T-cell-specific deletion of a polypeptide N-acetylgalactosaminyl-transferase gene by site-directed recombination.

Hennet, Thierry; Hagen, Fred K.; Tabak, Lawrence A.; Marth, Jamey D.

doi:10.1073/pnas.92.26.12070

Cited by 248 publications

(192 citation statements)

References 28 publications

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“…The Tak1 flox/flox mice were born and lived normally, and they expressed TAK1 protein as expected (data not shown). To delete the Tak1 allele specifically in T cells, we crossed Tak1 flox/flox mice with the Lck-Cre transgenic mice, which express the Cre recombinase under the control of the T cell-specific Lck promoter (21). Southern blotting and PCR showed that the floxed Tak1 alleles were excised in thymocytes, but not in the tail (Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Essential role of TAK1 in thymocyte development and activation

Liu¹,

Xie

Schneider

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

151

158

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The protein kinase TAK1 mediates the activation of NF-B in response to stimulation by proinflammatory cytokines and microbial pathogens in the innate immunity pathways. However, the physiological function of TAK1 in the adaptive immunity pathways is unclear. By engineering mice lacking TAK1 in T cells, here, we show that TAK1 is essential for thymocyte development and activation in vivo. Deletion of TAK1 prevented the maturation of single-positive thymocytes displaying CD4 or CD8, leading to reduction of T cells in the peripheral tissues. Thymocytes lacking TAK1 failed to activate NF-B and JNK and were prone to apoptosis upon stimulation. Our results provide the genetic evidence that TAK1 is required for the activation of NF-B in thymocytes and suggest that TAK1 plays a central role in both innate and adaptive immunity.T he Rel͞NF-B family of transcription factors regulates the expression of a plethora of genes involved in inflammation, immunity, and apoptosis (1, 2). NF-B is normally sequestered in the cytoplasm of unstimulated cells through its association with the I B family of inhibitory proteins. Stimulation of cells with a variety of agents leads to the rapid phosphorylation and subsequent degradation of I B by the ubiquitin-proteasome pathway, thus allowing NF-B to enter the nucleus to turn on various target genes.Phosphorylation of I B is catalyzed by a large kinase complex consisting of I B kinase (IKK)␣, IKK␤, and NEMO (also known as IKK␥ or IKKAP). The IKK complex integrates signals from diverse pathways, including those emanating from the receptors for TNF␣ and IL-1␤, Toll-like receptors (TLRs), and T cell receptors (TCRs) (3-6). Stimulation of IL-1R and some TLRs leads to the recruitment of several proteins, including the adaptor MyD88, the kinases IRAK4 and IRAK1, and the ubiquitin ligase TRAF6. TRAF6 functions in conjunction with the ubiquitin-conjugating enzyme (E2) complex Ubc13-Uev1A to catalyze the synthesis of Lys-63-linked polyubiquitin chains on certain protein targets, including TRAF6 itself (7,8). Polyubiquitinated TRAF6 activates a protein kinase complex consisting of the TAK1 kinase and the adaptor proteins TAB1 and TAB2 (8, 9). The activation of TAK1 by TRAF6 requires the binding between the K63 polyubiquitin chains and a conserved novel zinc finger (NZF) domain of TAB2 or its homologue TAB3 (10). After TAK1 is activated, it phosphorylates IKK␤ within the activation loop, resulting in the activation of IKK. TAK1 also phosphorylates and activates MKK6 and MKK7, leading to the activation of p38 and JNK kinase pathways.Recent studies have shown that TRAF-mediated polyubiquitination and the TAK1 kinase complex also play an important role in NF-B activation in T cells (11). Stimulation of TCR by an antigenic peptide and its cognate MHC activates a tyrosine kinase phosphorylation cascade, which, in turn, leads to the activation of protein kinase (PK)C . PKC then triggers the recruitment of the CARD domain proteins CARMA1 and BCL10 and the paracaspase MALT1 to lipid rafts (12)(13)(14). MALT1 ...

