Metabolic labelling of membrane microdomains/rafts in Jurkat cells indicates the presence of glycerophospholipids implicated in signal transduction by the CD3 T-cell receptor

2002 ◽  
Vol 363 (3) ◽  
pp. 645-655 ◽  
Author(s):  
Alexandre K. ROUQUETTE-JAZDANIAN ◽  
Claudette PELASSY ◽  
Jean-Philippe BREITTMAYER ◽  
Jean-Louis COUSIN ◽  
Claude AUSSEL
Keyword(s):  
Fatty Acids ◽  
T Cells ◽  
T Cell ◽  
Protein Kinase ◽  
Lipid Rafts ◽  
T Cell Activation ◽  
Cell Activation ◽  
Saturated Fatty Acids ◽  
Membrane Microdomains ◽  
Metabolic Labelling

Cell membranes contain sphingolipids and cholesterol, which cluster together in distinct domains called rafts. The outer-membrane leaflet of these peculiar membrane domains contains glycosylphosphatidylinositol-anchored proteins, while the inner leaflet contains proteins implicated in signalling, such as the acylated protein kinase p56lck and the palmitoylated adaptator LAT (linker for activation of T-cells). We present here an approach to study the lipid composition of rafts and its change upon T-cell activation. Our method is based on metabolic labelling of Jurkat T-cells with different precursors of glycerophospholipid synthesis, including glycerol and fatty acids with different lengths and degrees of saturation as well as phospholipid polar head groups. The results obtained indicate that lipid rafts isolated by the use of sucrose density-gradient centrifugation after Triton X-100 extraction in the cold, besides sphingolipids and cholesterol, contain unambiguously all classes of glycerophospholipids: phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine. Fatty acid labelling shows that lipid rafts are labelled preferentially with saturated fatty acids while the rest of the plasma membrane incorporates mostly long-chained polyunsaturated fatty acids. To see whether the raft composition as measured by metabolic labelling of phospholipids is involved in T-cell activation, we investigated the production of sn-1,2-diacylglycerol (DAG) in CD3-activated cells. DAG production occurs within rafts, confirming previous demonstration of protein kinase C translocation into membrane microdomains. Our data demonstrate that raft disorganization by methyl-β-cyclodextrin impairs both CD3-induced DAG production and changes in cytosolic Ca2+ concentration. These lines of evidence support the conclusion that the major events in T-cell activation occur within or due to lipid rafts.

Association of CD26 with CD45RA outside lipid rafts attenuates cord blood T-cell activation

Blood ◽  
2004 ◽  
Vol 103 (3) ◽  
pp. 1002-1010 ◽  
Author(s):  
Seiji Kobayashi ◽  
Kei Ohnuma ◽  
Masahiko Uchiyama ◽  
Kouichi Iino ◽  
Satoshi Iwata ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cord Blood ◽  
Lipid Rafts ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cell Receptor ◽  
Cell Protein ◽  
Membrane Microdomains

AbstractCD26 is a T-cell activation antigen that contains dipeptidyl peptidase IV activity and binds adenosine deaminase. Recent work showed that specialized membrane microdomains, also known as lipid rafts, play a key role in T-cell signaling. In this study, we investigate the role of CD26 in cord blood T-cell activation and signal transduction. We demonstrated that different expression levels of CD26 were observed between cord blood T cells (CBTCs) and peripheral blood T cells (PBTCs) and that CD26+CD45RA+ CBTCs were different compared with CD26+CD45RA+ PBTCs. Moreover, the comitogenic effect of CD26 was not as pronounced in CBTCs as in PBTCs. We also showed that CD26 cross-linking induced less phosphorylation of T-cell receptor-signaling molecules, lymphoid T-cell protein tyrosine kinase (Lck), zeta-associated protein 70 (ZAP-70), T-cell receptor ζ (TCRζ), and linker for activator of T cells (LAT) in CBTCs than in PBTCs. Furthermore, CD26 molecules associated with CD45RA molecules outside lipid rafts in CBTCs. Our results suggest that strong physical linkage of CD26 with CD45RA outside lipid rafts may be responsible for the attenuation of T-cell activation signaling through CD26, which may be responsible for immature immune response and the low incidence of severe graft-versus-host disease in cord blood transplantation. (Blood. 2004;103:1002-1010)

scholarly journals Modulating p56Lck in T-Cells by a Chimeric Peptide Comprising Two Functionally Different Motifs of Tip fromHerpesvirus saimiri

