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T Curran

Publications and source records attributed to T Curran.

At least 19 recordsLinked to original sources
PubMed1996

An enhanced immune response in mice lacking the transcription factor NFAT1.

Transcription factors of the NFAT family are thought to play a major role in regulating the expression of cytokine genes and other inducible genes during the immune response. The role of NFAT1 was investigated by targeted disruption of the NFAT1 gene. Unexpectedly, cells from NFAT1 -/- mice showed increased primary responses to Leishmania major and mounted increased secondary responses to ovalbumin in vitro. In an in vivo model of allergic inflammation, the accumulation of eosinophils and levels of serum immunoglobulin E were increased in NFAT1 -/- mice. These results suggest that NFAT1 exerts a negative regulatory influence on the immune response.

Amino Acid Sequence↗
PubMed1996

Phosphorylation of c-Fos at the C-terminus enhances its transforming activity.

c-Fos is phosphorylated by MAP kinase and the 90 kDa-ribosomal S6 kinase (RSK) in vitro at serines 362 and 374 (rat) which we demonstrate are major in vivo phosphorylation sites in early G1. We have constructed c-Fos mutants with these serines changed to aspartic acid residues (FosD) to mimic phosphorylation or to alanine residues (FosA) to prevent phosphorylation. Cells expressing FosD exhibited a more extensive transformed phenotype than those expressing either FosA or wild type c-Fos (FosWT). We also observed that FosA has a reduced half-life in comparison with FosD in G1. Furthermore, we observed enhanced AP-1 transactivation activity in cells expressing FosD. These results indicate that phosphorylation of c-Fos at its extreme carboxyterminus, possibly by MAP kinase and RSK, supports the proliferative response by increasing c-Fos stability and/or by increasing its transactivation activity. Under conditions in which the MAP kinase pathway is constitutively activated, c-Fos phosphorylation probably contributes to cellular transformation. The highly conserved nature of these phosphorylation sites in other c-fos family members suggests that these may also be targets of MAP kinase and RSK.

3T3 Cells↗
PubMed1996

Ubiquitinylation of transcription factors c-Jun and c-Fos using reconstituted ubiquitinylating enzymes.

Recombinant c-Jun and c-Fos were ubiquitinylated by the ubiquitin carrier enzymes E214K, E220K, or E232K in the presence of the ubiquitin-activating enzyme, E1. Addition of ubiquitin protein ligase E3 substantially enhanced the E214K-mediated ubiquitinylation of c-Jun and c-Fos. Truncated c-Jun and c-Fos mutant proteins including wbJun and wbFos were also ubiquitinylated under the same conditions, suggesting the sites of ubiquitinylation are located within the dimerization and DNA binding domains of c-Jun and c-Fos. The E3-dependent ubiquitinylation of c-Jun was inhibited upon the heterodimerization of c-Jun with c-Fos. Further addition of E220K significantly enhanced ubiquitinylation of c-Jun in the heterodimer suggesting a regulatory role of E220K. Polyubiquitinylated c-Jun, wbFos, and wbJun, but not E220K-ubiquitinylated c-Jun, were readily degraded by the ATP-dependent 26 S multicatalytic proteases. These results suggest that the temporal control of c-Jun and c-Fos may be regulated through the ubiquitinylation pathways, and the ubiquitinylation of c-Jun and c-Fos may in turn be regulated in response to the heterodimerization between them and the cooperation between E220K and E3 mediated polyubiquitinylation.

Adenosine Triphosphate↗
PubMed1996

Fos-Jun dimerization promotes interaction of the basic region with TFIIE-34 and TFIIF.

The regulation of RNA polymerase II-mediated transcription involves both direct and indirect interactions among regulatory proteins and the general transcription factors (GTFs) that assemble at TATA-containing promoters. Here we show that the oncogenic transcription factors Fos and Jun make direct physical contacts with three proteins of the basal transcription apparatus, TFIIE-34 (TFIIE-beta), TFIIF-30 (RAP30), and TFIIF-74 (RAP74). The interactions among the activator proteins and these three GTFs were not detected with other transcription factors, including some bZIP protein family members. Both coimmunoprecipitation and protein blotting experiments demonstrated that the interactions were strongly favored by dimerization of Fos and Jun and that they involved the basic region and basic region-proximal domain of both proteins. Mutations within the DNA-binding domains of Fos and Jun abolished binding to GTFs, although the presence of DNA was not required for the association. Surprisingly, only a single basic region in the context of a protein dimer was sufficient for the interaction. Squelching of AP-1-dependent transcription in vitro by an excess of Fos-Jun dimers was relieved by the addition of TFIIE, indicating that it is a direct functional target of Fos and Jun. These results suggest that dimerization induces a conformational alteration in the basic region of Fos and Jun that promotes an association with TFIIE-34 and TFIIF, thus contributing to transcription initiation.

