interact with the AP-1 consensus sequence. It was found that two Jun leucine zipper regions could mediate protein dimerization, whereas two Fos leucine zipper regions could not. The dimers formed between two Jun leucine repeats were less stable than those formed between a Fos and a Jun leucine zipper. A conserved His residue seven amino acids C-terminal of the last leucine of the zipper region contributed to the stability of protein-protein interactions. The basic region of both Fos and Jun was found to interact with DNA without the presence of the other, i.e. the combination of two Fos- or two Jun-DNA binding domains could bind to the AP-1 site. However, replacement of the Jun N-terminus with that of Fos resulted in a decrease in DNA binding, indicating that residues outside of the Jun basic region contribute to DNA binding. The results also suggest that the dimerization and DNA binding functions of each protein are not completely independent properties, but that each exerts an influence on the other.

Amino Acid Sequence↗
PubMed1990

A ubiquitous nuclear protein stimulates the DNA-binding activity of fos and jun indirectly.

The protooncogenes c-fos and c-jun encode nuclear proteins (fos and jun, respectively) that function cooperatively as a heterodimeric protein complex in the regulation of gene transcription. These proteins dimerize via a structural motif known as the leucine zipper and bind to activator protein-1 sites via a conserved domain that is rich in basic amino acids. Previously, we demonstrated that while fos and jun polypeptides expressed in Escherichia coli dimerize efficiently, they exhibit only a low level of DNA-binding activity. Here we show that the DNA-binding activity of fos-jun heterodimers and jun-jun homodimers is stimulated dramatically by a ubiquitous nuclear protein. This protein does not appear to participate in the DNA-protein complex, and it does not affect the specificity of the interaction with DNA. These results suggest that a nuclear protein regulates the DNA-binding activity of fos and jun indirectly.

Amino Acid Sequence↗
PubMed1990

Encounters with Fos and Jun on the road to AP-1.

The nuclear proto-oncogenes, c-fos and c-jun, are induced in response to a diverse array of extracellular stimuli. Their protein products, Fos and Jun, form a heterodimeric complex that interacts with the DNA regulatory element known as the AP-1 binding site. Protein dimerization occurs via a parallel interaction of leucine zipper domains and is required for DNA binding. In addition to the leucine zipper, DNA binding requires two clusters of basic amino acids adjacent to the leucine zipper domains of both Fos and Jun. The leucine zipper and DNA-binding regions are highly conserved among the c-fos and c-jun families of related inducible genes. Thus, multiple protein complexes can be formed that may interact with AP-1 binding sites in numerous genes to affect gene expression in response to environmental signals.

Amino Acid Sequence↗
PubMed1990

Direct cloning of leucine zipper proteins: Jun binds cooperatively to the CRE with CRE-BP1.

The proto-oncogene products Fos and Jun form a stable heterodimeric complex that functions in transcriptional regulation by interacting with the DNA sequence known as the AP-1 site. Dimer formation occurs through the leucine zipper, a structural motif involving a heptad repeat of leucine residues that is conserved in several fos- and jun-related genes. We have employed a novel cloning strategy to isolate genes encoding proteins capable of forming complexes with Jun. The procedure involves direct screening of a lambda gt11 cDNA library with a biotinylated Jun polypeptide. One clone isolated in this manner encodes CRE-BP1, a leucine zipper-containing protein that binds to the cyclic AMP response element (CRE) as a homodimer. CRE-BP1 also forms heterodimers with Jun but not with Fos. Jun binds cooperatively to the CRE in association with CRE-BP1. Thus, the DNA-binding specificity and affinity of Jun are modulated by association with Fos or with CRE-BP1.

Animals↗