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B Pierrat

Publications and source records attributed to B Pierrat.

12 recordsLinked to original sources
PubMed2001

SH3GLB, a new endophilin-related protein family featuring an SH3 domain.

A new cDNA encoding a protein of 362 amino acids designated SH3GLB1, for SH3 domain GRB2-like endophilin B1, was identified in a yeast two-hybrid screen devoted to the identification of new partners interacting with the apoptosis inducer Bax. SH3GLB1 shows strong similarities to the SH3 domain-containing proteins of the endophilin family and presumably represents the human homologue of the potential Caenorhabditis elegans SH3 containing-protein identified by systematic translation of the C. elegans genome (GenBank Accession No. U46675). Reversing prey to bait in the yeast screen, a second protein, SH3GLB2, of 395 amino acids showing 65% identity to SH3GLB1 was identified as an interacting partner of SH3GLB1. The discovery of SH3GLB1 itself in the screening with SH3GLB1 as a bait and further mapping experiments demonstrated that a core coiled-coil-type region is required for the formation of SH3GLB homo- and/or heterodimers, whereas the SH3 domain is not involved in these interactions. Interestingly, the similarities with the endophilin proteins cover the entire sequence of the SH3GLB family, suggesting a common fold and presumably a common mode of action. Furthermore, SH3GLB members colocalize to the cytoplasmic compartment of the cell together with Bax and are excluded from the nucleus. SH3GLB1 and SH3GLB2 do not significantly influence the onset and time course of Bax-mediated apoptosis in HeLa or 293T cells.

Adaptor Proteins, Signal Transducing↗
PubMed2000

Comparative microarray analysis of gene expression during apoptosis-induction by growth factor deprivation or protein kinase C inhibition.

The transcriptional response of mouse pro-B cells to two different apoptotic stimuli was investigated. First, interleukin-3 (IL-3) deprivation was used to trigger programmed cell death in IL-3 dependent FL5.12 cells. Alternatively, cells were treated with the protein kinase C (PKC) inhibitor staurosporine. The temporal pattern of gene expression was followed with cDNA microarrays, covering over 8700 different mouse cDNA sequences corresponding to approximately 7900 unique genes. Messenger RNA levels of 315 genes were found to be regulated by more than twofold upon IL-3 removal, while 125 genes reacted to staurosporine treatment. Cross-comparison revealed an intersection of 34 genes similarly regulated in both pathways and thus representing candidates for common apoptosis regulators. For many expressed sequence tags (ESTs) our data suggest for the first time functions in the control of apoptosis, stress response or the cell cycle. IL-3 removal led to the repression of genes required for proliferation and to the induction of genes, linked to apoptotic and signaling pathways. Staurosporine caused predominantly activation of genes, some of which had previously been described to be involved in inflammation. Our findings indicate that cellular responses to both apoptotic stimuli influence various physiological pathways which had not previously been known to be linked.

Animals↗
PubMed2000

Uncoupling proteins 2 and 3 interact with members of the 14.3.3 family.

Uncoupling proteins (UCPs) are members of the superfamily of the mitochondrial anion carrier proteins (MATP). Localized in the inner membrane of the organelle, they are postulated to be regulators of mitochondrial uncoupling. UCP2 and 3 may play an important role in the regulation of thermogenesis and, thus, on the resting metabolic rate in humans. To identify interacting proteins that may be involved in the regulation of the activity of UCPs, the yeast two-hybrid system was applied. Segments of hUCP2 containing the hydrophilic loops facing the intermembrane space, or combinations of these, were used to screen an adipocyte activation domain (AD) fusion library. The 14.3.3 protein isoforms theta, beta, zeta were identified as possible interacting partners of hUCP2. Screening of a human skeletal muscle AD fusion library, on the other hand, yielded several clones all of them encoding the gamma isoform of the 14.3.3 family. Mapping experiments further revealed that all these 14.3.3 proteins interact specifically with the C-terminal intermembrane space domain of both hUCP2 and hUCP3 whereas no interactions could be detected with the C-terminal part of hUCP1. Direct interaction between UCP3 and 14.3.3 theta could be demonstrated after in vitro translation by coimmunoprecipitation. When coexpressed in a heterologous yeast system, 14.3.3 proteins potentiated the inhibitory effect of UCP3 overexpression on cell growth. These findings suggest that 14.3.3 proteins could be involved in the targeting of UCPs to the mitochondria.

