21 results on '"Tavernier, J"'
Search Results
2. Dimerization of the interferon type I receptor IFNaR2-2 is sufficient for induction of interferon effector genes but not for full antiviral activity.
- Author
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Pattyn, E, Van Ostade, X, Schauvliege, L, Verhee, A, Kalai, M, Vandekerckhove, J, and Tavernier, J
- Abstract
We constructed chimeric receptors wherein the extracellular domain of the erythropoietin receptor (EpoR) was fused to the transmembrane and intracellular domains of the interferon (IFN) type I receptor subunits, IFNaR1 or IFNaR2-2. Transfection into 2fTGH and Tyk2-deficient 11,1 cells showed that EpoR/IFNaR2-2 alone was able to transduce a signal upon stimulation with erythropoietin (Epo), as judged by induction of the interferon type I-inducible 6-16 promoter. In contrast, protection against infection with encephalomyocarditis virus or vesicular stomatitis virus was reduced or absent, respectively. To further investigate the role of IFNaR1 in the induction of an antiviral state, we analyzed the Epo- versus IFNalpha-induced transcription of a set of genes, involved in antiviral protection. Up to 24 h after stimulation with Epo or IFNalpha, comparable transcription of the p56, dsRNA-dependent protein kinase, 2'-5'A synthetase, and MxA genes was seen. However, at later time points, only in the case of Epo induction, a sharp decrease of mRNA levels was observed. Western blotting analysis of dsRNA-dependent protein kinase showed a similar pattern at the protein level. Taken together, our results imply a role for IFNaR1 in the induction of sustained mRNA and protein levels that are likely required for optimal antiviral activity.
- Published
- 1999
3. A single STAT recruitment module in a chimeric cytokine receptor complex is sufficient for STAT activation.
- Author
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Behrmann, I, Janzen, C, Gerhartz, C, Schmitz-Van de Leur, H, Hermanns, H, Heesel, B, Graeve, L, Horn, F, Tavernier, J, and Heinrich, P C
- Abstract
We established a system of receptor chimeras that enabled us to induce heterodimerization of different cytoplasmic tails. Fusion constructs were created that are composed of the extracellular parts of the interleukin-5 receptor alpha and beta chains, respectively, and the transmembrane and intracellular parts of gp130, the signal transducing chain of the interleukin-6 receptor complex. In COS-7 transfectants we observed a dose-dependent interleukin-5-inducible STAT1 activation for which the presence of both the alpha and the beta chain chimera was needed. No STAT activity was detected if one of the cytoplasmic tails of the receptor complex was deleted, indicating that STAT activity resulted from a receptor dimer rather than from higher receptor aggregates. We further investigated whether dimerization of STAT1 depends on the juxtaposition of two STAT recruitment modules in a receptor complex. We show that a receptor dimer with only a single STAT1 docking site was still able to lead to STAT1 activation. This indicates that the formation of a paired set of STAT binding sites in a receptor complex is not the prerequisite for STAT factor dimerization. Our findings are discussed in view of alternative STAT dimerization models.
- Published
- 1997
4. Identification and characterization of a functional promoter region in the human eosinophil IL-5 receptor alpha subunit gene.
- Author
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Sun, Z, Yergeau, D A, Tuypens, T, Tavernier, J, Paul, C C, Baumann, M A, Tenen, D G, and Ackerman, S J
- Abstract
The molecular basis for the commitment of multipotential myeloid progenitors to the eosinophil lineage, and the transcriptional mechanisms by which eosinophil-specific genes are subsequently expressed and regulated during eosinophil development are currently unknown. Interleukin-5 (IL-5) is a T cell and mast cell-derived cytokine with actions restricted to the eosinophil and closely related basophil lineages in humans. The high affinity receptor for IL-5 (IL-5R) is composed of an alpha subunit (IL-5R alpha) expressed by the eosinophil lineage, that associates with a beta c subunit shared with the receptors for IL-3 and granulocyte-macrophage colony stimulating factor (GM-CSF). As a prerequisite to studies of the transcriptional regulation of the IL-5R alpha subunit gene, we used three different methods, including primer extension, RNase protection, and 5'-RACE to precisely map the transcriptional start site to a position 15 base pairs (bp) upstream of the 5' end of the published sequence of IL-5R alpha exon 1. To initially identify the IL-5R alpha promoter, 3.5 kilobases (kb) and 561 bp of the 5' sequence flanking the transcriptional start site were subcloned into the promoterless pXP2-luciferase vector. Transient transfection of these constructs into an eosinophil-committed HL-60 subline, clone HL-60-C15, induced the expression of approximately 240-fold greater luciferase activity than the promoterless vector, identifying a strong functionally active promoter region within the 561 bp of sequence proximal to the transcriptional start site and with activity equivalent to pXP2 constructs containing the entire 3.5 kb of upstream sequence. To more precisely localize the cis-acting regulatory elements in this region important for promoter activity, a series of 5' deletion mutants of the 561-bp region were generated in the pXP2-luciferase vector. Deletion of the region between bp -432 and -398 reduced promoter activity by more than 80% in the HL-60-C15 cell line. Further analyses of the activity of the IL-5R alpha promoter constructs in various other eosinophil, myeloid, and non-myeloid cell lines indicated that the promoter was relatively myeloid and eosinophil lineage-specific in its expression. Consensus sequences for known transcription factor binding sites were not present in the 34-bp region of the promoter required for maximal activity, suggesting unique myeloid- and possibly eosinophil-specific regulatory elements.(ABSTRACT TRUNCATED AT 400 WORDS)