2015 ◽  
Vol 2015 ◽  
pp. 1-12
Author(s):  
Jean-Paul Vernot ◽  
Ana María Perdomo-Arciniegas ◽  
Luis Alberto Pérez-Quintero ◽  
Diego Fernando Martínez
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lipid Rafts ◽  
T Cell Activation ◽  
Cell Activation ◽  
Jurkat Cells ◽  
Chimeric Peptide ◽  
Vector Targeting

The Lck interacting protein Tip ofHerpesvirus saimiriis responsible for T-cell transformation bothin vitroandin vivo. Here we designed the chimeric peptide hTip-CSKH, comprising the Lck specific interacting motif CSKH of Tip and its hydrophobic transmembrane sequence (hTip), the latter as a vector targeting lipid rafts. We found that hTip-CSKH can induce a fivefold increase in proliferation of human andAotussp. T-cells. Costimulation with PMA did not enhance this proliferation rate, suggesting that hTip-CSKH is sufficient and independent of further PKC stimulation. We also found that human Lck phosphorylation was increased earlier after stimulation when T-cells were incubated previously with hTip-CSKH, supporting a strong signalling and proliferative effect of the chimeric peptide. Additionally, Lck downstream signalling was evident with hTip-CSKH but not with control peptides. Importantly, hTip-CSKH could be identified in heavy lipid rafts membrane fractions, a compartment where important T-cell signalling molecules (LAT, Ras, and Lck) are present during T-cell activation. Interestingly, hTip-CSKH was inhibitory to Jurkat cells, in total agreement with the different signalling pathways and activation requirements of this leukemic cell line. These results provide the basis for the development of new compounds capable of modulating therapeutic targets present in lipid rafts.

scholarly journals A Model System for Activation-Induced Alternative Splicing of CD45 Pre-mRNA in T Cells Implicates Protein Kinase C and Ras

2000 ◽  
Vol 20 (1) ◽  
pp. 70-80 ◽  
Author(s):  
Kristen W. Lynch ◽  
Arthur Weiss
Keyword(s):  
T Cells ◽  
Protein Kinase C ◽  
Alternative Splicing ◽  
T Cell ◽  
Protein Kinase ◽  
Cell Line ◽  
T Cell Activation ◽  
Cell Activation ◽  
Model System ◽  
Kinase C

ABSTRACT Multiple isoforms of the protein tyrosine phosphatase CD45 are expressed on the surface of human T cells. Interestingly, the expression of these isoforms has been shown to vary significantly upon T-cell activation. In this report, we describe a novel cell line-based model system in which we can mimic the activation-induced alternative splicing of CD45 observed in primary T cells. Of the many proximal signaling events induced by T-cell stimulation, we show that activation of protein kinase C and activation of Ras are important for the switch toward the exclusion of CD45 variable exons, whereas events related to Ca2+ flux are not. In addition, the ability of cycloheximide to block the activation-induced alternative splicing of CD45 suggests a requirement for de novo protein synthesis. We further demonstrate that sequences which have previously been implicated in the tissue-specific regulation of CD45 variable exons are likewise necessary and sufficient for activation-induced splicing. These results provide an initial understanding of the requirements for CD45 alternative splicing upon T-cell activation, and they confirm the importance of this novel cell line in facilitating a more detailed analysis of the activation-induced regulation of CD45 than has been previously possible.

scholarly journals The coreceptor CD2 uses plasma membrane microdomains to transduce signals in T cells

2009 ◽  
Vol 185 (3) ◽  
pp. 521-534 ◽  
Author(s):  
Yoshihisa Kaizuka ◽  
Adam D. Douglass ◽  
Santosh Vardhana ◽  
Michael L. Dustin ◽  
Ronald D. Vale
Keyword(s):  
T Cells ◽  
Plasma Membrane ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Adaptor Protein ◽  
Cell Receptor ◽  
Signaling Molecules ◽  
Membrane Microdomains ◽  
Receptor Interaction

The interaction between a T cell and an antigen-presenting cell (APC) can trigger a signaling response that leads to T cell activation. Prior studies have shown that ligation of the T cell receptor (TCR) triggers a signaling cascade that proceeds through the coalescence of TCR and various signaling molecules (e.g., the kinase Lck and adaptor protein LAT [linker for T cell activation]) into microdomains on the plasma membrane. In this study, we investigated another ligand–receptor interaction (CD58–CD2) that facilities T cell activation using a model system consisting of Jurkat T cells interacting with a planar lipid bilayer that mimics an APC. We show that the binding of CD58 to CD2, in the absence of TCR activation, also induces signaling through the actin-dependent coalescence of signaling molecules (including TCR-ζ chain, Lck, and LAT) into microdomains. When simultaneously activated, TCR and CD2 initially colocalize in small microdomains but then partition into separate zones; this spatial segregation may enable the two receptors to enhance signaling synergistically. Our results show that two structurally distinct receptors both induce a rapid spatial reorganization of molecules in the plasma membrane, suggesting a model for how local increases in the concentration of signaling molecules can trigger T cell signaling.