Animals↗
PubMed1995

Use of fluorescence resonance energy transfer to estimate intramolecular distances in the Msx-1 homeodomain.

We have utilized fluorescence resonance energy transfer (FRET) to investigate the spatial proximities of segments in the Msx-1 homeodomain (Msx). This strategy makes use of a single, invariant tryptophan (Trp-48) in helix III as the donor for FRET. The acceptor molecule, 5-[[[(iodoacetyl)amino]-ethyl]amino]naphthalene-1-sulfonic acid (AEDANS), was incorporated into Msx at positions 6, 10, or 27 which are within the N-terminal arm, and helices I and II since these segments have been implicated in interactions with helix III. Specific incorporation of AEDANS was achieved by using a two-step strategy consisting of site-directed mutagenesis for introducing unique cysteine residues at the selected positions followed by covalent modification of these cysteine residues with AEDANS. Using this approach, we demonstrated energy transfer between Trp-48 and the AEDANS-labeled cysteines at positions 6, 10, and 27 and estimated the distances between the Trp-48 and AEDANS pairs to be 19, 23, and 16 A, respectively. We further demonstrated that FRET provides a strategy for detecting subtle alterations in protein conformation that result from replacement of specific residues in helix III and the N-terminal arm. Together, these findings show that FRET provides a useful approach for estimating intramolecular distances and for examining the conformation of Msx. Moreover, given the fact that Trp-48 is invariant among all homeodomain sequences, we propose that FRET will provide a general approach for facilitating comparative analyses of homeodomain conformations.

Amino Acid Sequence↗
PubMed1995

A protein related to extracellular matrix proteins deleted in the mouse mutant reeler.

The autosomal recessive mouse mutation reeler leads to impaired motor coordination, tremors and ataxia. Neurons in affected mice fail to reach their correct locations in the developing brain, disrupting the organization of the cerebellar and cerebral cortices and other laminated regions. Here we use a previously characterized reeler allele (rl(tg)) to close a gene, reelin, deleted in two reeler alleles. Normal but not mutant mice express reelin in embryonic and postnatal neurons during periods of neuronal migration. The encoded protein resembles extracellular matrix proteins involved in cell adhesion. The reeler phenotype thus seems to reflect a failure of early events associated with brain lamination which are normally controlled by reelin.

Alleles↗
PubMed1995

Fos: an immediate-early transcription factor in neurons.

In the past several years a great deal of evidence has accumulated linking neuronal activation events to the regulation of gene expression. We have pursued an analysis of c-fos regulation in the nervous system to elucidate the molecular mechanisms involved in stimulus-transcription coupling. The c-fos gene can be viewed as an archetype of the set of cellular immediate-early genes encoding transcription factors. These genes are believed to function in coupling short-term signals elicited by extracellular to long-term changes in cellular phenotype by orchestrating alterations in target gene expression. Several animal seizure models have been used to demonstrate the activation of gene expression in specific populations of neurons. Using a transgenic mouse approach, based on a foslacZ fusion gene, we now demonstrate an association between c-fos expression and cell death in the nervous system. A delayed and protracted induction was observed following surgical lesion and in response to neurotoxin exposure. This system allows us to determine, for the first time, the DNA regulatory sequences that are responsible for the induction of gene expression in neurons in vivo. Furthermore, foslacZ transgenic mice provide a unique resource for identifying cell populations that respond to specific stimuli or that are susceptible to particular toxins.

Animals↗
PubMed1995

Regulation of c-fos expression in transgenic mice requires multiple interdependent transcription control elements.

Transcription control regions of eukaryotic genes contain multiple sequence elements proposed to function independently to regulate transcription. We developed transgenic mice carrying fos-lacZ fusion genes with clustered point mutations in each of several distinct regulatory sequences: the sis-inducible element, the serum response element, the fos AP-1 site, and the calcium/cAMP response element. Analysis of Fos-lacZ expression in the CNS and in cultured cells demonstrated that all of the regulatory elements tested were required in concert for tissue- and stimulus-specific regulation of the c-fos promoter. This implies that the regulation of c-fos expression requires the concerted action of multiple control elements that direct the assembly of an interdependent transcription complex.

Animals↗
PubMed1995

Violations of the independence assumption in process dissociation.

L. L. Jacoby, J. P. Toth, and A. P. Yonelinas (1993) advocated a process-dissociation procedure for estimating the contributions to task performance of consciously controlled (R) versus automatic (A) memory processes. The procedure relies on the strong assumption that memory-guided performance attributable to R is stochastically independent of that attributable to A. Violations of this independence assumption can produce artifactual dissociations between estimates of R and A. Such artifactual dissociations were obtained in a series of word-stem completion experiments: R increased with presentation duration, whereas A, paradoxically, decreased. Direct evidence for nonindependence was obtained from correlations between R and A in each of the experiments. These results suggest that the independence assumption was violated, and other applications of process dissociation should not be taken at face value without a thorough evaluation of independence.