- Published
- 1995
5. Multiple effects of tumor necrosis factor on lipoprotein lipase in vivo.
- Author
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Semb, H, Peterson, J, Tavernier, J, and Olivecrona, T
- Abstract
A single dose of recombinant murine tumor necrosis factor (TNF) suppressed lipoprotein lipase activity in adipose tissue of fed rats, mice, and guinea pigs for 48 h, even though TNF itself is rapidly metabolized in vivo. Immunoprecipitation of [35S]lipoprotein lipase from fat pads pulse-labeled with [35S]methionine showed a decrease in relative synthesis of the enzyme, which correlated to the decrease in activity. There was no decrease in general protein synthesis and no change in distribution of the enzyme between adipocytes and extracellular locations in the tissue. This is in contrast to fasting in which case there is redistribution of the enzyme within the tissue, decrease in general protein synthesis, but no change in relative synthesis of lipoprotein lipase. TNF did not decrease lipoprotein lipase activity in any tissue other than the adipose but increased the activity in several cases, most markedly in the liver. No [35S]methionine was incorporated into lipoprotein lipase by liver slices from normal or TNF-treated animals. Thus, the increased activity can not be ascribed to enhanced hepatic synthesis of the enzyme. There was an increase in lipoprotein lipase activity in plasma, which correlated to the increase in liver. Thus, TNF suppresses lipoprotein lipase synthesis in adipocytes, but not in other tissues, and has some as yet undefined effect on lipoprotein lipase turnover in extrahepatic tissues, which results in increased transport of active lipase through plasma to the liver.
- Published
- 1987
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6. Binding of human tumor necrosis factor to high affinity receptors on HeLa and lymphoblastoid cells sensitive to growth inhibition.
- Author
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Baglioni, C, primary, McCandless, S, additional, Tavernier, J, additional, and Fiers, W, additional
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- 1985
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7. Ubiquitin D Regulates IRE1α/c-Jun N-terminal Kinase (JNK) Protein-dependent Apoptosis in Pancreatic Beta Cells.
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Brozzi F, Gerlo S, Grieco FA, Juusola M, Balhuizen A, Lievens S, Gysemans C, Bugliani M, Mathieu C, Marchetti P, Tavernier J, and Eizirik DL
- Subjects
- Aged, Aged, 80 and over, Animals, Blotting, Western, Cell Line, Cell Line, Tumor, Cells, Cultured, Cytokines pharmacology, Endoribonucleases genetics, Female, Gene Expression drug effects, HEK293 Cells, Humans, Insulin-Secreting Cells drug effects, Male, Middle Aged, Protein Binding, Protein Serine-Threonine Kinases genetics, RNA Interference, Rats, Reverse Transcriptase Polymerase Chain Reaction, Ubiquitins genetics, Young Adult, Apoptosis, Endoribonucleases metabolism, Insulin-Secreting Cells metabolism, JNK Mitogen-Activated Protein Kinases metabolism, Protein Serine-Threonine Kinases metabolism, Ubiquitins metabolism
- Abstract
Pro-inflammatory cytokines contribute to pancreatic beta cell apoptosis in type 1 diabetes at least in part by inducing endoplasmic reticulum (ER) stress and the consequent unfolded protein response (UPR). It remains to be determined what causes the transition from "physiological" to "apoptotic" UPR, but accumulating evidence indicates that signaling by the ER transmembrane protein IRE1α is critical for this transition. IRE1α activation is regulated by both intra-ER and cytosolic cues. We evaluated the role for the presently discovered cytokine-induced and IRE1α-interacting protein ubiquitin D (UBD) on the regulation of IRE1α and its downstream targets. UBD was identified by use of a MAPPIT (mammalian protein-protein interaction trap)-based IRE1α interactome screen followed by comparison against functional genomic analysis of human and rodent beta cells exposed to pro-inflammatory cytokines. Knockdown of UBD in human and rodent beta cells and detailed signal transduction studies indicated that UBD modulates cytokine-induced UPR/IRE1α activation and apoptosis. UBD expression is induced by the pro-inflammatory cytokines interleukin (IL)-1β and interferon (IFN)-γ in rat and human pancreatic beta cells, and it is also up-regulated in beta cells of inflamed islets from non-obese diabetic mice. UBD interacts with IRE1α in human and rodent beta cells, modulating IRE1α-dependent activation of JNK and cytokine-induced apoptosis. Our data suggest that UBD provides a negative feedback on cytokine-induced activation of the IRE1α/JNK pro-apoptotic pathway in cytokine-exposed beta cells., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2016
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8. A combined "omics" approach identifies N-Myc interactor as a novel cytokine-induced regulator of IRE1 protein and c-Jun N-terminal kinase in pancreatic beta cells.