14-3-3 Proteins↗
PubMed1998

RSK-B, a novel ribosomal S6 kinase family member, is a CREB kinase under dominant control of p38alpha mitogen-activated protein kinase (p38alphaMAPK).

A novel ribosomal S6 kinase (RSK) family member, RSK-B, was identified in a p38alphaMAPK-baited intracellular interaction screen. RSK-B presents two catalytic domains typical for the RSK family. The protein kinase C-like N-terminal and the calcium/calmodulin kinase-like C-terminal domains both contain conserved ATP-binding and activation consensus sequences. RSK-B is a p38alphaMAPK substrate, and activated by p38alphaMAPK and, more weakly, by ERK1. RSK-B phosphorylates the cAMP response element-binding protein (CREB) and c-Fos peptides. In intracellular assays, RSK-B drives cAMP response element- and AP1-dependent reporter expression. RSK-B locates to the cell nucleus and co-translocates p38alphaMAPK. In conclusion, RSK-B is a novel CREB kinase under dominant p38alphaMAPK control, also phosphorylating additional substrates.

Amino Acid Sequence↗
PubMed1998

Retinoic acid receptors interact physically and functionally with the T:G mismatch-specific thymine-DNA glycosylase.

The pleiotropic effects of retinoids are mediated by nuclear receptors that are activated by 9-cis- or all-trans-retinoic acid to function as ligand-dependent transcription factors. In a yeast one-hybrid screen for proteins capable of interacting with native retinoic acid receptor (RAR), we have isolated the T:G mismatch-specific thymine-DNA glycosylase (TDG), which initiates the repair of T:G mismatches caused by spontaneous deamination of methylated cytosines. Here, we report that TDG can interact with RAR and the retinoid X receptor (RXR) in a ligand-independent manner, both in yeast and in vitro. Mapping of the binding sites revealed interaction with a region of the ligand binding domain harboring alpha-helix 1 in both RAR and RXR. In transient transfection experiments, TDG potentiated transactivation by RXR from a direct repeat element spaced by one nucleotide (DR1) and by RXR/RAR heterodimers from a direct repeat element spaced by five nucleotides (DR5). In vitro, TDG enhanced RXR and RXR/RAR binding to their response elements. These data indicate that TDG is not only a repair enzyme, but could also function in the control of transcription.

Amino Acid Sequence↗
PubMed1997

Blockade of p38 mitogen-activated protein kinase pathway inhibits inducible nitric-oxide synthase expression in mouse astrocytes.

Treatment of mouse astrocyte cultures with combined interleukin (IL)-1alpha and tumor necrosis factor (TNF)-alpha induced expression of inducible nitric-oxide synthase (iNOS), resulting in sustained release of large amounts of nitric oxide, whereas TNF-alpha and IL-1alpha individually were unable to induce iNOS expression in astrocytes. The role of MAPK cascades and of NF-kappaB activation in the early intracellular signal transduction involved in iNOS transcription in TNF-alpha/IL-1alpha-stimulated astrocytes was investigated. TNF-alpha and IL-1alpha activated all p42/44(MAPK), p38(MAPK), and p54(JNK) pathways as determined by immunoprecipitation kinase assays using specific antibodies and substrates. The p38(MAPK) pathway is specifically involved in TNF-alpha/IL-1alpha-induced iNOS expression, since iNOS protein and nitric oxide release in the presence of a specific inhibitor of p38(MAPK), 4-(4-fluorophenyl)-2-2-(4-hydroxyphenyl)-5-(4-pyridyl)-imidazole (FHPI), were dramatically diminished. In contrast, PD98059, a specific inhibitor of MEK1 had no effect on iNOS expression. p38(MAPK) did not couple NF-kappaB to iNOS transcription, but NF-kappaB had a clear role in iNOS transcription regulation. Northern blot analysis showed that the p38(MAPK) pathway controlled iNOS expression at the transcriptional level, since iNOS mRNA was reduced in the presence of FHPI in TNF-alpha/IL-1alpha-stimulated astrocytes. iNOS expression was investigated with TNF receptor (TNFR)-1- and TNFR-2-deficient mice. The TNF-alpha activity in TNF-alpha/IL-1alpha-stimulated astrocytes was exclusively mediated through TNFR-1, most likely because TNFR-2-mediated signals in astrocytes did not connect to the p38(MAPK) pathway. These data suggest that TNF-alpha/IL-1alpha-induced iNOS expression depends on a yet undetermined second pathway in addition to p38(MAPK).

