Biomedical subjects

J Lechner

Publications and source records attributed to J Lechner.

At least 19 recordsLinked to original sources

PubMed2001

Recruitment to Golgi membranes of ADP-ribosylation factor 1 is mediated by the cytoplasmic domain of p23.

Binding to Golgi membranes of ADP ribosylation factor 1 (ARF1) is the first event in the initiation of COPI coat assembly. Based on binding studies, a proteinaceous receptor has been proposed to be critical for this process. We now report that p23, a member of the p24 family of Golgi-resident transmembrane proteins, is involved in ARF1 binding to membranes. Using a cross-link approach based on a photolabile peptide corresponding to the cytoplasmic domain of p23, the GDP form of ARF1 (ARF1-GDP) is shown to interact with p23 whereas ARF1-GTP has no detectable affinity to p23. The p23 binding is shown to localize specifically to a 22 amino acid C-terminal fragment of ARF1. While a monomeric form of a non-photolabile p23 peptide does not significantly inhibit formation of the cross-link product, the corresponding dimeric form does compete efficiently for this interaction. Consistently, the dimeric p23 peptide strongly inhibits ARF1 binding to native Golgi membranes suggesting that an oligomeric form of p23 acts as a receptor for ARF1 before nucleotide exchange takes place.

ADP-Ribosylation Factor 1↗

PubMed2001

The budding yeast proteins Spc24p and Spc25p interact with Ndc80p and Nuf2p at the kinetochore and are important for kinetochore clustering and checkpoint control.

Here, we show that the budding yeast proteins Ndc80p, Nuf2p, Spc24p and Spc25p interact at the kinetochore. Consistently, Ndc80p, Nuf2p, Spc24p and Spc25p associate with centromere DNA in chromatin immunoprecipitation experiments, and SPC24 interacts genetically with MCM21 encoding a kinetochore component. Moreover, although conditional lethal spc24-2 and spc25-7 cells form a mitotic spindle, the kinetochores remain in the mother cell body and fail to segregate the chromosomes. Despite this defect in chromosome segregation, spc24-2 and spc25-7 cells do not arrest in metaphase in response to checkpoint control. Furthermore, spc24-2 cells showed a mitotic checkpoint defect when microtubules were depolymerized with nocodazole, indicating that Spc24p has a function in checkpoint control. Since Ndc80p, Nuf2p and Spc24p are conserved proteins, it is likely that similar complexes are part of the kinetochore in other organisms.

Amino Acid Sequence↗

PubMed2001

Identification of a 60S preribosomal particle that is closely linked to nuclear export.

A nuclear GTPase, Nug1p, was identified in a genetic screen for components linked to 60S ribosomal subunit export. Nug1p cosedimented with nuclear 60S preribosomes and was required for subunit export to the cytoplasm. Tagged Nug1p coprecipitated with proteins of the 60S subunit, late precursors to the 25S and 5.8S rRNAs, and at least 21 nonribosomal proteins. These included a homologous nuclear GTPase, Nug2p, the Noc2p/Noc3p heterodimer, Rix1p, and Rlp7p, each of which was implicated in 60S subunit export. Other known ribosome synthesis factors and proteins of previously unknown function, including the 559 kDa protein Ylr106p, also copurified. Eight of these proteins were copurified with nuclear pore complexes, suggesting that this complex represents the transport intermediate for 60S subunit export.

Active Transport, Cell Nucleus↗

PubMed2001

Expression level-dependent contribution of glucocorticoid receptor domains for functional interaction with STAT5.

The action of the glucocorticoid receptor (GR) on beta-casein gene transcription serves as a well-studied example of a case where the action of the GR is dependent on the activity of another transcription factor, STAT5. We have investigated the domain-requirement of the GR for this synergistic response in transfection experiments employing GR mutants and CV-1 or COS-7 cells. The results were influenced by the expression levels of the GR constructs. At low expression, STAT5-dependent transactivation by mutants of the GR DNA binding domain or N-terminal transactivation domain was impaired and the antiglucocorticoid RU486 exhibited a weak agonistic activity. When the N-terminal region of the GR was exchanged with the respective domain of the progesterone receptor, STAT5-dependent transactivation was reduced at low and high expression levels. Only at high expression levels did the GR exhibit the properties of a coactivator and enhanced STAT5 activity in the absence of a functional DNA binding domain and of GR binding sites in the proximal region of the beta-casein gene promoter. Furthermore, at high GR expression levels RU486 was nearly as efficient as dexamethasone in activating transcription via the STAT5 dependent beta-casein gene promoter. The results reconcile the controversial issue regarding the DNA binding-independent action of the GR together with STAT5 and provide evidence that the mode of action of the GR depends not only on the type of the particular promoter at which it acts but also on the concentration of the GR. GR DNA binding function appears to be mandatory for beta-casein gene expression in mammary epithelial cells, since the promoter function is completely dependent on the integrity of GR binding sites in the promoter.