- Author
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Brozzi F, Gerlo S, Grieco FA, Nardelli TR, Lievens S, Gysemans C, Marselli L, Marchetti P, Mathieu C, Tavernier J, and Eizirik DL
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- Aged, Animals, Apoptosis physiology, Endoribonucleases genetics, HEK293 Cells, Humans, Insulin-Secreting Cells cytology, Interferon-gamma genetics, Interleukin-1beta genetics, Intracellular Signaling Peptides and Proteins genetics, JNK Mitogen-Activated Protein Kinases genetics, Mice, Middle Aged, Multienzyme Complexes genetics, Protein Serine-Threonine Kinases genetics, Rats, Rats, Wistar, Endoribonucleases metabolism, Insulin-Secreting Cells metabolism, Interferon-gamma metabolism, Interleukin-1beta metabolism, Intracellular Signaling Peptides and Proteins metabolism, JNK Mitogen-Activated Protein Kinases metabolism, Multienzyme Complexes metabolism, Protein Serine-Threonine Kinases metabolism, Unfolded Protein Response physiology
- Abstract
Type 1 diabetes is an autoimmune disease with a strong inflammatory component. The cytokines interleukin-1β and interferon-γ contribute to beta cell apoptosis in type 1 diabetes. These cytokines induce endoplasmic reticulum stress and the unfolded protein response (UPR), contributing to the loss of beta cells. IRE1α, one of the UPR mediators, triggers insulin degradation and inflammation in beta cells and is critical for the transition from "physiological" to "pathological" UPR. The mechanisms regulating inositol-requiring protein 1α (IRE1α) activation and its signaling for beta cell "adaptation," "stress response," or "apoptosis" remain to be clarified. To address these questions, we combined mammalian protein-protein interaction trap-based IRE1α interactome and functional genomic analysis of human and rodent beta cells exposed to pro-inflammatory cytokines to identify novel cytokine-induced regulators of IRE1α. Based on this approach, we identified N-Myc interactor (NMI) as an IRE1α-interacting/modulator protein in rodent and human pancreatic beta cells. An increased expression of NMI was detected in islets from nonobese diabetic mice with insulitis and in rodent or human beta cells exposed in vitro to the pro-inflammatory cytokines interleukin-1β and interferon-γ. Detailed mechanistic studies demonstrated that NMI negatively modulates IRE1α-dependent activation of JNK and apoptosis in rodent and human pancreatic beta cells. In conclusion, by using a combined omics approach, we identified NMI induction as a novel negative feedback mechanism that decreases IRE1α-dependent activation of JNK and apoptosis in cytokine-exposed beta cells
- Published
- 2014
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9. The small GTPase Arf6 is essential for the Tram/Trif pathway in TLR4 signaling.
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Van Acker T, Eyckerman S, Vande Walle L, Gerlo S, Goethals M, Lamkanfi M, Bovijn C, Tavernier J, and Peelman F
- Subjects
- ADP-Ribosylation Factor 6, ADP-Ribosylation Factors genetics, Animals, Cell Line, Endocytosis drug effects, Endocytosis physiology, Humans, Interferon Regulatory Factor-3 genetics, Interferon Regulatory Factor-3 metabolism, Lipopolysaccharides pharmacology, Macrophages cytology, Mice, Mice, Knockout, Myelin and Lymphocyte-Associated Proteolipid Proteins genetics, Myelin and Lymphocyte-Associated Proteolipid Proteins metabolism, Myeloid Differentiation Factor 88 genetics, Myeloid Differentiation Factor 88 metabolism, Receptors, Interleukin genetics, Signal Transduction drug effects, Toll-Like Receptor 4 genetics, Transcription, Genetic drug effects, Transcription, Genetic physiology, ADP-Ribosylation Factors metabolism, Macrophages metabolism, Receptors, Interleukin metabolism, Signal Transduction physiology, Toll-Like Receptor 4 metabolism
- Abstract
Recognition of lipopolysaccharides (LPS) by Toll-like receptor 4 (TLR4) at the plasma membrane triggers NF-κB activation through recruitment of the adaptor proteins Mal and MyD88. Endocytosis of the activated TLR4 allows recruitment of the adaptors Tram and Trif, leading to activation of the transcription factor IRF3 and interferon production. The small GTPase ADP-ribosylation factor 6 (Arf6) was shown to regulate the plasma membrane association of Mal. Here we demonstrate that inhibition of Arf6 also markedly reduced LPS-induced cytokine production in Mal(-/-) mouse macrophages. In this article, we focus on a novel role for Arf6 in the MyD88-independent TLR4 pathway. MyD88-independent IRF3 activation and IRF3-dependent gene transcription were strictly dependent on Arf6. Arf6 was involved in transport of Tram to the endocytic recycling compartment and internalization of LPS, possibly explaining its requirement for LPS-induced IRF3 activation. Together, these results show a critical role for Arf6 in regulating Tram/Trif-dependent TLR4 signaling.
- Published
- 2014
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10. Identification of binding sites for myeloid differentiation primary response gene 88 (MyD88) and Toll-like receptor 4 in MyD88 adapter-like (Mal).
