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  • 1
    Keywords: PROTEINS ; ACTIVATION ; COMPLEXES ; BINDING ; CRYSTAL-STRUCTURE ; sensitivity ; STOICHIOMETRY ; RAFTS ; membrane microdomains ; MULTIVALENT STRUCTURE
    Abstract: The T-cell antigen receptor (TCR) exists in monomeric and nanoclustered forms independently of antigen binding. Although the clustering is involved in the regulation of T-cell sensitivity, it is unknown how the TCR nanoclusters form. We show that cholesterol is required for TCR nanoclustering in T cells and that this clustering enhances the avidity but not the affinity of the TCR-antigen interaction. Investigating the mechanism of the nanoclustering, we found that radioactive photocholesterol specifically binds to the TCRbeta chain in vivo. In order to reduce the complexity of cellular membranes, we used a synthetic biology approach and reconstituted the TCR in liposomes of defined lipid composition. Both cholesterol and sphingomyelin were required for the formation of TCR dimers in phosphatidylcholine-containing large unilamellar vesicles. Further, the TCR was localized in the liquid disordered phase in giant unilamellar vesicles. We propose a model in which cholesterol and sphingomyelin binding to the TCRbeta chain causes TCR dimerization. The lipid-induced TCR nanoclustering enhances the avidity to antigen and thus might be involved in enhanced sensitivity of memory compared with naive T cells. Our work contributes to the understanding of the function of specific nonannular lipid-membrane protein interactions.
    Type of Publication: Journal article published
    PubMed ID: 23091059
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  • 2
    Keywords: RECEPTOR ; EXPRESSION ; GROWTH ; IN-VIVO ; PATHWAYS ; PROTEIN ; SACCHAROMYCES-CEREVISIAE ; COMPLEX ; COMPLEXES ; BINDING ; IDENTIFICATION ; Saccharomyces cerevisiae ; YEAST ; lifestyle ; BETA ; CONSERVATION ; EGG EXTRACTS ; GTPASE RAN ; IMPORTIN-ALPHA ; LOCALIZATION ; MESSENGER-RNA EXPORT ; NUCLEAR EXPORT RECEPTOR ; NUCLEUS ; TRANSPORT FACTOR ; XENOPUS
    Abstract: The small Ras-like GTPase Ran plays an essential role in the transport of macromolecules in and out of the nucleus and has been implicated in spindle (1, 2) and nuclear envelope formation (3, 4) during mitosis in higher eukaryotes. We identified Saccharomyces cerevisiae open reading frame YGL164c encoding a novel RanGTP-binding protein, termed Yrb30p. The protein competes with yeast RanBP1 (Yrb1p) for binding to the GTP-bound form of yeast Ran (Gsp1p) and is, like Yrb1p, able to form trimeric complexes with RanGTP and some of the karyopherins. In contrast to Yrb1p, Yrb30p does not coactivate but inhibits RanGAP1(Rna1p)-mediated GTP hydrolysis on Ran, like the karyopherins. At steady state, Yrb30p localizes exclusively to the cytoplasm, but the presence of a functional nuclear export signal and the localization of truncated forms of Yrb30p suggest that the protein shuttles between nucleus and cytoplasm and is exported via two alternative pathways, dependent on the nuclear export receptor Xpo1p/Crm1p and on RanGTP binding. Whereas overproduction of the full-length protein and complete deletion of the open reading frame reveal no obvious phenotype, overproduction of C-terminally truncated forms of the protein inhibits yeast vegetative growth. Based on these results and the exclusive conservation of the protein in the fungal kingdom, we hypothesize that Yrb30p represents a novel modulator of the Ran GTPase switch related to fungal lifestyle
    Type of Publication: Journal article published
    PubMed ID: 12578832
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  • 3
    Keywords: IN-VITRO ; IONIZING-RADIATION ; IRRADIATION ; CELL ; Germany ; human ; IN-VIVO ; KINASE ; PATHWAY ; VITRO ; VIVO ; SITE ; PROTEIN ; radiation ; ACTIVATION ; COMPLEX ; COMPLEXES ; DNA ; CARCINOGENESIS ; cell cycle ; CELL-CYCLE ; CYCLE ; PHOSPHORYLATION ; MUTANT ; LESIONS ; PROGRESSION ; CYCLE PROGRESSION ; DAMAGE ; DNA-DAMAGE ; DEGRADATION ; ATR ; CHK1 ; CYCLE CONTROL ; G(1)/S TRANSITION ; PROTEASOME ; S-PHASE ; serine