scholarly journals Protein Kinase C-Mediated Phosphorylation of BCL11B at Serine 2 Negatively Regulates Its Interaction with NuRD Complexes during CD4+T-Cell Activation

2016 ◽  
Vol 36 (13) ◽  
pp. 1881-1898 ◽  
Author(s):  
Marion Dubuissez ◽  
Ingrid Loison ◽  
Sonia Paget ◽  
Han Vorng ◽  
Saliha Ait-Yahia ◽  
...  
Keyword(s):  
T Cells ◽  
Protein Kinase C ◽  
T Cell ◽  
Protein Kinase ◽  
T Cell Activation ◽  
Cell Activation ◽  
Regulatory Protein ◽  
Mitogen Activated Protein Kinase ◽  
Kinase C ◽  
Tcr Activation

The transcription factor BCL11B/CTIP2 is a major regulatory protein implicated in various aspects of development, function and survival of T cells. Mitogen-activated protein kinase (MAPK)-mediated phosphorylation and SUMOylation modulate BCL11B transcriptional activity, switching it from a repressor in naive murine thymocytes to a transcriptional activator in activated thymocytes. Here, we show that BCL11B interacts via its conserved N-terminal MSRRKQ motif with endogenous MTA1 and MTA3 proteins to recruit various NuRD complexes. Furthermore, we demonstrate that protein kinase C (PKC)-mediated phosphorylation of BCL11B Ser2 does not significantly impact BCL11B SUMOylation but negatively regulates NuRD recruitment by dampening the interaction with MTA1 or MTA3 (MTA1/3) and RbAp46 proteins. We detected increased phosphorylation of BCL11B Ser2 uponin vivoactivation of transformed and primary human CD4+T cells. We show that following activation of CD4+T cells, BCL11B still binds toIL-2andId2promoters but activates their transcription by recruiting P300 instead of MTA1. Prolonged stimulation results in the direct transcriptional repression ofBCL11Bby KLF4. Our results unveil Ser2 phosphorylation as a new BCL11B posttranslational modification linking PKC signaling pathway to T-cell receptor (TCR) activation and define a simple model for the functional switch of BCL11B from a transcriptional repressor to an activator during TCR activation of human CD4+T cells.

The molecular machinery for cAMP-dependent immunomodulation in T-cells

2006 ◽  
Vol 34 (4) ◽  
pp. 476-479 ◽  
Author(s):  
K. Taskén ◽  
A.J. Stokka
Keyword(s):  
T Cell ◽  
Immune Responses ◽  
Lipid Rafts ◽  
T Cell Activation ◽  
Negative Feedback ◽  
Cell Activation ◽  
Cell Receptor ◽  
Membrane Microdomains ◽  
Type I ◽  
Anchoring Protein

cAMP inhibits Src-family kinase signalling by PKA (protein kinase A)-mediated phosphorylation and activation of Csk (C-terminal Src kinase). The PKA type I–Csk pathway is assembled and localized in membrane microdomains (lipid rafts) and regulates immune responses activated through the TCR (T-cell receptor). PKA type I is targeted to the TCR–CD3 complex during T-cell activation via an AKAP (A-kinase-anchoring protein) that serves as a scaffold for the cAMP–PKA/Csk pathway in lipid rafts of the plasma membrane during T-cell activation. Displacement of PKA by anchoring disruption peptides prevents cAMP/PKA type I-mediated inhibition of T-cell activation. These findings provide functional evidence that PKA type I regulation of T-cell responses is dependent on AKAP anchoring. Furthermore, we show that upon TCR/CD28 co-ligation, β-arrestin in complex with PDE4 (phosphodiesterase 4) is recruited to lipid rafts. The CD28-mediated recruitment of PDE4 to lipid rafts potentiates T-cell immune responses and counteracts the local, TCR-induced production of cAMP that produces negative feedback in the absence of a co-receptor stimulus. The specific recruitment of PDE4 thus serves to abrogate the negative feedback by cAMP which is elicited in the absence of a co-receptor stimulus.

scholarly journals Recruitment of Slp-76 to the Membrane and Glycolipid-Enriched Membrane Microdomains Replaces the Requirement for Linker for Activation of T Cells in T Cell Receptor Signaling

