Opposing roles of serine/threonine kinases MEKK1 and LOK in regulating the CD28 responsive element in T-cells

2002 ◽  
Vol 363 (1) ◽  
pp. 175-182 ◽  
Author(s):  
Li TAO ◽  
Scott WADSWORTH ◽  
Jason MERCER ◽  
Cynthia MUELLER ◽  
Kirsten LYNN ◽  
...  
Keyword(s):  
T Cell ◽  
Reporter Gene ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Jurkat Cells ◽  
Dominant Negative Mutant ◽  
Nuclear Factor ◽  
Response Element ◽  
Enhancer Element

T-cell activation requires signals from both the T-cell receptor (TcR) and other co-stimulatory molecules such as CD28. TcR- and CD28-mediated signals are integrated during T-cell activation resulting in the expression of cytokine genes such as interleukin-2 (IL-2). An enhancer element (CD28RE) of the IL-2 gene specifically responsive to CD28 signals has been previously identified and characterized. This response element and an adjacent Activated Protein-1 (nuclear factor-interleukin-2B) site together (RE/AP1) were shown to complex with c-rel, AP-1 and other factors. However, details of the signal transduction pathways leading from CD28 to the composite response element remain poorly understood. We present data showing that overexpression of the serine threonine kinase, mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase kinase-1 (MEKK1), but not nuclear factor-κB inducing kinase, or MAP kinase/ERK kinase-1 (MEK1), can significantly increase the level of CD28RE/AP1-driven luciferase (Luc) reporter gene expression in Jurkat E6-1 cells. A MEKK1 dominant negative mutant blocked such activation induced by stimulation with Raji B cells and the superantigen staphylococcus enterotoxin E (SEE), as well as via CD3/CD28. Mutations in either site of the RE/AP1 element abolished MEKK1-induced Luc expression. Calcineurin inhibitors, CsA and FK520, or inhibitors of p38 kinase (SB 203580), or MEK1 (PD 098059), did not affect MEKK1-induced reporter activation. These results directly implicate MEKK1 in the CD28 signalling pathway that activates the CD28 response element. Co-expression of the lymphocyte-oriented kinase (LOK) kinase attenuated Raji/SEE-induced IL-2 production in Jurkat cells, as well as MEKK1 and Raji/SEE-induced reporter gene activation. These data suggest that MEKK1 and LOK may have opposing roles in regulating the CD28RE/AP1 element.

scholarly journals Signalling through CD28 T-cell activation pathway involves an inositol phospholipid-specific phospholipase C activity

1993 ◽  
Vol 293 (3) ◽  
pp. 835-842 ◽  
Author(s):  
J Nunes ◽  
S Klasen ◽  
M D Franco ◽  
C Lipcey ◽  
C Mawas ◽  
...  
Keyword(s):  
T Cell ◽  
Phospholipase C ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Jurkat Cells ◽  
Cd28 Molecule ◽  
Human T Cell ◽  
Protein Kinase C Inhibitor ◽  
Human T Cell Line

Stimulation of the human T-cell line, Jurkat, by a monoclonal antibody (mAb) directed against the CD28 molecule leads to sustained increases in intracellular levels of Ca2+ ([Ca2+]i); the initial rise in Ca2+ comes from internal stores, followed by Ca2+ entry into the cells. The CD28 molecule also appears to activate polyphosphoinositide (InsPL)-specific phospholipase C (PLC) activity in Jurkat cells, as demonstrated by PtdInsP2 breakdown, InsP3 and 1,2-diacylglycerol generation and PtdIns resynthesis. We also observed that interleukin-2 (IL2) production induced via CD28 triggering was sensitive to a selective protein kinase C inhibitor. Of the four other anti-CD28 mAbs (CD28.2, CD28.4, CD28.5, CD28.6) tested, only one (CD28.5) was unable to generate any InsPL-specific PLC or IL2 secretion. However, the cross-linking of cell-bound CD28.5 with anti-mouse Ig antibodies led to an increase in [Ca2+]i. CD28-molecule clustering in itself appears to be a sufficient signal for induction of PLC activity.

scholarly journals Submicromolar La3+ concentrations block the calcium release-activated channel, and impair CD69 and CD25 expression in CD3- or thapsigargin-activated Jurkat cells

1996 ◽  
Vol 313 (3) ◽  
pp. 909-913 ◽  
Author(s):  
Claude AUSSEL ◽  
Rachid MARHABA ◽  
Claudette PELASSY ◽  
Jean-Philippe BREITTMAYER
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Calcium Release ◽  
Cell Signalling ◽  
Interleukin 2 ◽  
Jurkat Cells ◽  
T Cell Signalling ◽  
A Chain ◽  
20 Nm

