The Wnt family of proteins is a group of extracellular signalling molecules that regulate cell-fate decisions in developing and adult tissues. It is presumed that all 19 mammalian Wnt family members contain two types of post-translational modification: the covalent attachment of fatty acids at two distinct positions, and the N-glycosylation of multiple asparagines. We examined how these modifications contribute to the secretion, extracellular movement and signalling activity of mouse Wnt1 and Wnt3a ligands. We revealed that O-linked acylation of serine is required for the subsequent S-palmitoylation of cysteine. As such, mutant proteins that lack the crucial serine residue are not lipidated. Interestingly, although double-acylation of Wnt1 was indispensable for signalling in mammalian cells, in Xenopus embryos the S-palmitoyl-deficient form retained the signalling activity. In the case of Wnt3a, the functional duality of the attached acyls was less prominent, since the ligand lacking S-linked palmitate was still capable of signalling in various cellular contexts. Finally, we show that the signalling competency of both Wnt1 and Wnt3a is related to their ability to associate with the extracellular matrix.

• MeSH
• buněčné linie MeSH
• cystein metabolismus   MeSH
• embryonální vývoj MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• lipoylace MeSH
• molekulární sekvence - údaje MeSH
• mutace MeSH
• myši MeSH
• protein Wnt1 genetika   metabolismus   MeSH
• protein Wnt3 MeSH
• protein Wnt3A MeSH
• proteiny Wnt genetika   metabolismus   MeSH
• proteiny Xenopus MeSH
• sekvence aminokyselin MeSH
• serin metabolismus   MeSH
• substituce aminokyselin MeSH
• Xenopus embryologie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Death receptor 6 (DR6/TNFRSF21) is a death domain-containing receptor of the TNFR superfamily with an apparent regulatory function in hematopoietic and neuronal cells. In this study we document that DR6 is an extensively posttranslationally modified transmembrane protein and that N- and O-glycosylations of amino acids in its extracellular part are mainly responsible for its approximately 40 kDa mobility shift in SDS polyacrylamide gels. Site-directed mutagenesis confirmed that all six extracellular asparagines are N-glycosylated and that the Ser/Thr/Pro cluster in the "stalk" domain juxtaposed to the cysteine-rich domains (CRDs) is a major site for the likely mucine-type of O-glycosylation. Deletion of the entire linker region between CRDs and the transmembrane domain, spanning over 130 amino acids, severely compromises the plasma membrane localization of DR6 and leads to its intracellular retention. Biosynthetic labeling with radiolabeled palmitate and side-directed mutagenesis also revealed that the membrane-proximal Cys368 in the intracellular part of DR6 is, similarly as cysteines in Fas/CD95 or DR4 ICPs, S-palmitoylated. However, palmitoylation of Cys368 is apparently not required for DR6 targeting into Brij-98 insoluble lipid rafts. In contrast, we show that N-glycosylation of the extracellular part might participate in directing DR6 into these membrane microdomains.

• MeSH
• buněčné linie MeSH
• glykosylace MeSH
• HeLa buňky MeSH
• HL-60 buňky MeSH
• Jurkat buňky MeSH
• lidé MeSH
• lipoylace MeSH
• membránové mikrodomény metabolismus   MeSH
• molekulová hmotnost MeSH
• mutageneze cílená MeSH
• nádorové buněčné linie MeSH
• posttranslační úpravy proteinů MeSH
• protein - isoformy fyziologie   genetika   chemie   MeSH
• receptory TNF genetika   chemie   metabolismus   MeSH
• rekombinantní proteiny genetika   chemie   metabolismus   MeSH
• sekvenční delece MeSH
• terciární struktura proteinů MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• Publikační typ
• práce podpořená grantem MeSH

A major outcome of the canonical Wnt/beta-catenin-signalling pathway is the transcriptional activation of a specific set of target genes. A typical feature of the transcriptional response induced by Wnt signalling is the involvement of Tcf/Lef factors that function in the nucleus as the principal mediators of signalling. Vertebrate Tcf/Lef proteins perform two well-characterized functions: in association with beta-catenin they activate gene expression, and in the absence of Wnt ligands they bind TLE/Groucho proteins to act as transcriptional repressors. Although the general characteristics of Tcf/Lef factors are well understood, the mechanisms that control their specific roles in various cellular backgrounds are much less defined. In this report we reveal that the evolutionary conserved Dazap2 protein functions as a TCF-4 interacting partner. We demonstrate that a short region proximal to the TCF-4 HMG box mediates the interaction and that all Tcf/Lef family members associate with Dazap2. Interestingly, knockdown of Dazap2 not only reduced the activity of Wnt signalling as measured by Tcf/beta-catenin reporters but additionally altered the expression of Wnt-signalling target genes. Finally, chromatin immunoprecipitation studies indicate that Dazap2 modulates the affinity of TCF-4 for its DNA-recognition motif.