Automatism↗
PubMed1995

N-terminal variants of thyroid hormone receptor beta: differential function and potential contribution to syndrome of resistance to thyroid hormone.

The human syndrome of resistance to thyroid hormone (RTH) is associated with dominant mutations in the thyroid hormone receptor beta (TR beta) gene that generate mutant receptors with impaired binding for T3. Although the TR beta gene differentially expresses two N-terminal variant receptors, TR beta 1 and TR beta 2, functional analyses of RTH mutants have focused exclusively on TR beta 1. Since TR beta 2 is expressed in tissues that are malfunctional in RTH, the role of mutations in the context of TR beta 2 was examined. We compared the functional properties of corresponding RTH mutations in the common C-terminal domain of both TR beta 1 and TR beta 2. Wild type TR beta 1 and TR beta 2 bound similarly as homodimers and as heterodimers with retinoid X receptors to T3-responsive elements consisting of a direct repeat with 4-base pair spacing or an everted repeat. Homodimers, but not monomers or heterodimers, of both receptor subtypes were dissociated by the addition of T3. However, TR beta 2 formed at least 10-fold more stable homodimers than TR beta 1 on a palindromic repeat element, indicating that the N termini of TR beta 1 and TR beta 2 differentially influence dimerization on DNA. The RTH-like mutants of both TR beta 1 and TR beta 2 were equally insensitive to T3. They were defective in T3 binding but still bound DNA like their wild type counterparts except that the T3-dependent dissociation of homodimers from DNA was severely reduced. Wild type TR beta 1 and TR beta 2 mediated T3-inducible transactivation in cotransfection assays; this, however, was abolished in both mutants. TR beta 1 mediated more sensitive T3-dependent transcriptional suppression than TR beta 2 through the negative T3 response region of the TSH beta gene. Again, the mutation abolished T3-dependent suppression by both mutants. Furthermore, both mutants inhibited T3-inducible transcriptional activation by different wild type TR alpha and beta variants. These results indicate that both mutants have the potential to contribute to the pathogenesis of RTH and suggest that a reassessment of previous models of RTH is required to take into account the inhibitory activity of both TR beta 2 and TR beta 1 mutants.

3T3 Cells↗
PubMed1995

When encoding fails: instructions, feedback, and registration without learning.

Four experiments replicated and extended the registration-without-learning effect, in which there is little improvement in the ability to discriminate an old target (X) from a highly similar test item (Y) after the first few presentations of X, even though judgments of frequency continue to rise in an open-ended fashion. Forced-choice testing revealed the anomalous form of the learning curve for X-Y discrimination (faster and then slower than the exponential). Effects of several different learning instructions were compared, but these appeared to affect only the level of initial learning, and to do little to promote X-Y discrimination learning on later presentations. The opportunity for self-testing with feedback during study provided no benefits when responding was covert, but did when overt anticipation was required. The findings are discussed in relation to the roles of bottom-up and top-down processing in memory encoding, and to the importance of error-correcting feedback in further structural learning of materials, once the materials have become familiar.

Adult↗
PubMed1995

Kainic acid-induced neuronal death is associated with DNA damage and a unique immediate-early gene response in c-fos-lacZ transgenic rats.

Previously, we established that persistent upregulation of c-fos expression preceded kainic acid (KA)-induced neuronal death in mice. To discriminate between events that are products of the seizures elicited by KA and those that are specifically associated with its neurotoxic actions, we have examined the expression of cellular immediate-early genes (cIEGs) following KA or pentylenetetrazol (PTZ) treatment in c-fos-lacZ transgenic rats. While both chemoconvulsants elicit seizures, only KA causes selective neuronal death. Following treatment of transgenic rats with KA there was a protracted expression of Fos-lacZ that lasted for 2-3 d. In contrast, PTZ elicited a transient increase in the transgene product that lasted about 6 hr. Normally, Fos and Fos-lacZ were detected only in neuronal nuclei. However, 6 hr following kainic acid (but not PTZ) administration, beta-galactosidase activity appeared in the cytoplasm of neurons within vulnerable regions (as determined by the terminal transferase biotinylated-UTP nick end labeling (TUNEL) procedure). Like c-fos, transcripts for other cIEGs were elevated for longer periods in the KA-treated rat hippocampus. In addition, fra-1 and fra-2 were only induced in the KA-treated rat. These changes in mRNA levels were paralleled by a sustained increase in AP-1 DNA binding activity. Thus, quantitative and qualitative changes in AP-1 DNA binding complexes accompany neurotoxic cell death that are not observed following seizures.

Animals↗