The calcium release-activated channel (CRAC) opened in Jurkat cells activated either with CD3 monoclonal antibody or the endoplasmic reticulum Ca2+-ATPase blocker, thapsigargin, is blocked by La3+ with an IC50 of 20 nM. Similarly, the entry of Mn2+, used as a surrogate for Ca2+, is also blocked by submicromolar La3+ concentrations. La3+ seems to play its role simply by plugging the CRAC because this ion does not penetrate the cells, as demonstrated by chelation experiments with EGTA. Blocking the Ca2+ influx in activated Jurkat cells results in a lack of expression of CD25, a chain of the interleukin-2 receptor and of CD69, a marker of T-cell activation. By contrast, the very early steps of the T-cell signalling pathway such as the release of Ca2+ from intracellular stores and the subsequent inhibition of phosphatidylserine synthesis are not affected by La3+.

scholarly journals p21ras and calcineurin synergize to regulate the nuclear factor of activated T cells.

1993 ◽  
Vol 178 (5) ◽  
pp. 1517-1522 ◽  
Author(s):  
M Woodrow ◽  
N A Clipstone ◽  
D Cantrell
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Protein Tyrosine Kinase ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Cytosolic Calcium ◽  
Nuclear Factor ◽  
Activated T Cells ◽  
Guanine Nucleotide Binding Protein

In T lymphocytes, triggering of the T cell receptor (TCR) induces several signaling cascades which ultimately synergize to induce the activity of the nuclear factor of activated T cells (NFAT), a DNA binding complex critical to the inducibility and T cell specificity of the T cell growth factor interleukin 2. One immediate consequence of T cell activation via the TCR is an increase in cytosolic calcium. Calcium signals are important for NFAT induction, and recent studies have identified calcineurin, a calcium-calmodulin dependent serine-threonine phosphatase, as a prominent component of the calcium signaling pathway in T cells. A second important molecule in TCR signal transduction is the guanine nucleotide binding protein, p21ras, which is coupled to the TCR by a protein tyrosine kinase dependent mechanism. The experiments presented here show that expression by transfection of mutationally activated calcineurin or activated p21ras alone is insufficient for NFAT transactivation. However, coexpression of the activated calcineurin with activated p21ras could mimic TCR signals in NFAT induction. These data identify calcineurin and p21ras as cooperative partners in T cell activation.

scholarly journals GABP factors bind to a distal interleukin 2 (IL-2) enhancer and contribute to c-Raf-mediated increase in IL-2 induction.

1997 ◽  
Vol 17 (8) ◽  
pp. 4381-4389 ◽  
Author(s):  
A Avots ◽  
A Hoffmeyer ◽  
E Flory ◽  
A Cimanis ◽  
U R Rapp ◽  
...  
Keyword(s):  
T Cell ◽  
Protein Kinase ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Published Data ◽  
Distal Enhancer ◽  
Binding Studies ◽  
Enhancer Element ◽  
Signal Cascades

Triggering of the T-cell receptor-CD3 complex activates two major signal cascades in T lymphocytes, (i) Ca2+-dependent signal cascades and (ii) protein kinase cascades. Both signal cascades contribute to the induction of the interleukin 2 (IL-2) gene during T-cell activation. Prominent protein kinase cascades are those that activate mitogen-activated protein (MAP) kinases. We show here that c-Raf, which is at the helm of the classic MAP-Erk cascade, contributes to IL-2 induction through a distal enhancer element spanning the nucleotides from positions -502 to -413 in front of the transcriptional start site of the IL-2 gene. Induction of this distal IL-2 enhancer differs from induction of the proximal IL-2 promoter-enhancer, since it is induced by phorbol esters alone and independent from Ca2+ signals. In DNA-protein binding studies, we detected the binding of transcription factors GABP alpha and -beta to a dyad symmetry element (DSE) of the distal enhancer, which is formed by palindromic binding sites of Ets-like factors. Introduction of point mutations suppressing GABP binding to the DSE interfered with the induction of the distal enhancer and the entire IL-2 promoter-enhancer, while overexpression of both GABP factors enhanced the IL-2 promoter-enhancer induction. Overexpression of BXB, a constitutive active version of c-Raf, and of further members of the Ras-Raf-Erk signal cascade exerted an increase of GABP-mediated promoter-enhancer induction. In conjunction with previously published data on c-Raf-induced phosphorylation of GABP factors (E. Flory, A. Hoffmeyer, U. Smola, U. R. Rapp, and J. T. Bruder, J. Virol. 70:2260-2268, 1996), these results indicate a contribution of GABP factors to the Raf-mediated enhancement of IL-2 induction during T-cell activation.

scholarly journals The Interleukin 2 Receptor α Chain/CD25 Promoter Is a Target for Nuclear Factor of Activated T Cells