- Author
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Bovijn C, Desmet AS, Uyttendaele I, Van Acker T, Tavernier J, and Peelman F
- Subjects
- Humans, Membrane Glycoproteins chemistry, Membrane Glycoproteins genetics, Membrane Glycoproteins metabolism, Mutation, Peptide Mapping methods, Protein Binding, Receptors, Interleukin-1 chemistry, Receptors, Interleukin-1 genetics, Receptors, Interleukin-1 metabolism, Binding Sites, Models, Biological, Myeloid Differentiation Factor 88 chemistry, Myeloid Differentiation Factor 88 genetics, Myeloid Differentiation Factor 88 metabolism, Protein Multimerization physiology, Toll-Like Receptor 4 chemistry, Toll-Like Receptor 4 genetics, Toll-Like Receptor 4 metabolism
- Abstract
Upon activation, Toll-like receptor 4 (TLR4) binds adapter proteins, including MyD88 (myeloid differentiation primary response gene 88) and Mal (MyD88 adapter-like) for its signal transduction. TLR4 and the adapter proteins each contain a Toll/Il-1 receptor domain (TIR domain). In this study we used random mutagenesis and the mammalian two-hybrid method MAPPIT (mammalian protein-protein interaction trap) to identify mutations in Mal that disrupt its interaction with TLR4 and/or MyD88. Our study shows that four potential binding sites and the AB-loop in the Mal TIR domain all contribute to formation of the TLR4-Mal-MyD88 complex. Mutations in the symmetrical back-to-back Mal homodimer interface affect Mal homodimerization and interaction with MyD88 and TLR4. Our data suggest that Mal dimerization may lead to formation of potential binding platforms on the top and the side of the Mal dimer that bind MyD88 or TLR4. Mutations that affect the interaction of Mal with MyD88 also affect NF-κB activation induced by Mal overexpression. In MAPPIT, co-expression of the MyD88 TIR domain enhances Mal dimerization and Mal binding to TLR4. Similarly, co-expression of Mal and the MyD88 TIR domain strongly promotes dimerization of the TLR4 intracellular domain in MAPPIT. The different types of TIR-TIR interactions in the TLR4-Mal-MyD88 complex thus show cooperative binding in MAPPIT. We present plausible models for the TIR-TIR interactions in the TLR4-Mal-MyD88 complex.
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- 2013
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11. Identification of interaction sites for dimerization and adapter recruitment in Toll/interleukin-1 receptor (TIR) domain of Toll-like receptor 4.
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Bovijn C, Ulrichts P, De Smet AS, Catteeuw D, Beyaert R, Tavernier J, and Peelman F
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- Animals, Humans, Mice, Mutagenesis, Site-Directed, Myeloid Differentiation Factor 88 chemistry, Myeloid Differentiation Factor 88 genetics, Myeloid Differentiation Factor 88 metabolism, Peptide Mapping, Protein Structure, Tertiary, Structural Homology, Protein, Toll-Like Receptor 10 chemistry, Toll-Like Receptor 10 genetics, Toll-Like Receptor 10 metabolism, Toll-Like Receptor 4 genetics, Toll-Like Receptor 4 metabolism, Models, Molecular, Protein Multimerization physiology, Toll-Like Receptor 4 chemistry
- Abstract
Toll-like receptor signaling requires interactions of the Toll/IL-1 receptor (TIR) domains of the receptor and adapter proteins. Using the mammalian protein-protein interaction trap strategy, homology modeling, and site-directed mutagenesis, we identify the interaction surfaces in the TLR4 TIR domain for the TLR4-TLR4, TLR4-MyD88 adapter-like (MAL), and TLR4-TRIF-related adapter molecule (TRAM) interaction. Two binding sites are equally important for TLR4 dimerization and adapter recruitment. In a model based on the crystal structure of the dimeric TLR10 TIR domain, the first binding site mediates TLR4-TLR4 TIR-TIR interaction. Upon dimerization, two identical second binding sites of the TLR4 TIR domain are juxtaposed and form an extended binding platform for both MAL and TRAM. In our mammalian protein-protein interaction trap assay, MAL and TRAM compete for binding to this platform. Our data suggest that adapter binding can stabilize the TLR4 TIR dimerization.
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- 2012
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12. New activation modus of STAT3: a tyrosine-less region of the interleukin-22 receptor recruits STAT3 by interacting with its coiled-coil domain.