Animals↗
PubMed1994

A highly conserved region in the hormone-binding domain of the human estrogen receptor functions as an efficient transactivation domain in yeast.

Human estrogen receptor (hER) mutants which activate transcription in the absence of hormone were isolated by random mutagenesis and genetic selection in the yeast Saccharomyces cerevisiae. Twenty constitutive hER mutants defining ten different alleles were selected. All sequence changes resulted in truncations of the receptor within a 123-amino-acid (aa) segment (aa 270 to 393) spanning the D region and the N-terminal part of region E which contains the hormone-binding domain (HBD). Transactivation assays using both the constitutive hER mutants and a series of deleted receptor derivatives generated in vitro revealed that the N-terminal part of region E, between aa 302 and 339, contains an efficient transcriptional activation function which is constitutively active in yeast. The location of this transactivation function in hER is similar to that of the tau 2 activation function of the glucocorticoid receptor and corresponds to a sequence which is highly conserved among the steroid hormone receptors. Thus, a conserved region exists in the HBD of the hER which can function as an autonomous transactivation domain.

Amino Acid Sequence↗
PubMed1994

Homo- and heterodimers of the retinoid X receptor (RXR) activated transcription in yeast.

The polymorphic nature of sequences which act as retinoic acid response elements (RAREs and RXREs) in transactivation assays in mammalian cells, suggests that elements consisting of a direct repetition of a half site motif, separated by 1 to 5 base pairs (DR1 to DR5), are targets for retinoic acid (RA) signalling. In a previous report we showed that in yeast cells, heterodimers of the retinoic acid receptors RAR alpha and RXR alpha were required for efficient transcription of a reporter gene containing a DR5 element [Heery et al., (1993); Proc. Natl. Acad. Sci. USA, 90: 4281-4285]. Here we report that DR1 to DR5 elements containing a direct repeat of the 5'-AGGTCA-3' motif, and an inverted repeat of the same sequence with no spacer (IR0), behave as RAREs in yeast cells coexpressing RAR alpha and RXR alpha, albeit with different efficacies. Heterodimer activity was strongest on a DR5 reporter gene, and the strength of activation of the reporter series (DR5 > DR1 > DR3 > DR2 = IR0 = DR4) correlated with the ability of the heterodimer to bind to the corresponding sequences in vitro. Significantly, a reporter containing a DR1 element was selectively and efficiently activated in yeast cells expressing only RXR alpha. This activity was dependent on the induction by 9-cis retinoic acid of an activation function (AF-2) located in the RXR alpha ligand binding domain. In addition, a strong synergistic activity of RXR alpha was observed on a reporter containing the putative RXR element (RXRE) from the rat CRBPII gene promoter. Thus, RXR alpha can function independently as a transcription factor, in the absence of RARs or other heteromeric partners. Similarly, homodimers of RAR alpha selectively stimulated the transcription of a DR5 reporter in a ligand-dependent manner, but less efficiently than RAR alpha/RXR alpha heterodimers.

Base Sequence↗
PubMed1993

The 5' untranslated region of the PPR1 regulatory gene dictates rapid mRNA decay in yeast.