    Abstract: The human Cdc25A phosphatase plays a pivotal role at the G(1)/S transition by activating cyclin E and A/Cdk2 complexes through dephosphorylation. In response to ionizing radiation, Cdc25A is phosphorylated by both Chk1 and Chk2 on Ser-123. This in turn leads to ubiquitylation and rapid degradation of Cdc25A by the proteasome resulting in cell cycle arrest. We found that in response to UV irradiation, Cdc25A is phosphorylated at a different serine residue, Ser-75. Significantly, Cdc25A mutants carrying alanine instead of either Ser-75 or Ser-123 demonstrate that only Ser-75 mediates protein stabilization in response to UV-induced DNA damage. As a consequence, cyclin E/Cdk2 kinase activity was high. Furthermore, we find that Cdc25A was phosphorylated by Chk1 on Ser-75 in vitro and that the same site was also phosphorylated in vivo. Taken together, these data strongly suggest that phosphorylation of Cdc25A on Ser-75 by Chk1 and its subsequent degradation is required to delay cell cycle progression in response to UV-induced DNA lesions
    Type of Publication: Journal article published
    PubMed ID: 12759351
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  • 4
    Keywords: APOPTOSIS ; CELLS ; EXPRESSION ; GROWTH ; INHIBITOR ; tumor ; KINASE ; DEATH ; PROTEIN ; MONOCLONAL-ANTIBODY ; DEATH DOMAIN ; TUMOR-NECROSIS-FACTOR ; COMPLEX ; LIGAND ; COMPLEXES ; DOMAIN ; mechanisms ; PHOSPHORYLATION ; protein kinase ; PROTEIN-KINASE ; treatment ; antibodies ; antibody ; INDUCED APOPTOSIS ; MODULATION ; PATHOGENESIS ; DISC ; SIGNALING COMPLEX ; ANTI-APOPTOTIC MOLECULE ; CASPASE-8 ACTIVATION ; DEATH-EFFECTOR DOMAIN ; FAS/TNFR1- INDUCED APOPTOSIS ; INHIBITORY PROTEIN ; INTEGRIN ACTIVATION ; INTRACELLULAR REGULATION ; SURFACE ANTIGEN
    Abstract: Fas, upon cross-linking with Fas ligand (FasL) or Fas agonistic antibody, transduces apoptotic yet also proliferative signals, which have been implicated in tumor pathogenesis. In this study, we investigated the molecular mechanisms that control Fas-mediated signaling in glioma cells. Fas agonistic antibody, CH-11, induced apoptosis in sensitive glioma cells through caspase-8 recruitment to the Fas-mediated death-inducing signaling complex (DISC) where caspase-8 was cleaved to initiate apoptosis through a systematic cleavage of downstream substrates. In contrast, CH-11 stimulated cell growth in resistant glioma cells through recruitment of c-FLIP (cellular Fas-associated death domain (FADD)-like interleukin-1beta- converting enzyme (FLICE)-inhibitory protein) to the Fas- mediated DISC. Three isoforms of long form c-FLIP were detected in glioma cells, but only the phosphorylated isoform was recruited to and cleaved into a p43 intermediate form in the Fas-mediated DISC in resistant cells. Calcium/calmodulin- dependent protein kinase II (CaMK II) activity was up-regulated in resistant cells. Treatment of resistant cells with the CaMK II inhibitor KN-93 inhibited CaMK II activity, reduced c-FLIP expression, inhibited c-FLIP phosphorylation, and rescued CH-11 sensitivity. Transfection of CaMK II cDNA in sensitive cells rendered them resistant to CH-11. These results indicated that CaMK 11 regulates c-FLIP expression and phosphorylation, thus modulating Fas-mediated signaling in glioma cells
    Type of Publication: Journal article published
    PubMed ID: 12496285
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  • 5
    Keywords: RECEPTOR ; CANCER CELLS ; CELLS ; EXPRESSION ; GROWTH ; tumor ; carcinoma ; CELL ; Germany ; human ; IN-VIVO ; KINASE ; MODEL ; PATHWAY ; CDNA ; CLONES ; GENE ; PROTEIN ; transcription ; ACTIVATION ; LIGAND ; MECHANISM ; FAMILY ; TRANSCRIPTION FACTOR ; AP-1 ; BINDING ; BIOLOGY ; C-JUN ; MEMBER ; MEMBERS ; MOLECULAR-BIOLOGY ; PHOSPHORYLATION ; protein kinase ; PROTEIN-KINASE ; ACID ; GROWTH-INHIBITION ; CARCINOMA-CELLS ; BETA ; Jun ; DEGRADATION ; HeLa cells ; DIMERIZATION ; AFFINITY ; MEDIATED REPRESSION ; N-TERMINAL KINASE ; CONSTITUTIVE EXPRESSION ; CROSS-TALK ; FACTOR-ALPHA ; F ; molecular biology ; ACTIVATED PROTEIN-KINASES ; ALL-TRANS ; cervical carcinoma cells ; HELA-CELLS ; PROTEASOME INHIBITORS ; X-RECEPTOR