- Author
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Dumoutier L, de Meester C, Tavernier J, and Renauld JC
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- Animals, Antineoplastic Agents, Hormonal pharmacology, Binding Sites, Blotting, Western, COS Cells, Cell Line, Cell Line, Tumor, Cell Proliferation drug effects, Chlorocebus aethiops, Dexamethasone pharmacology, Dose-Response Relationship, Drug, Flow Cytometry, Humans, Immunoprecipitation, Interleukin-9 pharmacology, Interleukins pharmacology, Mutation, Protein Binding drug effects, Receptors, Interleukin genetics, STAT3 Transcription Factor genetics, Transfection, Tyrosine genetics, Interleukin-22, Receptors, Interleukin metabolism, STAT3 Transcription Factor metabolism, Tyrosine metabolism
- Abstract
Activation of STAT proteins by cytokines is initiated by their Src homology 2 domain-mediated association with phosphotyrosine residues from the cytoplasmic domain of a receptor. Here, we show that the C terminus of the interleukin-22 receptor (IL-22R) recruits in a tyrosine-independent manner the coiled-coil domain of STAT3. Mutation of all IL-22R cytoplasmic tyrosines did not abolish activation of STAT3, in contrast to that of STAT1 and STAT5. Coimmunoprecipitation and glutathione S-transferase pulldown experiments showed that the coiled-coil domain of STAT3 is constitutively associated with the C-terminal part of IL-22R, and a chimeric STAT3-STAT5 protein containing the coiled-coil domain of STAT3 could be activated by this tyrosine-independent mechanism. Deletion of the C-terminal part of IL-22R dramatically decreased its ability to activate STAT3 and to mediate IL-22 activity in cell lines, demonstrating that preassociation of STAT3 with this cytokine receptor, independent from the interaction between the Src homology 2 domain and phosphotyrosines, is required for its full activity.
- Published
- 2009
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13. Elongin B/C recruitment regulates substrate binding by CIS.
- Author
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Piessevaux J, De Ceuninck L, Catteeuw D, Peelman F, and Tavernier J
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- Amino Acid Sequence, Animals, Elongin, Humans, Mice, Mice, Knockout, Molecular Sequence Data, Myeloid Differentiation Factor 88 metabolism, Proteasome Endopeptidase Complex metabolism, Protein Processing, Post-Translational, Protein Structure, Tertiary, Sequence Homology, Amino Acid, Suppressor of Cytokine Signaling 1 Protein, Suppressor of Cytokine Signaling Proteins chemistry, Suppressor of Cytokine Signaling Proteins metabolism, Suppressor of Cytokine Signaling Proteins physiology, Transcription Factors chemistry
- Abstract
SOCS proteins play a major role in the regulation of cytokine signaling. They are recruited to activated receptors and can suppress signaling by different mechanisms including targeting of the receptor complex for proteasomal degradation. The activity of SOCS proteins is regulated at different levels including transcriptional control and posttranslational modification. We describe here a novel regulatory mechanism for CIS, one of the members of this protein family. A CIS mutant deficient in recruitment of the Elongin B/C complex completely failed to suppress STAT5 activation. This deficiency was not caused by altered turnover of CIS but by loss of cytokine receptor interaction. Intriguingly, no such effect was seen for binding to MyD88. The interaction between CIS and the Elongin B/C complex, which depends on the levels of uncomplexed Elongin B/C, was easily disrupted. This regulatory mechanism may be unique for CIS, as similar mutations in SOCS1, -2, -3, -6, and -7 had no functional impact. Our findings indicate that the SOCS box not only plays a role in the formation of E3 ligase complexes but, at least for CIS, can also regulate the binding modus of SOCS box-containing proteins.
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- 2008
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14. Functional cross-modulation between SOCS proteins can stimulate cytokine signaling.
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Piessevaux J, Lavens D, Montoye T, Wauman J, Catteeuw D, Vandekerckhove J, Belsham D, Peelman F, and Tavernier J
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- Animals, Cell Line, Elongin, Humans, Mice, Protein Binding, Suppressor of Cytokine Signaling Proteins antagonists & inhibitors, Suppressor of Cytokine Signaling Proteins genetics, Transcription Factors metabolism, Cytokines metabolism, Signal Transduction, Suppressor of Cytokine Signaling Proteins metabolism
- Abstract
SOCS (suppressors of cytokine signaling) proteins are negative regulators of cytokine signaling that function primarily at the receptor level. Remarkably, in vitro and in vivo observations revealed both inhibitory and stimulatory effects of SOCS2 on growth hormone signaling, suggesting an additional regulatory level. In this study, we examined the possibility of direct cross-modulation between SOCS proteins and found that SOCS2 could interfere with the inhibitory actions of other SOCS proteins in growth hormone, interferon, and leptin signaling. This SOCS2 effect was SOCS box-dependent, required recruitment of the elongin BC complex, and coincided with degradation of target SOCS proteins. Detailed mammalian protein-protein interaction trap (MAPPIT) analysis indicated that SOCS2 can interact with all members of the SOCS family. SOCS2 may thus function as a molecular bridge between a ubiquitin-protein isopeptide ligase complex and SOCS proteins, targeting them for proteasomal turnover. We furthermore extended these observations to SOCS6 and SOCS7. Our findings point to a unique regulatory role for SOCS2, SOCS6, and SOCS7 within the SOCS family and provide an explanation for the unexpected phenotypes observed in SOCS2 and SOCS6 transgenic mice.
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- 2006
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15. Mapping of binding site III in the leptin receptor and modeling of a hexameric leptin.leptin receptor complex.