Amino Acid Sequence↗

PubMed2000

Interaction of yeast kinetochore proteins with centromere-protein/transcription factor Cbf1.

The centromere-kinetochore complex of Saccharomyces cerevisiae is a specialized chromosomal substructure that mediates attachment of duplicated chromosomes to the mitotic spindle by a regulated network of protein-DNA and protein-protein interactions. We have used in vitro assays to analyze putative molecular interactions between components of the yeast centromerekinetochore complex. Glutathione S-transferase pull-down experiments showed the direct interaction of in vitro translated p110, p64, and p58 of the essential CBF3 kinetochore protein complex with Cbf1p, a basic region helix-loop-helix zipper protein (bHLHzip) that specifically binds to the CDEI region on the centromere DNA. Furthermore, recombinant p64 and p23 each stimulated the in vitro DNA binding activity of Cbf1p. The N-terminal 70 amino acids of p23 were sufficient to mediate this effect. P64 could also promote the multimerization activity of Cbf1p in the presence of centromere DNA in vitro. These results show the direct physical interaction of Cbf1p and CBF3 subunits and provide evidence that CBF3 components can promote the binding of Cbf1p to its binding site in the yeast kinetochore. A functional comparison of the centromere binding proteins with transcription factors binding at MET16 promoters reveals the strong analogy between centromeres and the MET16 promoter.

Basic Helix-Loop-Helix Leucine Zipper Transcriptio↗

PubMed2000

Differential expression of cell-cell adhesion proteins and cyclin D in MEK1-transdifferentiated MDCK cells.

Overexpression of a constitutively active mutant of the mitogen-activated protein kinase kinase MEK1 (caMEK1) in epithelial Madin-Darby canine kidney (MDCK)-C7 cells disrupts morphogenesis, induces an invasive phenotype, and is associated with a reduced rate of cell proliferation. The role of cell-cell adhesion molecules and cell cycle proteins in these processes, however, has not been investigated. We now report loss of E-cadherin expression as well as a marked reduction of beta- and alpha-catenin expression in transdifferentiated MDCK-C7 cells stably expressing caMEK1 (C7caMEK1) compared with epithelial mock-transfected MDCK-C7 (C7Mock1) cells. At least part of the remaining alpha-catenin was coimmunoprecipitated with beta-catenin, whereas no E-cadherin was detected in beta-catenin immunoprecipitates. In both cell types, the proteasome-specific protease inhibitors N-acetyl-Leu-Leu-norleucinal (ALLN) and lactacystin led to a time-dependent accumulation of beta-catenin, including the appearance of high-molecular-weight beta-catenin species. Quiescent as well as serum-stimulated C7caMEK1 cells showed a higher cyclin D expression than epithelial C7Mock1 cells. The MEK inhibitor U-0126 inhibited extracellular signal-regulated kinase phosphorylation and cyclin D expression in C7caMEK1 cells and almost abolished their already reduced cell proliferation rate. We conclude that the transdifferentiated and invasive phenotype of C7caMEK1 cells is associated with a diminished expression of proteins involved in cell-cell adhesion. Although beta-catenin expression is reduced, C7caMEK1 cells show a higher expression of U-0126-sensitive cyclin D protein.

Animals↗

PubMed1999

Cloning of a centromere binding factor 3d (CBF3D) gene from Candida glabrata.

The gene encoding centromere binding factor 3d (CBF3D) of the human pathogenic yeast Candida glabrata has been isolated by hybridization of Saccharomyces cerevisiae CBF3D (ScCBF3D) DNA to a C. glabrata partial genomic library. Sequence analysis revealed a 540 bp open reading frame encoding a protein of 179 amino acids with a calculated molecular mass of 20.9 kDa. The amino acid sequence is highly homologous (78.6% identity) to ScCbf3d and 48.3% identical to the human homologue p19 (SKP1). Southern blot analysis indicates that CgCbf3d is encoded by an unique nuclear gene. The cloned CgCBF3D gene can functionally substitute the S. cerevisiae homologue in a S. cerevisiae CBF3D-deletion mutant. The GenBank Accession No. for this gene is AF 072472.

Amino Acid Sequence↗

PubMed1999

A putative protein complex consisting of Ctf19, Mcm21, and Okp1 represents a missing link in the budding yeast kinetochore.