    Abstract: Expression of the nuclear retinoic acid receptor beta2 (RARbeta2) gene is often disturbed in cervical carcinoma cells. One important mechanism by which RARbeta2 can exert growth inhibitory function is based on its ability to repress the AP-1 transcription factor in a ligand-dependent manner. Because less is known about the biological effects of RARbeta in the absence of ligand, the corresponding cDNA was stably introduced into HPV18-positive HeLa cervical carcinoma cells. In the present study we describe a novel mechanism by which AP-1 becomes inactivated. Constitutive expression of nonliganded RARbeta abrogated both AP-1 binding affinity and activity by a selective degradation of the c-Jun protein as major dimerization partner, without substitution by other members of the Jun family. Blockage of the proteasomal pathway completely rescued c-Jun and reconstituted the AP-1 function. Moreover, HeLa RARbeta2 clones treated either with tumor necrosis factor-alpha or transfected with a constitutive active upstream mitogen-activated protein kinase (MEKK1Delta) also resulted in c-Jun phosphorylation and restoration of AP-1 affinity and functionality similar to that found in nontransfected parental HeLa cells. These data revealed an important cross-talk between trans-repression of AP-1 and nonliganded RARbeta in human papillomavirus-positive cells. Because AP-1 activity was not irreversibly disturbed, but could be switched on through activation of the Jun N-terminal kinase pathway, a model for the transient activation of AP-1 even in the presence of RARbeta as repressor is suggested
    Type of Publication: Journal article published
    PubMed ID: 15308638
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  • 6
    Keywords: PEPTIDE ; CANCER ; INHIBITOR ; Germany ; KINASE ; TOOL ; DISEASE ; SITE ; PROTEIN ; DRUG ; TRANSDUCTION ; COMPLEX ; COMPLEXES ; MECHANISM ; DOMAIN ; INDUCTION ; BINDING ; BIOLOGY ; PROTEIN-KINASE ; signal transduction ; SIGNAL ; VARIANTS ; DISCOVERY ; MOLECULE ; CATALYTIC SUBUNIT ; SIGNAL-TRANSDUCTION ; ATP ; CRYSTAL-STRUCTURE ; PKA ; PROTEIN-KINASE-C ; FLEXIBILITY ; max ; DOMAINS ; INHIBITORS ; POTENT ; VARIANT ; PHASE-III ; SELECTIVE INHIBITORS ; STAUROSPORINE ; STRUCTURAL BASIS ; UCN-01
    Abstract: As the key mediators of eukaryotic signal transduction, the protein kinases often cause disease, and in particular cancer, when disregulated. Appropriately selective protein kinase inhibitors are sought after as research tools and as therapeutic drugs; several have already proven valuable in clinical use. The AGC subfamily protein kinase C (PKC) was identified early as a cause of cancer, leading to the discovery of a variety of PKC inhibitors. Despite its importance and early discovery, no crystal structure for PKC has yet been reported. Therefore, we have co-crystallized PKC inhibitor bisindolyl maleimide 2 (BIM2) with PKA variants to study its binding interactions. BIM2 co-crystallized as an asymmetric pair of kinase-inhibitor complexes. In this asymmetric unit, the two kinase domains have different lobe configurations, and two different inhibitor conformers bind in different orientations. One kinase molecule ( A) is partially open with respect to the catalytic conformation, the other ( B) represents the most open conformation of PKA reported so far. In monomer A, the BIM2 inhibitor binds tightly via an induced fit in the ATP pocket. The indole moieties are rotated out of the plane with respect to the chemically related but planar inhibitor staurosporine. In molecule B a different conformer of BIM2 binds in a reversed orientation relative to the equivalent maleimide atoms in molecule A. Also, a critical active site salt bridge is disrupted, usually indicating the induction of an inactive conformation. Molecular modeling of the clinical phase III PKC inhibitor LY333531 into the electron density of BIM2 reveals the probable binding mechanism and explains selectivity properties of the inhibitor