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Peelman F, Iserentant H, De Smet AS, Vandekerckhove J, Zabeau L, and Tavernier J
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- Amino Acid Sequence, Animals, Binding Sites genetics, Cell Line, Conserved Sequence, Humans, In Vitro Techniques, Leptin chemistry, Mice, Models, Molecular, Molecular Sequence Data, Multiprotein Complexes, Mutagenesis, Peptide Mapping, Protein Structure, Quaternary, Protein Structure, Secondary, Protein Structure, Tertiary, Receptors, Cell Surface genetics, Receptors, Leptin, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Sequence Deletion, Sequence Homology, Amino Acid, Transfection, Leptin metabolism, Receptors, Cell Surface chemistry, Receptors, Cell Surface metabolism
- Abstract
The leptin.leptin receptor (LR) system shows strong similarities to the long chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) cytokine.cytokine receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites (I-III). We demonstrated previously that leptin has similar binding sites I-III and mapped the interactions between binding site II and cytokine receptor homology domain II (CRH2) (Peelman, F., Van Beneden, K., Zabeau, L., Iserentant, H., Ulrichts, P., Defeau, D., Verhee, A., Catteeuw, D., Elewaut, D., and Tavernier, J. (2004) J. Biol. Chem. 279, 41038-41046). In this study, we built homology models for the CRH1 and Ig-like domains of the LR. The Ig-like domain shows a large conserved surface patch in the beta-sheet formed by beta-strands 3, 6, and 7. Mutations in this patch almost completely abolished the leptin-induced STAT3-dependent reporter activity. We propose that a conserved cluster of residues Leu370, Ala407, Tyr409, His417, and His418 forms the center of binding site III of the LR. We built a hexameric leptin.LR complex model based on the hexameric IL-6 complex. In this model, a conserved hydrophobic protuberance of Val36, Thr37, Phe41, and Phe43 in the A-B loop of leptin fits perfectly in the CRH2 domain, corresponding to the IL-6 alpha-receptor, and forms the center of binding site I. The 2:4 hexameric leptin.LR complex offers a rational explanation for mutagenesis studies and residue conservation.
- Published
- 2006
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16. Leptin receptor activation depends on critical cysteine residues in its fibronectin type III subdomains.
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Zabeau L, Defeau D, Iserentant H, Vandekerckhove J, Peelman F, and Tavernier J
- Subjects
- Animals, Blotting, Western, COS Cells, Cell Line, Cell Membrane metabolism, Cluster Analysis, Cytokines chemistry, DNA-Binding Proteins chemistry, Dimerization, Disulfides chemistry, Dose-Response Relationship, Drug, Fibronectins metabolism, Gene Deletion, Genes, Reporter, Genetic Vectors, Humans, Immunoprecipitation, Leptin chemistry, Ligands, Mice, Mutation, Phosphorylation, Plasmids metabolism, Protein Binding, Protein Structure, Tertiary, Receptors, Leptin, STAT3 Transcription Factor, Signal Transduction, Trans-Activators chemistry, Transfection, Cysteine chemistry, Fibronectins chemistry, Receptors, Cell Surface metabolism
- Abstract
The leptin receptor (LR) complex is composed of a single subunit belonging to the class I cytokine receptor family and exists as a preformed complex. The extracellular portion contains two cytokine receptor homology (CRH) domains, separated by an Ig-like domain and followed by two membrane-proximal fibronectin type III (FNIII) domains. The mechanisms underlying ligand-induced receptor activation are still poorly understood. LRs can exist as disulfide-linked dimers at the cell surface, even in the absence of leptin. We evaluated the role of the two unpaired cysteine residues (Cys-672 and Cys-751) in the FNIII domains in receptor clustering, leptin binding, and biological activity. Although mutation of cysteine on position 751 to serine has hardly any effect on ligand binding and receptor activation, the C672S mutant exhibits a marked reduction in STAT3-dependent signaling. The double mutant was completely devoid of biological activity, although leptin binding remained unaffected. Mutation of both residues resulted in complete loss of disulfide bridge formation of FNIII domains in solution. In contrast, no difference was observed in ligand-independent oligomerization of the membrane-bound receptor, suggesting a role for cysteines in the CRH2 domain in formation of the preformed LR complex. We propose a model wherein leptin-induced clustering of two preformed dimers forms the activated LR complex. Disulfide bridge formation involving Cys-672 and Cys-751 may be necessary for JAK activation and hence signaling.
- Published
- 2005
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17. Mapping of the leptin binding sites and design of a leptin antagonist.
- Author
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Peelman F, Van Beneden K, Zabeau L, Iserentant H, Ulrichts P, Defeau D, Verhee A, Catteeuw D, Elewaut D, and Tavernier J
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Binding, Competitive, COS Cells, Cell Line, Dose-Response Relationship, Drug, Genes, Reporter, Humans, Luciferases metabolism, Mice, Mice, Inbred DBA, Models, Biological, Models, Molecular, Molecular Sequence Data, Mutation, Protein Binding, Protein Structure, Tertiary, Recombinant Proteins chemistry, Sequence Homology, Amino Acid, Signal Transduction, Time Factors, Transfection, Drug Design, Leptin antagonists & inhibitors, Leptin chemistry
- Abstract
The leptin/leptin receptor system shows strong similarities to the long-chain cytokine interleukin-6 (IL-6) and granulocyte colony-stimulating factor cytokine/receptor systems. The IL-6 family cytokines interact with their receptors through three different binding sites I-III. The leptin structure was superposed on the crystal structures of several long-chain cytokines, and a series of leptin mutants was generated focusing on binding sites I-III. The effect of the mutations on leptin receptor (LR) signaling and on binding to the membrane proximal cytokine receptor homology domain (CRH2) of the LR was determined. Mutations in binding site I at the C terminus of helix D show a modest effect on signaling and do not affect binding to CRH2. Binding site II is composed of residues at the surface of helices A and C. Mutations in this site impair binding to CRH2 but have only limited effect on signaling. Site III mutations around the N terminus of helix D impair receptor activation without affecting binding to CRH2. We identified an S120A/T121A mutant in binding site III, which lacks any signaling capacity, but which still binds to CRH2 with wild type affinity. This leptin mutant behaves as a potent leptin antagonist both in vitro and in vivo., (Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2004
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18. Phosphorylation by protein kinase CK2 modulates the activity of the ATP binding cassette A1 transporter.