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Section: Resultsmentioning

confidence: 99%

“…Blastocyst injection was performed at Baylor College of Medicine. Lck-Cre transgenic mice were obtained from The Jackson Laboratory (21). Floxed Tak1 mice were crossed to Lck-Cre mice at University of Texas Southwestern Medical Center.…”

Section: Methodsmentioning

confidence: 99%

Essential role of TAK1 in thymocyte development and activation

Liu¹,

Xie

Schneider

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

151

158

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“…1). Lck-Cre transgenic mice express the Cre recombinase under the control of the Lck proximal promoter, which drives the Cre expression specifically in immature thymocytes (27,29).…”

Section: Methodsmentioning

confidence: 99%

Expression of the Sphingosine 1-Phosphate Receptor, S1P1, on T-cells Controls Thymic Emigration

Allende

Dreier

Mandala

et al. 2004

Journal of Biological Chemistry

415

381

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S1P 1 is a widely distributed G protein-coupled receptor whose ligand, sphingosine 1-phosphate, is present in high concentrations in the blood. The sphingosine 1-phosphate receptor-signaling pathway is believed to have potent effects on cell trafficking in the immune system. To determine the precise role of the S1P 1 receptor on T-cells, we established a T-cell-specific S1P 1 knock-out mouse. The mutant mice showed a block in the egress of mature T-cells into the periphery. The expression of the S1P 1 receptor was up-regulated in mature thymocytes, and its deletion altered the chemotactic responses of thymocytes to sphingosine 1-phosphate. The results indicated that the expression of the S1P 1 receptor on T-cells controls their exit from the thymus and entry into the blood and, thus, has a central role in regulating the numbers of peripheral T-cells.Sphingolipids are important signaling molecules in a variety of biologic contexts (1-3). Sphingosine 1-phosphate, in one paradigm, binds to members of a family of G protein-coupled receptors (S1P 1-15 ) triggering diverse effects including proliferation, survival, migration, morphogenesis, adhesion molecule expression, and cytoskeletal changes (4 -8).S1P 1 /Edg 1 , the first of these receptors described (9, 10), couples to a G i pathway. During embryonic development, the S1P 1 receptor is highly expressed in the vascular system. Gene disruption in mice has demonstrated an essential function of the S1P 1 receptor in endothelial cells for the formation of a stable vascular network (11,12).The S1P 1 receptor is widely expressed in the adult, in particular in endothelial cells, the brain, and the heart, and also in the cells of the immune system (13, 14). S1P 1 and S1P 4 receptors have been detected in T-lymphocytes (15-17). Stimulation of receptor signaling has been found to mediate and regulate cell migration and suppress proliferation and cytokine production (17, 18). Recently, S1P 1 receptors have also been implicated in lymphocyte trafficking and homing as the result of studies using FTY720, a potent immunosuppressive agent. FTY720, a sphingosine analogue, is phosphorylated by sphingosine kinase type I and more efficiently by sphingosine kinase type II (19 -21) and functions as an agonist ligand for S1P 1 , S1P 3 , S1P 4 , and S1P 5 receptors (19,22). FTY720 causes lymphopenia through sequestration of circulating lymphocytes within lymph nodes and Peyer's patches (19,(22)(23)(24) and also blocks the egress of T-cells from the thymus (25, 26). However, it is not known at which S1P receptor and cell type FTY720 exerts its effects.To begin to address the physiologic function of the S1P 1 receptor and sphingosine 1-phosphate signaling in T-cells, we have generated a T-cell-specific mouse knock-out of the S1P 1 receptor using the Cre/loxP system. Our results with these mice reveal a crucial role for the S1P 1 receptor in the egress of mature T-cells from the thymus into the circulation. EXPERIMENTAL PROCEDURESGeneration of the S1P 1 loxP/loxP Lck-Cre Mice-We previously g...