In Saccharomyces cerevisiae, the mRNA encoded by the PPR1 gene is very unstable (t1/2 = 1 min), whereas the mRNA encoded by the URA3 gene is relatively stable (t1/2 = 10 min). To identify cis-acting sequences that dictate mRNA decay rates in yeast, we have constructed PPR1/URA3 gene fusions and measured the half-lives of the resulting chimeric transcripts. The mRNA containing the URA3 coding region fused to the untranslated regions (UTR) of PPR1 decayed at a rate similar to the native PPR1 mRNA, suggesting that the instability of the PPR1 mRNA is not linked to its coding sequence. When the 5'-UTR of PPR1 was replaced by the 5'-UTR of URA3, the chimeric transcript was strongly stabilized, indicating that the 5'-UTR of PPR1 is required for the rapid decay of its mRNA. Fusion of this PPR1 5'-UTR to the URA3 coding region was sufficient to destabilize the chimeric mRNA. We conclude that the PPR1 5'-UTR contains sequence(s) that can promote rapid mRNA decay in yeast.

Base Sequence↗
PubMed1993

Efficient transactivation by retinoic acid receptors in yeast requires retinoid X receptors.

All-trans and 9-cis retinoic acids are natural derivatives of vitamin A that modulate gene expression as a consequence of binding to nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RXRs form heterodimers with RARs in vitro, and such complexes display enhanced binding affinities for cognate DNA response elements. As yeast is devoid of endogenous RARs and RXRs, we used this organism to investigate whether transactivation in vivo requires RAR/RXR heterodimers. Using a domain-swapping approach, we demonstrate that chimeric RAR alpha 1 and RXR alpha containing the DNA-binding domain of the estrogen receptor activate transcription of a cognate reporter gene in yeast, independently of each other. These activities result from an inducible transcription activation function located in the ligand-binding domains of RAR alpha 1 and RXR alpha and a constitutive activation function located in the A/B region of RAR alpha 1. The inducible activation function of RXR alpha is induced exclusively by 9-cis-retinoic acid in this system. Transactivation of a reporter gene containing a retinoic acid response element by RAR alpha was considerably increased by RXR alpha, even in the absence of ligand. Optimal induction was achieved with 9-cis-retinoic acid, which stimulates the activity of both receptors. This study illustrates the utility of yeast to investigate signal transduction by retinoids in the absence of endogenous RARs, RXRs, and detectable retinoic acid isomerization.

Animals↗
PubMed1992

Functional analysis of the human estrogen receptor using a phenotypic transactivation assay in yeast.

We have constructed yeast strains in which the expression of the Saccharomyces cerevisiae URA3 gene is induced by the human estrogen receptor (hER). Promoter sequences required for both basal and activated transcription of URA3 were replaced with one or three estrogen-response elements (EREs) positioned upstream of the native TATA box. These constructs were each integrated at the TRP1 locus of a yeast strain in which the natural URA3 gene had been deleted, and the integrants were transformed with low- or high-copy-number shuttle plasmids expressing wild-type or truncated derivatives of hER. Transformants were assayed for growth on uracil-deficient medium plus or minus estradiol (E2), for resistance to 5-fluoroorotic acid (5-FOA) and for activity of OMPdecase (orotidine-5'-monophosphate decarboxylase), the product of the URA3 gene. We show that the growth and 5-FOA-resistance (5-FOAR) phenotypes of these strains are strictly dependent upon the function of the receptor derivatives. Induction of URA3, measured by OMPdecase activity, was observed over a 20- to 2500-fold range depending on the receptor derivative, its expression level and the number of EREs in the responsive promoter. Both one- and three-ERE reporter strains expressing the full-length receptor are completely E2-dependent for growth, and display a 5-FOAR phenotype in the absence of the hormone. We demonstrate that the individual hER transactivation functions, TAF1 and TAF2, are both functional in yeast, and that the hormone-dependent TAF2 is the more potent activator on our reporters. We show that hER displays strong homosynergism in yeast, and discuss the contributions of the two TAFs in hER synergism.(ABSTRACT TRUNCATED AT 250 WORDS)

Blotting, Western↗