1998 ◽  
Vol 188 (7) ◽  
pp. 1369-1373 ◽  
Author(s):  
Kai Schuh ◽  
Thomas Twardzik ◽  
Burkhard Kneitz ◽  
Jörg Heyer ◽  
Anneliese Schimpl ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Receptor Expression ◽  
Transcriptional Level ◽  
Nuclear Factor ◽  
Close Link ◽  
Α Chain

The expression of the murine interleukin (IL)-2 receptor α chain/CD25 is strongly induced at the transcriptional level after T cell activation. We show here that nuclear factor of activated T cell (NF-AT) factors are involved in the control of CD25 promoter induction in T cells. NF-ATp and NF-ATc bind to two sites around positions −585 and −650 located upstream of the proximal CD25 promoter. Immediately 3′ from these NF-AT motifs, nonconsensus sites are located for the binding of AP-1–like factors. Mutations of sites that suppress NF-AT binding impair the induction and strong NF-ATp–mediated transactivation of the CD25 promoter in T cells. In T lymphocytes from NF-ATp–deficient mice, the expression of CD25 is severely impaired, leading to a delayed IL-2 receptor expression after T cell receptor (TCR)/CD3 stimulation. Our data indicate an important role for NF-AT in the faithful expression of high affinity IL-2 receptors and a close link between the TCR-mediated induction of IL-2 and IL-2 receptor α chain promoters, both of which are regulated by NF-AT factors.

scholarly journals Antiapoptotic Function of Cdk9 (TAK/P-TEFb) in U937 Promonocytic Cells

2001 ◽  
Vol 75 (3) ◽  
pp. 1220-1228 ◽  
Author(s):  
Shannon M. Foskett ◽  
Romi Ghose ◽  
Derek Ng Tang ◽  
Dorothy E. Lewis ◽  
Andrew P. Rice
Keyword(s):  
T Cell Activation ◽  
Transcriptional Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Dominant Negative ◽  
Jurkat Cells ◽  
Dominant Negative Mutant ◽  
U937 Cells ◽  
Hiv 1 ◽  
Promonocytic Cells

ABSTRACT Cdk9 is the catalytic subunit of TAK (cyclinT1/P-TEFb), a cellular protein kinase that mediates human immunodeficiency virus type 1 (HIV-1) Tat transcriptional activation function. To examine Cdk9 function in cells relevant to HIV-1 infection, we used a murine leukemia virus retrovirus vector to transduce and overexpress the cDNA of a dominant negative mutant Cdk9 protein (Cdk9-dn) in Jurkat T cells and U937 promonocytic cells. In Jurkat cells, overexpression of Cdk9-dn specifically inhibited Tat transactivation and HIV-1 replication but had no inhibitory effect on induction of CD69, CD25, and interleukin-2 following T-cell activation. In U937 cells, overexpression of Cdk9-dn sensitized cells to apoptosis, especially after phorbol myristate acetate (PMA) treatment to induce differentiation to macrophage-like cells. Because Cdk9 function is induced in PMA-treated U937 cells, Cdk9 may play an antiapoptotic role during monocyte differentiation.

Thiopental Inhibits the Activation of Nuclear Factor κB

2002 ◽  
Vol 96 (5) ◽  
pp. 1202-1213 ◽  
Author(s):  
Torsten Loop ◽  
Zhiheng Liu ◽  
Matjaz Humar ◽  
Alexander Hoetzel ◽  
Albert Benzing ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Reporter Gene ◽  
T Cell Activation ◽  
Cell Activation ◽  
Target Genes ◽  
Interleukin 2 ◽  
Gene Activity ◽  
Jurkat Cells ◽  
Reporter Gene Activity ◽  
Nf Kappab

Background Thiopental is frequently used for the treatment of intracranial hypertension after severe head injury. Its long-term administration increases the incidence of nosocomial infections, which contributes to the high mortality rate of these patients. However, the mechanism of its immunosuppressing effect remains unknown. Methods The effect of thiopental (200-1000 microg/ml) on the activation of the nuclear transcription factor kappaB (NF-kappaB; electrophoretic mobility shift assays), on NF-kappaB-driven reporter gene activity (transient transfection assays), on the expression of NF-kappaB target genes (enzyme-linked immunoassays), on T-cell activation (flow cytometric analyses of CD69 expression), and on the content of the NF-kappaB inhibitor IkappaB-alpha (Western blotting) was studied in human T lymphocytes in vitro. Results Thiopental inhibited the activation of the transcription factor NF-kappaB but did not alter the activity of the cyclic adenosine monophosphate response element binding protein. Other barbiturates (methohexital), anesthetics (etomidate, propofol, ketamine), or opioids (fentanyl, morphine) did not affect NF-kappaB activation. Thiopental-mediated suppression of NF-kappaB could be observed in Jurkat cells and in primary CD3+ lymphocytes from healthy volunteers, was time- and concentration-dependent, occurred at concentrations that are clinically achieved, and persisted for hours after the incubation. It was associated with an inhibition of NF-kappaB-driven reporter gene activity, of the expression of interleukin-2, -6, and -8, and interferon gamma, and of the activation of CD3+ lymphocytes. Suppression of NF-kappaB appeared to involve reduced degradation of IkappaB-alpha. Conclusion The results demonstrate that thiopental inhibits the activation of NF-kappaB and may thus provide a molecular mechanism for some of the immunosuppressing effects associated with thiopental therapy.

scholarly journals RelA is a potent transcriptional activator of the CD28 response element within the interleukin 2 promoter.