We have established a one-hybrid screen that allows the in vivo localization of proteins at a functional Saccharomyces cerevisiae centromere. Applying this screen we have identified three proteins-Ctf19, Mcm21, and the product of an unspecified open reading frame that we named Okp1-as components of the budding yeast centromere. Ctf19, Mcm21, and Okp1 most likely form a protein complex that links CBF3, a protein complex directly associated with the CDE III element of the centromere DNA, with further components of the budding yeast centromere, Cbf1, Mif2, and Cse4. We demonstrate that the CDE III element is essential and sufficient to localize the established protein network to the centromere and propose that the interaction of the CDE II element with the CDE III localized protein complex facilitates a protein-DNA conformation that evokes the active centromere.

Amino Acid Sequence↗

PubMed1999

Probing the Saccharomyces cerevisiae centromeric DNA (CEN DNA)-binding factor 3 (CBF3) kinetochore complex by using atomic force microscopy.

Yeast centromeric DNA (CEN DNA) binding factor 3 (CBF3) is a multisubunit protein complex that binds to the essential CDEIII element in CEN DNA. The four CBF3 proteins are required for accurate chromosome segregation and are considered to be core components of the yeast kinetochore. We have examined the structure of the CBF3-CEN DNA complex by atomic force microscopy. Assembly of CBF3-CEN DNA complexes was performed by combining purified CBF3 proteins with a DNA fragment that includes the CEN region from yeast chromosome III. Atomic force microscopy images showed DNA molecules with attached globular bodies. The contour length of the DNA containing the complex is approximately 9% shorter than the DNA alone, suggesting some winding of DNA within the complex. The measured location of the single binding site indicates that the complex is located asymmetrically to the right of CDEIII extending away from CDEI and CDEII, which is consistent with previous data. The CEN DNA is bent approximately 55 degrees at the site of complex formation. A significant fraction of the complexes are linked in pairs, showing three to four DNA arms, with molecular volumes approximately three times the mean volumes of two-armed complexes. These multi-armed complexes indicate that CBF3 can bind two DNA molecules together in vitro and, thus, may be involved in holding together chromatid pairs during mitosis.

Centromere↗

PubMed1999

Effects of interferon alpha-2b on barrier function and junctional complexes of renal proximal tubular LLC-PK1 cells.

BACKGROUND: Interferon alpha-2b (IFNalpha) treatment of diseases can be accompanied by impaired renal function and capillary leak syndrome. To explore potential mechanisms of IFNalpha-induced renal dysfunction, an in vitro cell culture model system was established to investigate the effects of IFNalpha on barrier function and junctional complexes. METHODS: LLC-PK1 cells were cultured on microporous membranes. Transepithelial resistance (TER) was measured, and the dose- and time-dependent effects of IFNalpha were assessed. The expression patterns of junctional proteins were examined by Western blot analysis and by confocal immunofluorescence microscopy. RESULTS: IFNalpha produced a dose- and time-dependent decrease in TER. The effect was reversible on removal of IFNalpha at doses up to 5 x 103 U/ml. Tyrphostin, an inhibitor of phosphotyrosine kinases, ameliorated the IFNalpha-induced decrease in TER. Increased expression of occludin and E-cadherin was detected by Western blot analysis after IFNalpha treatment. Immunofluorescence confocal microscopy revealed a broader staining of occludin and E-cadherin following IFNalpha treatment, with prominent staining at the basal cell pole in addition to localization at the junctional region. A marked increase in phosphotyrosine staining along the apico-lateral cell border was detected after IFNalpha treatment. CONCLUSIONS: These findings provide evidence that IFNalpha can directly affect barrier function in renal epithelial cells. The mechanisms involve enhanced tyrosine phosphorylation and overexpression and possibly displacement or missorting of the junctional proteins occludin and E-cadherin.

Animals↗

PubMed1998

Selective coupling of STAT factors to the mouse prolactin receptor.