    Type of Publication: Journal article published
    PubMed ID: 14996846
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  • 7
    Keywords: RECEPTOR ; CELLS ; CELL ; Germany ; DENSITY ; PROTEIN ; cell line ; ACTIVATION ; LIGAND ; IMPACT ; DOMAIN ; BINDING ; ACID ; ACIDS ; MUTANT ; MEMBRANE ; MUTATION ; REQUIRES ; SIGNAL-TRANSDUCTION ; CELL-LINE ; LINE ; INTERFACE ; CONSTITUTIVE ACTIVATION ; AMINO-ACIDS ; mutagenesis ; point mutation ; DIMERIZATION ; MEMBRANE PROTEIN ; MEMBRANE-PROTEIN ; LIGAND-BINDING ; COILED COILS ; signaling ; SEGMENT ; RECEPTOR ACTIVATION ; PATTERN ; LEADS ; INFECTED-CELLS ; FUNCTIONAL-CHARACTERIZATION ; AMINO-ACID ; GLYCOPHORIN-A ; VIRUS GP55 GLYCOPROTEIN
    Abstract: Structural and functional studies recently indicated that the erythropoietin receptor exists as a preassembled homodimer whose activation by ligand binding requires self-interaction of its transmembrane segment. Here, we probed the interface formed by the transmembrane segments by asparagine-scanning mutagenesis in a natural membrane. We show that this interface is based on a leucine zipper-like heptad repeat pattern of amino acids. The strongest impact of asparagine was observed at position 241, suggesting the highest packing density around this position, which is in agreement with results obtained upon mutation to alanine. Interestingly, the same face of the transmembrane helix had previously been shown to enter a heterophilic interaction with the transmembrane segment of gp55-P, a viral membrane protein that leads to ligand-independent receptor activation in infected cells. Further, functional characterization of an erythropoietin receptor mutant with asparagine at position 241 in a hematopoietic cell line showed that this protein could still be activated by erythropoietin yet was not constitutively active. This suggests that forced self-interaction of the transmembrane segments does not suffice to induce signaling of the erythropoietin receptor
    Type of Publication: Journal article published
    PubMed ID: 14602718
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  • 8
    Keywords: SPECTRA ; SITE ; ENZYMES ; PROTEIN ; COMPLEX ; COMPLEXES ; DNA ; DOMAIN ; BIOLOGY ; TYPE-1 ; FREQUENCY ; virus ; MUTANT ; SUBUNIT ; NUCLEOTIDES ; WILD-TYPE ; MUTATIONS ; REPLICATION FIDELITY ; PRIMER ; EXONUCLEASE ACTIVITY ; MISMATCH EXTENSION ; PROCESSIVITY ; UL42
    Abstract: Nucleotide incorporation by the herpes simplex virus type 1 DNA polymerase catalytic subunit (pol) is less faithful than for most replicative DNA polymerases, despite the presence of an associated 3'- to 5'-exonuclease (exo) activity. To determine the aspects of fidelity affected by the exo activity, nucleotide incorporation and mismatch extension frequency for purified wild-type and an exo-deficient mutant (D368A) pol were compared using primer/templates that varied at only a single position. For both enzymes, nucleotide discrimination during incorporation occurred predominantly at the level of Km for nucleotide and was the major contributor to fidelity. The contribution of the exo activity to reducing the efficiency of formation of half of all possible mispairs was 6-fold or less, and 30-fold when averaged for the formation of all possible mispairs. In steady-state reactions, mismatches imposed a significant kinetic barrier to extension independent of exo activity. However, during processive DNA synthesis in the presence of only three nucleotides, misincorporation and mismatch extension were efficient for both exo-deficient and wildtype pol catalytic subunits, although slower kinetics of mismatch extension by the exo-deficient pol were observed. The UL42 processivity factor decreased the extent of misincorporation by both the wild-type and the exo-deficient