- Author
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Roosbeek S, Peelman F, Verhee A, Labeur C, Caster H, Lensink MF, Cirulli C, Grooten J, Cochet C, Vandekerckhove J, Amoresano A, Chimini G, Tavernier J, and Rosseneu M
- Subjects
- ATP Binding Cassette Transporter 1, ATP-Binding Cassette Transporters chemistry, ATP-Binding Cassette Transporters genetics, Amino Acid Sequence, Animals, Cell Line, Cholesterol metabolism, Humans, Molecular Sequence Data, Mutation, Phospholipids metabolism, Phosphorylation, Recombinant Proteins metabolism, Sequence Homology, Amino Acid, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, ATP-Binding Cassette Transporters metabolism, Casein Kinase II metabolism
- Abstract
In a previous characterization of the ABCA subfamily of the ATP-binding cassette (ABC) transporters, we identified potential protein kinase 2 (CK2) phosphorylation sites, which are conserved in eukaryotic and prokaryotic members of the ABCA transporters. These phosphorylation residues are located in the conserved cytoplamic R1 and R2 domains, downstream of the nucleotide binding domains NBD1 and NBD2. To study the possible regulation of the ABCA1 transporter by CK2, we expressed the recombinant cytoplasmic domains of ABCA1, NBD1+R1 and NBD2+R2. We demonstrated that in vitro ABCA1 NBD1+R1, and not NBD2+R2, is phosphorylated by CK2, and we identified Thr-1242, Thr-1243, and Ser-1255 as the phosphorylated residues in the R1 domain by mass spectrometry. We further investigated the functional significance of the threonine and serine phosphorylation sites in NBD1 by site-directed mutagenesis of the entire ABCA1 followed by transfection into Hek-293 Tet-Off cells. The ABCA1 flippase activity, apolipoprotein AI and AII binding, and cellular phospholipid and cholesterol efflux were enhanced by mutations preventing CK2 phosphorylation of the threonine and serine residues. This was confirmed by the effect of specific protein kinase CK2 inhibitors upon the activity of wild type and mutant ABCA1 in transfected Hek-293 Tet-Off cells. The activities of the mutants mimicking threonine phosphorylation were close to that of wild type ABCA1. Our data, therefore, suggest that besides protein kinase A and C, protein kinase CK2 might play an important role in vivo in regulating the function and transport activity of ABCA1 and possibly of other members of the ABCA subfamily.
- Published
- 2004
- Full Text
- View/download PDF
19. Down-modulation of type 1 interferon responses by receptor cross-competition for a shared Jak kinase.
- Author
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Dondi E, Pattyn E, Lutfalla G, Van Ostade X, Uzé G, Pellegrini S, and Tavernier J
- Subjects
- Animals, Blotting, Western, Cell Line, Cell Separation, Cell Survival, Dose-Response Relationship, Drug, Enzyme Activation, Flow Cytometry, Humans, Interferon-alpha metabolism, Interferon-beta metabolism, Janus Kinase 1, Kinetics, Ligands, Luciferases metabolism, Membrane Proteins, Phosphorylation, Plasmids metabolism, Precipitin Tests, Protein Binding, Protein Biosynthesis, Protein Structure, Tertiary, Receptor, Interferon alpha-beta, Receptors, Erythropoietin metabolism, Receptors, Interferon metabolism, Receptors, Interleukin metabolism, Receptors, Interleukin-12, Reverse Transcriptase Polymerase Chain Reaction, Signal Transduction, TYK2 Kinase, Transcription, Genetic, Transfection, Tyrosine metabolism, Down-Regulation, Protein-Tyrosine Kinases metabolism, Proteins metabolism
- Abstract
In contrast to the large number of class I and II cytokine receptors, only four Janus kinase (Jak) proteins are expressed in mammalian cells, implying the shared use of these kinases by many different receptor complexes. Consequently, if receptor numbers exceed the amount of available Jak, cross-interference patterns can be expected. We have engineered two model cellular systems expressing two different exogenous Tyk2-interacting receptors. A receptor chimera was generated wherein the extracellular part of the interferon type 1 receptor (Ifnar1) component of the interferon-alpha/beta receptor is replaced by the equivalent domain of the erythropoietin receptor. Despite Tyk2 activation, erythropoietin treatment of cells expressing this erythropoietin receptor/Ifnar1 chimera did not evoke any detectable IFN-type response. However, a dose-dependent interference with signal transduction via the endogenous Ifnar complex was found for STAT1, STAT2, STAT3, Tyk2, and Jak1 activation, for gene induction, and for antiviral activity. In a similar approach, cells expressing the beta1 chain of the interleukin-12 receptor showed a reduced transcriptional response to IFN-alpha as well as reduced STAT and kinase activation. In both model systems, titration of the Tyk2 kinase away from the Ifnar1 receptor chain accounts for the observed cross-interference.