Prolactin has been reported to induce distinct sets of signal transducers and activators of transcription (STAT) in a cell-type-specific fashion. In the mammary epithelium, although STAT1, STAT3, STAT5A, STAT5B and STAT6 are present in a latent form, only STAT5A and STAT5B are activated. This selective activation of STAT5 by prolactin was also observed in COS-7 cells cotransfected with the long form of the mouse prolactin receptor (PRL-R) and expression vectors for STAT1, STAT3, STAT5 and STAT6. Mutated PRL-Rs and chimeric erythropoietin/gp130 (EPO/gp130) receptors with a tyrosine-containing motif attached at the carboxy terminus were employed to determine the sites in the PRL-R required for the specific activation of STAT5. The experiments revealed the importance of two motifs containing Y477 and Y578 in the PRL-R. When linked to the EPO/gp130 receptor, these sequences were sufficient to specifically induce DNA binding of STAT5 and to activate transcription from the beta-casein gene promoter. By contrast, only weakly they induced DNA binding of STAT6 and STAT3 and did not induce STAT1. A synthetic nonapeptide with phosphorylated Y477 was able to disrupt STAT5 DNA binding in vitro. Our results define structural domains within the carboxy terminus of the PRL-R which recruit STAT5 for signalling and which are capable of distinguishing STAT5 from other STAT proteins. The activity of STAT5 forms with deletions of the carboxy terminus was induced more strongly than that of their full-length counterparts 2 min after activation of the PRL-R. This effect was not dependent on the presence of Y477 and Y578 in the PRL-R, indicating that facilitated activation of short STAT5 isoforms relies on mechanisms other than increased coupling to specific regions of the PRL-R.

Animals↗

PubMed1998

A distal enhancer region in the human beta-casein gene mediates the response to prolactin and glucocorticoid hormones.

The 5' flanking region of the human beta-casein gene was investigated for the presence of regulatory sequences mediating the action of the lactogenic hormones prolactin and dexamethasone. DNA encompassing 9389 base pairs of the flanking region was isolated and a sequence comparison performed with regulatory regions previously identified in the beta-casein gene of rodents and ruminants. The analysis revealed the presence of a distal region between -4700 and -4550 with a high percentage of identity to the bovine beta-casein enhancer region, and a proximal region between -1 and -200 similar to the proximal promoter regions found in rodents and ruminants. Reporter gene constructs under the control of the distal or the proximal region of the human beta-casein gene were tested for their responsiveness to prolactin and dexamethasone. In transfection experiments, the distal region functioned as a lactogenic hormone inducible enhancer, whereas the proximal region exhibited low activity. In electromobility shift assays, multiple binding sites for Stat5, CCAAT/enhancer-binding proteins, and Ets domain proteins were identified in the distal human enhancer. These transcription factors have already been demonstrated as important regulators of the transcription of milk protein genes in rodents. Thus, a common set of transcription factors appears to be required for the expression of the human beta-casein gene and of milk protein genes in other species.

Animals↗

PubMed1998

Toxicity and bioaccumulation of thallium in Hyalella azteca, with comparison to other metals and prediction of environmental impact.

Thallium (Tl) is an extremely toxic but little studied metal. For Hyalella azteca exposed in Lake Ontario water, a 25% reduction in survival (the LC25) occurred at about 48 nmol litre(-1) after 4 weeks. Body concentrations of Tl, which were proportional to water concentrations, averaged 290 nmol g(-1) dry mass at the LC25. Growth was reduced at slightly lower concentrations. Concentrations affecting reproduction were variable at < 50% of the LC25. On a water-concentration basis Tl was more toxic than Ni, Cu or Zn, but less toxic than Cd or Hg to Hyalella; toxicity to Pb was similar. On a body-concentration basis, the toxicities of Tl, Cd, Hg and Pb were all similar. Unlike Cd, Tl toxicity and uptake was affected by K concentrations in the water, and not by Ca, Mg, Na or other ions. Toxicity was proportional to uptake, and body concentrations were better predictors of toxicity than water concentrations in media with varying K concentrations. Preliminary measurements of Tl and Cd uptake by Hyalella from Hamilton Harbour and Lake Ontario sediments suggested that total bioavailable metal concentrations were greater in deep-water sediments from Lake Ontario than in sediments from the harbour. The ratio of bioavailable metal to the toxic threshold was slightly higher for Cd than for Tl, but well below 1 for both metals.

Journal Article↗

PubMed1997

Promoter-dependent synergy between glucocorticoid receptor and Stat5 in the activation of beta-casein gene transcription.

Steroid hormone receptors and Stat factors comprise two distinct families of inducible transcription factors. Activation of a member of each family, namely the glucocorticoid receptor by glucocorticoids and Stat5 by prolactin, is required for the efficient induction of the expression of milk protein genes in the mammary epithelium. We have studied the mode of interaction between Stat5 and the glucocorticoid receptor in the activation of beta-casein gene transcription. The functional role of potential half-palindromic glucocorticoid receptor-binding sites mapped previously in the promoter region was investigated. beta-Casein gene promoter chloramphenicol acetyltransferase constructs containing mutations and deletions in these sites were tested for their responsiveness to the synergistic effect of prolactin and dexamethasone employing COS-7 cells or HC11 mammary epithelial cells. Synergism depended on promoter regions containing intact binding sites for the glucocorticoid receptor and Stat5. The carboxyl-terminal transactivation domains of Stat5a and Stat5b were not required for this synergism. Our results suggest that in lactogenic hormone response elements glucocorticoid receptor molecules bound to nonclassical half-palindromic sites gain competence as transcriptional activators by the interaction with Stat5 molecules binding to vicinal sites.