1995 ◽  
Vol 15 (8) ◽  
pp. 4260-4271 ◽  
Author(s):  
J H Lai ◽  
G Horvath ◽  
J Subleski ◽  
J Bruder ◽  
P Ghosh ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Interleukin 2 ◽  
Specific Antigen ◽  
Transcriptional Activator ◽  
Nf Kappa B ◽  
Enhancer Activity ◽  
Response Element

T-cell activation requires two different signals. The T-cell receptor's recognition of a specific antigen on antigen-presenting cells provides one, and the second signal comes from costimulatory molecules such as CD28. In contrast, T cells that are stimulated with antigen in the absence of the CD28 costimulatory signal can become anergic (nonresponsive). The CD28 response element (CD28RE) has been identified as the DNA element mediating interleukin 2 (IL-2) gene activation by CD28 costimulation. Our previous work demonstrates that the Rel/NF-kappa B family proteins c-Rel, RelA (p65), and NFKB1 (p50) are involved in the complex that binds to the CD28RE. We also showed that c-Rel, but not NFKB1 (p50), can bind to the CD28RE and activate CD28RE-driven transcription in cotransfection assays. However, the role of RelA (p65) in CD28 signaling has not yet been addressed. We provide evidence that RelA (p65) itself bound directly to the CD28RE of the IL-2 promoter and other lymphokine promoters. In addition, RelA (p65) was a potent transcriptional activator of the CD28RE in vivo. We show that a RelA (p65)-c-Rel heterodimer bound to the CD28RE and synergistically activated the CD28RE enhancer activity. We also demonstrate that activated Raf-1 kinase synergized with RelA (p65) in activating the CD28RE enhancer activity. Interestingly, a soluble anti-CD28 monoclonal antibody alone, in the absence of other stimuli, also synergized with RelA (p65) in activating the CD28RE. Furthermore, we show that RelA (p65) activated expression of the wild-type IL-2 promoter but not the CD28RE-mutated IL-2 promoter. A combination of RelA (p65) and NFKB1 (p50) also activated the IL-2 promoter through the CD28RE site. These results demonstrate the functional regulation of the CD28RE, within the IL-2 promoter, by Rel/NF-kappa B transcription factors.

scholarly journals Phosphorylation of two cytosolic proteins. An early event of T-cell activation

1989 ◽  
Vol 258 (2) ◽  
pp. 505-510 ◽  
Author(s):  
J F Peyron ◽  
C Aussel ◽  
B Ferrua ◽  
H Häring ◽  
M Fehlmann
Keyword(s):  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Peptide Mapping ◽  
Interleukin 2 ◽  
Jurkat Cells ◽  
Early Event ◽  
Ionophore A23187 ◽  
Receptor Complex ◽  
Cytosolic Proteins

T lymphocytes can be activated to proliferate by triggering the T-cell antigen-receptor complex (CD3-Ti) with anti-CD3 (Cluster of Differentiation 3) monoclonal antibody (mAb) or with the mitogenic lectin phytohaemagglutinin A (PHA). We have investigated the relationship between lymphocyte activation and protein phosphorylation in the human leukaemic T-cell line Jurkat. Incubation of 32P-labelled Jurkat cells with anti-CD3 mAb or PHA induced the phosphorylation of two cytosolic proteins that migrate with apparent Mr values of 21,000 (pp21) and 23,000 (pp23) and pI values of 5.1 and 5.0 respectively. Peptide mapping of the two proteins produced the same phosphopeptides pattern, suggesting that pp21 and pp23 are closely related. The phosphorylation of pp21 and pp23 induced by anti-CD3 mAb appeared to be transient, since it was already detected 2 min after the addition of the mAb, reached a maximum at 10 min and recovered its basal level after 1 h. Phosphorylation of pp21 and pp23 could also be elicited by the Ca2+ ionophore A23187 and sodium orthovanadate (Na3VO4), two agents that bypass the T-cell-receptor complex and produced an increase in cytosolic Ca2+ concentration. In addition, we found that vanadate, like the Ca2+ ionophore, induced the secretion of interleukin-2 (IL-2) when used in combination with a submitogenic concentration of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate. These results show that the Ca2+-dependent phosphorylation of pp21 and pp23 represents an early event in the process of signal transduction through the CD3-Ti receptor complex.

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