- Published
- 2001
- Full Text
- View/download PDF
20. A hydrophobic cluster at the surface of the human plasma phospholipid transfer protein is critical for activity on high density lipoproteins.
- Author
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Desrumaux C, Labeur C, Verhee A, Tavernier J, Vandekerckhove J, Rosseneu M, and Peelman F
- Subjects
- Amino Acid Sequence, Binding Sites, Carrier Proteins genetics, Humans, Membrane Proteins genetics, Models, Molecular, Molecular Sequence Data, Mutation, Protein Engineering, Recombinant Proteins metabolism, Sequence Alignment, Static Electricity, Acute-Phase Proteins, Carrier Proteins blood, Lipoproteins, HDL metabolism, Membrane Glycoproteins, Membrane Proteins blood, Phospholipid Transfer Proteins, Phospholipids metabolism
- Abstract
The plasma phospholipid transfer protein (PLTP) belongs to the lipid transfer/lipopolysaccharide binding protein (LT/LBP) family, together with the cholesteryl ester transfer protein, the lipopolysaccharide binding protein (LBP) and the bactericidal permeability increasing protein (BPI). In the present study, we used the crystallographic data available for BPI to build a three-dimensional model for PLTP. Multiple sequence alignment suggested that, in PLTP, a cluster of hydrophobic residues substitutes for a cluster of positively charged residues found on the surface of LBP and BPI, which is critical for interaction with lipopolysaccharides. According to the PLTP model, these hydrophobic residues are situated on an exposed hydrophobic patch at the N-terminal tip of the molecule. To assess the role of this hydrophobic cluster for the functional activity of PLTP, single point alanine mutants were engineered. Phospholipid transfer from liposomes to high density lipoprotein (HDL) by the W91A, F92A, and F93A PLTP mutants was drastically reduced, whereas their transfer activity toward very low density lipoprotein and low density lipoprotein did not change. The HDL size conversion activity of the mutants was reduced to the same extent as the PLTP transfer activity toward HDL. Based on these results, we propose that a functional solvent-exposed hydrophobic cluster in the PLTP molecule specifically contributes to the PLTP transfer activity on HDL substrates.
- Published
- 2001
- Full Text
- View/download PDF
21. ESM-1 is a novel human endothelial cell-specific molecule expressed in lung and regulated by cytokines.
- Author
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Lassalle P, Molet S, Janin A, Heyden JV, Tavernier J, Fiers W, Devos R, and Tonnel AB
- Subjects
- Amino Acid Sequence, Base Sequence, DNA, Complementary genetics, Genes, Humans, Molecular Sequence Data, Molecular Weight, Proteins metabolism, RNA, Messenger genetics, Sequence Alignment, Sequence Homology, Amino Acid, Tissue Distribution, Up-Regulation, Cytokines physiology, Endothelium, Vascular physiology, Gene Expression Regulation, Lung physiology, Neoplasm Proteins, Proteins genetics, Proteoglycans
- Abstract
We here report the identification of a novel human endothelial cell-specific molecule (called ESM-1) cloned from a human umbilical vein endothelial cell (HUVEC) cDNA library. Constitutive ESM-1 gene expression (as demonstrated by Northern blot and reverse transcription-polymerase chain reaction analysis) was found in HUVECs but not in the other human cell lines tested. The cDNA sequence contains an open reading frame of 552 nucleotides and a 1398-nucleotide 3'-untranslated region including several domains involved in mRNA instability and five putative polyadenylation consensus sequences. The deduced 184-amino acid sequence defines a cysteine-rich protein with a functional NH2-terminal hydrophobic signal sequence. Searches in several data bases confirmed the unique identity of this sequence. A rabbit immune serum raised against the 14-kDa COOH-terminal peptide of ESM-1 immunoprecipitated a 20-kDa protein only in ESM-1-transfected COS cells. Immunoblotting and immunoprecipitation of HUVEC lysates revealed a specific 20-kDa band corresponding to ESM-1. In addition, constitutive ESM-1 gene expression was shown to be tissue-restricted to the human lung. Southern blot analysis suggests that a single gene encodes ESM-1. A time-dependent up-regulation of ESM-1 mRNA was seen after addition of tumor necrosis factor alpha (TNFalpha) or interleukin (IL)-1beta but not with IL-4 or interferon gamma (IFNgamma) alone. In addition, when IFNgamma was combined with TNFalpha, IFNgamma inhibited the TNFalpha-induced increase of ESM-1 mRNA level. These data suggest that ESM-1 may have potent implications in the areas of vascular cell biology and human lung physiology.
- Published
- 1996
- Full Text
- View/download PDF
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