Animals↗

PubMed1997

Granulocyte-macrophage colony-stimulating factor induces a unique set of STAT factors in murine dendritic cells.

Cytokine-mediated signaling pathways were studied in mouse dendritic cells (DC) by analysis of the activation pattern of STAT factors. Electrophoretic mobility shift assays were performed to detect STAT isoform-specific complexes. Granulocyte-macrophage colony-stimulating factor (GM-CSF) simultaneously induced complexes containing STAT1, STAT3, STAT5A, STAT5B and STAT6. In non-DC, a similar broad activation pattern of STAT factors by GM-CSF or other cytokines has not been observed so far. By comparison, in peritoneal macrophages, GM-CSF induced a complex with the properties of a truncated form of STAT5. Other cytokines tested on DC either failed to induce STAT factors [interleukin (IL)-1 beta, IL-2, IL-15], or activated the same STAT factors as observed in peritoneal macrophages (IL-4, IFN-gamma). Our results implicate a specific effect of GM-CSF on STAT signaling in DC which might account for the cell type-specific effect of this cytokine on development and function.

Animals↗

PubMed1997

Mechanism of interaction between the glucocorticoid receptor and Stat5: role of DNA-binding.

The functional interaction between the glucocorticoid receptor (GR) and the signal transducer and activator of transcription-5 (Stat5) was investigated by studying the synergistic activation of beta-cascin gene transcription by prolactin and glucocorticoids. The synergism was shown to be mediated by a complex hormone response region with multiple binding sites for Stat5, the glucocorticoid receptor, and CCAAT/enhancer binding proteins (C/EBP). HC11 mammary epithelial cells, which contain physiological levels of GR and Stat5, and COS-7 cells overexpressing GR and Stat5 were employed. In both cell types intact binding sites for Stat5 and the GR were a prerequisite for the synergism, whereas C/EBP sites were only required in HC11 cells. Interestingly, the GR sites employed for the synergism were nonclassical, half palindromic sites, which did not function in the absence of activated Stat5 to mediate the action of the GR on transcription. The interaction of GR and Stat5 triggered by the unusual configuration of binding sites appears to represent a novel mechanism by which these two distinct types of transcription factors cooperate. The mode of interaction provides an efficient means to restrict gene expression to conditions where both Stat5 and the GR are activated.

Animals↗

PubMed1996

The Saccharomyces cerevisiae kinetochore contains a cyclin-CDK complexing homologue, as identified by in vitro reconstitution.

We have developed methods to reconstitute the centromere DNA (CEN)-bound Saccharomyces cerevisiae kinetochore complex, CBF3, from isolated CBF3 components in vitro. This revealed that cooperation of at least three CBF3 components is imperatively required to form an activity that specifically binds to the centromere DNA in vitro. Two of the CBF3 proteins, Cbf3a and Cbf3b, that were used in the reconstitution were obtained from heterologous systems. In contrast, Cbf3c, the third CBF3 component known, had to be purified from S. cerevisiae to obtain a Cbf3c preparation that was competent to reconstitute the CBF3-CEN complex in combination with Cbf3a and Cbf3b. This led to the identification of a 29 kDa protein that co-purified with Cbf3c. The 29 kDa protein was shown to be a fourth component of CBF3 and therefore was named Cbf3d. Analysing the Cbf3d gene revealed that Cbf3d exhibits strong homology to p19SKP1, a human protein that is part of active cyclin A-CDK2 complexes. Therefore, Cbf3d is the only CBF3 protein that has a known homologue in higher eukaryotes and may provide the anchor that directs cell cycle-regulated proteins to the kinetochore.

Amino Acid Sequence↗

PubMed1996

The Saccharomyces cerevisiae kinetochore.

Accurate chromosome segregation is dependent on a specialized chromosomal structure, the kinetochore/centromere. The only essential constituent of the S. cerevisiae kinetochore established today is CBF3, a multisubunit complex that binds to S. cerevisiae centromere DNA. Therefore CBF3 and its four components, Cbf3a, Cbf3b, Cbf3c and Cbf3d, will form the centerpiece of this review. In addition, we will describe proteins that are putatively involved in kinetochore function specifically in the context with CBF3 interaction. Furthermore, we discuss the role of the S. cerevisiae kinetochores in a putative cell cycle checkpoint control and in microtubule attachment.

Base Sequence↗