pol to similar levels, but mismatch extension by the wild-type pol . UL42 complex was much less efficient than by the mutant pol . UL42. Thus, despite relatively frequent ( 1 in 300) misincorporation events catalyzed by wild-type herpes simplex virus pol . UL42 holoenzyme, mismatch extension occurs only rarely, prevented in part by the kinetic barrier to extending a mismatch. The kinetic barrier also increases the probability that a mismatched primer terminus will be transferred to the exo site where it can be excised by the associated exo activity and subsequently extended with correct nucleotide
    Type of Publication: Journal article published
    PubMed ID: 14982924
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  • 9
    Keywords: APOPTOSIS ; CELLS ; EXPRESSION ; Germany ; DEATH ; PROTEIN ; PROTEINS ; LINES ; NF-KAPPA-B ; ACTIVATION ; COMPLEX ; COMPLEXES ; T-CELLS ; CELL-LINES ; VARIANTS ; UP-REGULATION ; NUMBER ; LINE ; cell lines ; REGULATOR ; SIGNALING COMPLEX DISC ; SIGNALING COMPLEX ; EFFECTOR ; CD95 APO-1/FAS ; CD95 ; HUMAN T-CELLS ; PROGRAM ; RE ; CASPASE-8 ; MEDIATED APOPTOSIS ; regulation ; CD95-MEDIATED APOPTOSIS ; SIGNALING COMPLEXES ; FLICE-INHIBITORY PROTEINS
    Abstract: c-FLIPs (c-FLICE inhibitory proteins) play an essential role in regulation of death receptor-induced apoptosis. Multiple splice variants of c-FLIP have been described on the mRNA level; so far only two of them, c-FLIPL and c-FLIPS, had been found to be expressed at the protein level. In this report, we reveal the endogenous expression of a third isoform of c-FLIP. We demonstrate its presence in a number of T and B cell lines as well as in primary human T cells. We identified this isoform as c-FLIPR, a death effector domain-only splice variant previously identified on the mRNA level. Importantly, c-FLIPR is recruited to the CD95 (Fas/APO-1) death-inducing signaling complex upon CD95 stimulation. Several properties of c-FLIPR are similar to c-FLIPS: both isoforms have a short half-life, a similar pattern of expression during activation of primary human T cells, and are strongly induced in T cells upon CD3/CD28 costimulation. Taken together, our data demonstrate endogenous expression of c-FLIPR and similar roles of c-FLIPR and c-FLIPS isoforms in death receptor-mediated apoptosis
    Type of Publication: Journal article published
    PubMed ID: 15701649
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  • 10
    Keywords: CELLS ; EXPRESSION ; Germany ; PATHWAY ; ENZYMES ; GENE ; GENES ; MICE ; MOLECULE ; ACID ; MOUSE ; PHENOTYPE ; sensitivity ; METHYLATION ; infertility ; FATTY-ACID ; SUBSET ; RE ; RESIDUES ; SYNTHASE ; glycosphingolipids ; LONG ; STRAINS ; SPHINGOMYELIN ; CHAIN FATTY-ACIDS ; GANGLIOSIDE ; glycosphingolipid ; GM3
    Abstract: Mice require testicular glycosphingolipids (GSLs) for proper spermatogenesis. Mutant mice strains deficient in specific genes encoding biosynthetic enzymes of the GSL pathway including Galgt1 ( encoding GM2 synthase) and Siat9 ( encoding GM3 synthase) have been established lacking various overlapping subsets of GSLs. Although male Galgt1 -/- mice are infertile, male Siat9 -/- mice are fertile. Interestingly, GSLs thought to be essential for male spermatogenesis are not synthesized in either of these mice strains. Hence, these GSLs cannot account for the different phenotypes. A novel class of GSLs was observed composed of eight fucosylated molecules present in fertile but not in infertile mutant mice. These GSLs contain polyunsaturated very long chain fatty acid residues in their ceramide moieties. GSLs of this class are expressed differentially in testicular germ cells. More importantly, the neutral subset of this new GSL class strictly correlates with male fertility. These data implicate polyunsaturated, fucosylated GSLs as essential for spermatogenesis and male mouse fertility
    Type of Publication: Journal article published
    PubMed ID: 15917254
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