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“…This resulted previously in the establishment of mice lacking an intact gene for pp-GalNAc-T13 by using the pp-GalNAc-T1 gene as a probe for cloning, and prior to evidence for multiple pp-GalNAc-T isozymes (26,27). In the previous study (27), the complete gene encoding pp-GalNAc-T13, which was partially identified and tentatively designated mouse pp-GalNAc-T8, was never cloned and characterized.…”

Section: The Nucleotide Sequence(s) Reported In This Paper Has Been Smentioning

confidence: 99%

Cloning and Characterization of a New Human UDP-N-Acetyl-α-d-galactosamine:PolypeptideN-Acetylgalactosaminyltransferase, Designated pp-GalNAc-T13, That Is Specifically Expressed in Neurons and Synthesizes GalNAc α-Serine/Threonine Antigen

Zhang

Iwasaki

Wang

et al. 2003

Journal of Biological Chemistry

129

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To date, 10 members of the UDP-N-acetyl-␣-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase (pp-GalNAc-T) family have been cloned and analyzed in human. In this study, we cloned and analyzed a novel human pp-GalNAc-T from an NT2 cell cDNA library, and we named it pp-GalNAc-T13. In amino acid sequences, pp-GalNAc-T13 was highly homologous, showing 84.3% identity, to pp-GalNAc-T1. Real time PCR analysis revealed pp-GalNAc-T13 to be highly and restrictively expressed in the brain and present at very low or undetectable levels in other tissues, in contrast to the ubiquitous expression of pp-GalNAc-T1. pp-GalNAc-T13 was abundantly expressed in all neuroblastoma cells examined and primary cultured neurons but not in glioblastoma cells and primary cultured astrocytes. pp-GalNAc-T13 exhibited much stronger activity to transfer GalNAc to mucin peptides, such as Muc5Ac and MUC7, than did pp-GalNAc-T1. In addition, pp-GalNAc-T13 differed in substrate specificity to pp-GalNAc-T1. pp-GalNAc-T13 was able to form a triplet Tn epitope, three consecutive GalNAc-Ser/Thr structures, on peptides encoded in syndecan-3, a proteoglycan expressed in neurons. pp-GalNAc-T13-deficient mice have been established in a previous work. Immunohistochemical study showed a remarkable decrease in Tn antigen expression in the cerebellum of the pp-GalNAc-T13 knockout mouse. pp-GalNAc-T13 would be a major enzyme responsible for the synthesis of O-glycan and specifically the Tn antigen epitope in neurons.O-Linked glycosylation of mucin is initiated by the transfer of N-acetylgalactosamine with an ␣-linkage to a serine or threonine residue in protein. The GalNAc residue attached to the peptide is usually extended to form more complex O-glycan structures by the action of multiple glycosyltransferases. The addition of GalNAc is controlled by multiple members of the pp-GalNAc-T 1 (EC 2.4.1.41) family. To date, 10 distinct members have been identified in human (pp-GalNAc-T1, -T2, -T3, -T4, -T6 to -T9, -T11, and -T12) (1-10) and 7 in rodent (ppGaN- Tase-T1, -T2, -T3, -T4, -T5, -T7, -T10 2 ) (11-17). All share a highly homologous primary sequence, 40 -60% homology at the amino acid level, to each other particularly in the predicted catalytic domain. Each member exhibits different substrate specificity toward peptide sequences. Thus, the positions of O-glycan in proteins are determined by the substrate specificity of each pp-GalNAc-T. The genes encoding these enzymes are distributed at different genomic localizations on chromosomes and have distinct genomic structures (4).pp-GalNAc-T1, which was the first pp-GalNAc-T to be cloned from bovine tissue (1), is the best characterized of the members. It shows activity toward Muc1, Muc2, Muc5Ac, and Muc7 peptides (4, 18). Peptide sequences derived from these mucins were used to determine the site of the O-glycosylation of the Muc2 and Muc5Ac mucin tandem repeat region by pp-GalNAc-T1 (19 -22).Many pp-GalNAc-Ts, including pp-GalNAc-T1, were found to * This work was performed as a part of the R&D Project o...

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T-cell-specific deletion of a polypeptide N-acetylgalactosaminyl-transferase gene by site-directed recombination.

Cited by 248 publications

References 28 publications

Essential role of TAK1 in thymocyte development and activation

Essential role of TAK1 in thymocyte development and activation

Expression of the Sphingosine 1-Phosphate Receptor, S1P1, on T-cells Controls Thymic Emigration

Cloning and Characterization of a New Human UDP-N-Acetyl-α-d-galactosamine:PolypeptideN-Acetylgalactosaminyltransferase, Designated pp-GalNAc-T13, That Is Specifically Expressed in Neurons and Synthesizes GalNAc α-Serine/Threonine Antigen

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