2000 ◽  
Vol 192 (7) ◽  
pp. 1047-1058 ◽  
Author(s):  
Nancy J. Boerth ◽  
Jeffrey J. Sadler ◽  
Daniel E. Bauer ◽  
James L. Clements ◽  
Shereen M. Gheith ◽  
...  
Keyword(s):  
Amino Acids ◽  
T Cells ◽  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Promoter Activity ◽  
Cell Activation ◽  
Cell Receptor ◽  
Membrane Microdomains ◽  
Tcr Signaling

Two hematopoietic-specific adapters, src homology 2 domain–containing leukocyte phosphoprotein of 76 kD (SLP-76) and linker for activation of T cells (LAT), are critical for T cell development and T cell receptor (TCR) signaling. Several studies have suggested that SLP-76 and LAT function coordinately to promote downstream signaling. In support of this hypothesis, we find that a fraction of SLP-76 localizes to glycolipid-enriched membrane microdomains (GEMs) after TCR stimulation. This recruitment of SLP-76 requires amino acids 224–244. The functional consequences of targeting SLP-76 to GEMs for TCR signaling are demonstrated using a LAT/SLP-76 chimeric protein. Expression of this construct reconstitutes TCR-inducted phospholipase Cγ1 phosphorylation, extracellular signal–regulated kinase activation, and nuclear factor of activated T cells (NFAT) promoter activity in LAT-deficient Jurkat T cells (J.CaM2). Mutation of the chimeric construct precluding its recruitment to GEMs diminishes but does not eliminate its ability to support TCR signaling. Expression of a chimera that lacks SLP-76 amino acids 224–244 restores NFAT promoter activity, suggesting that if localized, SLP-76 does not require an association with Gads to promote T cell activation. In contrast, mutation of the protein tyrosine kinase phosphorylation sites of SLP-76 in the context of the LAT/SLP-76 chimera abolishes reconstitution of TCR function. Collectively, these experiments show that optimal TCR signaling relies on the compartmentalization of SLP-76 and that one critical function of LAT is to bring SLP-76 and its associated proteins to the membrane.

scholarly journals Cutting Edge: Localization of Linker for Activation of T Cells to Lipid Rafts Is Not Essential in T Cell Activation and Development

2004 ◽  
Vol 174 (1) ◽  
pp. 31-35 ◽  
Author(s):  
Minghua Zhu ◽  
Shudan Shen ◽  
Yan Liu ◽  
Olivia Granillo ◽  
Weiguo Zhang
Keyword(s):  
T Cells ◽  
T Cell ◽  
Lipid Rafts ◽  
T Cell Activation ◽  
Cell Activation ◽  
Cutting Edge

scholarly journals Ionotrophic 5-hydroxytryptamine type 3 receptor activates the protein kinase C-dependent phospholipase D pathway in human T-cells

1999 ◽  
Vol 344 (1) ◽  
pp. 199-204 ◽  
Author(s):  
Naim Akhtar KHAN ◽  
Aziz HICHAMI
Keyword(s):  
T Cells ◽  
Protein Kinase C ◽  
T Cell ◽  
Protein Kinase ◽  
T Cell Activation ◽  
Phospholipase D ◽  
Cell Activation ◽  
Jurkat T Cells ◽  
Kinase C ◽  
Gf 109203X

The present study was undertaken to investigate the role of the 5-hydroxytryptamine (5-HT) ionotrophic receptor 5-HT3 in the activation of human Jurkat T-cells. 5-HT and 2-methyl-5-HT (2Me-5-HT), an agonist of the 5-HT3 receptor, induced increases in intracellular free Na+ concentrations, [Na+]i, via opening of the ionotrophic receptor in these cells. These two serotonergic (5-hydroxytryptaminergic) agents potentiated phytohaemagglutinin (PHA)-induced T-cell activation. However, they failed to potentiate dioctanoglycerol-plus-ionomycin-stimulated T-cell blastogenesis. Interestingly, an inhibitor of protein kinase C (PKC), GF 109203X, curtailed significantly 5-HT and 2Me-5-HT-potentiated T-cell activation. These results demonstrate that the opening of the 5-HT3 ionotrophic receptor is implicated in T-cell activation via the PKC pathway. Furthermore, 5-HT and 2Me-5-HT stimulated phospholipase D (PLD) activity, as measured by the production of phosphatidylethanol and phosphatidylbutanol at the expense of phosphatidic acid (PA). GF 109203X significantly curtailed the 5-HT- and 2Me-5-HT-induced PLD activity and T-cell activation. The PLD/PA pathway stimulated by these two serotonergic agents resulted in the production of 1,2-diacylglycerol (DAG) mass in Jurkat T-cells. These results altogether suggest that 5-HT and 2Me-5-HT potentiate T-cell activation via increases in [Na+]i and the activation of the PKC-dependent PLD pathway.

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