• MeSH
• beta-katenin metabolismus   MeSH
• buněčné linie MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• genetická transkripce MeSH
• genový knockdown MeSH
• lidé MeSH
• myši MeSH
• promotorové oblasti (genetika) MeSH
• proteiny vázající RNA antagonisté a inhibitory   genetika   metabolismus   MeSH
• proteiny Wnt metabolismus   MeSH
• regulace genové exprese MeSH
• transkripční faktory BHLH-Zip MeSH
• transkripční faktory chemie   metabolismus   MeSH
• vazebná místa MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• práce podpořená grantem MeSH

Wnt signaling enhances cell proliferation and the maintenance of hematopoietic cells. In contrast, cytotoxic ligand Apo2L/TRAIL induces the apoptosis of various transformed cells. We observed that co-culture of human pre-B leukemia cells KM3 and REH with Wnt1- or Wnt3a-producing rat embryonic fibroblasts efficiently suppressed Apo2L/TRAIL-induced apoptosis of the lymphoid cells. This suppression occurs at the early stages of the Apo2L/TRAIL apoptotic cascade and, interestingly, the activation of the Wnt pathway alone in human leukemia cells is not sufficient for their full anti-apoptotic protection. We hypothesize that a stimulus emanating specifically from Wnt1- or Wnt3a-expressing rat fibroblasts is responsible for the observed resistance to Apo2L/TRAIL. This anti-apoptotic signaling was significantly hampered by the inhibition of the MEK1/ERK1/2 or NFkappaB pathways in KM3 and REH cells. Our results imply that paracrine Wnt-related signals could be important for the survival of pre-B cell-derived malignancies.

• MeSH
• apoptóza fyziologie   MeSH
• beta-katenin fyziologie   MeSH
• cykloheximid farmakologie   MeSH
• daktinomycin farmakologie   MeSH
• financování organizované MeSH
• kokultivační techniky MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• nádorové buněčné linie MeSH
• pre-B-buněčná leukemie MeSH
• protein TRAIL antagonisté a inhibitory   MeSH
• protein Wnt1 biosyntéza   MeSH
• proteiny regulující apoptózu fyziologie   MeSH
• proteiny Wnt biosyntéza   MeSH
• signální transdukce fyziologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH

• Publikační typ
• abstrakt z konference MeSH

The hypermethylated in cancer 1 (HIC1) gene is epigenetically inactivated in cancer, and in addition, the haploinsufficiency of HIC1 is linked to the development of human Miller-Dieker syndrome. HIC1 encodes a zinc-finger transcription factor that acts as a transcriptional repressor. Additionally, the HIC1 protein oligomerizes via the N-terminal BTB/POZ domain and forms discrete nuclear structures known as HIC1 bodies. Here, we provide evidence that HIC1 antagonizes the TCF/beta-catenin-mediated transcription in Wnt-stimulated cells. This appears to be due to the ability of HIC1 to associate with TCF-4 and to recruit TCF-4 and beta-catenin to the HIC1 bodies. As a result of the recruitment, both proteins are prevented from association with the TCF-binding elements of the Wnt-responsive genes. These data indicate that the intracellular amounts of HIC1 protein can modulate the level of the transcriptional stimulation of the genes regulated by canonical Wnt/beta-catenin signaling.

• MeSH
• beta-katenin genetika   metabolismus   MeSH
• buněčné jádro metabolismus   MeSH
• cytoskeletální proteiny genetika   metabolismus   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• financování organizované MeSH
• genetická transkripce MeSH
• kultivační média speciální MeSH
• lidé MeSH
• myši MeSH
• promotorové oblasti (genetika) MeSH
• proteiny Wnt genetika   metabolismus   MeSH
• regulace genové exprese MeSH
• RNA interference MeSH
• signální transdukce fyziologie   MeSH
• transkripční faktory Krüppel-like MeSH
• transkripční faktory TCF genetika   metabolismus   MeSH
• transkripční faktory genetika   metabolismus   MeSH
• vazba proteinů MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH