Phosphorylation of the ETS-2 protein: regulation by the T-cell antigen receptor-CD3 complex

1990 ◽  
Vol 10 (3) ◽  
pp. 1249-1253
Author(s):  
S Fujiwara ◽  
S Koizumi ◽  
R J Fisher ◽  
N K Bhat ◽  
T S Papas
Keyword(s):  
T Cell ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Antigen Receptor ◽  
Jurkat Cells ◽  
Mobility Shift ◽  
T Cell Antigen Receptor ◽  
Cell Antigen Receptor ◽  
Cell Antigen

Phosphorylation of the human ets-2 protein in response to mitogenic signals to T lymphocytes was investigated in Jurkat cells. Activation of the cells by antibodies against the T-cell antigen receptor-CD3 complex or by concanavalin A was followed within 5 min by increased phosphorylation of the protein, as shown by a mobility shift of the protein from 54 to 56 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and increased incorporation of 32P. The Ca2+ ionophores A23187 and ionomycin were able to mimic this effect, suggesting that this phosphorylation is mediated by Ca2+.

scholarly journals Phosphorylation of the ETS-2 protein: regulation by the T-cell antigen receptor-CD3 complex.

1990 ◽  
Vol 10 (3) ◽  
pp. 1249-1253 ◽  
Author(s):  
S Fujiwara ◽  
S Koizumi ◽  
R J Fisher ◽  
N K Bhat ◽  
T S Papas
Keyword(s):  
T Cell ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Antigen Receptor ◽  
Jurkat Cells ◽  
Mobility Shift ◽  
T Cell Antigen Receptor ◽  
Cell Antigen Receptor ◽  
Cell Antigen

Phosphorylation of the human ets-2 protein in response to mitogenic signals to T lymphocytes was investigated in Jurkat cells. Activation of the cells by antibodies against the T-cell antigen receptor-CD3 complex or by concanavalin A was followed within 5 min by increased phosphorylation of the protein, as shown by a mobility shift of the protein from 54 to 56 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and increased incorporation of 32P. The Ca2+ ionophores A23187 and ionomycin were able to mimic this effect, suggesting that this phosphorylation is mediated by Ca2+.

scholarly journals A 275 basepair fragment at the 5' end of the interleukin 2 gene enhances expression from a heterologous promoter in response to signals from the T cell antigen receptor.

1987 ◽  
Vol 165 (2) ◽  
pp. 395-407 ◽  
Author(s):  
D B Durand ◽  
M R Bush ◽  
J G Morgan ◽  
A Weiss ◽  
G R Crabtree
Keyword(s):  
T Cell ◽  
Cell Line ◽  
Antigen Receptor ◽  
Calcium Ionophore ◽  
Jurkat Cells ◽  
T Cell Antigen Receptor ◽  
Linked Gene ◽  
Cell Antigen Receptor ◽  
Cell Antigen ◽  
Flanking Region

Using a transient transfection assay, we have defined the sequences required for the activation of the IL-2 gene in response to signals from the T cell antigen receptor. To do so we have transfected the human T cell line Jurkat with hybrid DNA constructs in which fragments from the IL-2 gene are linked to an indicator gene. The indicator gene product, as well as IL-2 production from the endogenous IL-2 gene were assayed after activation of the transfected Jurkat cells by various stimuli. We have demonstrated that a 275 bp fragment stretching from 52 to 326 bp upstream of the IL-2 gene transcription initiation site is required for expression of the linked indicator gene. This IL-2 gene fragment has several of the characteristics of a transcriptional enhancer element, in that it functions in both orientations and will enhance the expression from the promoter of an unrelated gene. Such enhancement occurred only after activation of Jurkat cells through the T cell antigen receptor. More specifically, this 275 bp fragment activated the expression of a linked gene after binding of a monoclonal antibody to the Jurkat T cell antigen receptor in the presence of PMA. In addition, calcium ionophore, which circumvents antigen receptor binding in T cell activation, induced the expression of the linked gene through this 275 bp sequence, in the presence of PMA. Finally, in a mutant Jurkat cell line lacking T3/antigen receptor complexes at the cell surface, no expression due to the IL-2 5' flanking region was seen after exposure to antibody to the T cell antigen receptor plus PMA or to PHA plus PMA. In contrast, calcium ionophore plus PMA did induce the expression of a linked gene through the IL-2 5' flanking region in the mutant Jurkat cell line. The responsiveness of the transfected hybrid genes containing the IL-2 regulatory region paralleled the expression of the endogenous IL-2 gene, as determined by IL-2 bioassays. We conclude that the 275 bp IL-2 sequence (-326 to -52 bp) is a target for the signal pathway originating at the T cell antigen receptor. Definition of this 275 bp target sequence should now permit the isolation of the molecules that bind to and activate the IL-2 gene.

scholarly journals ZAP-70-Independent Ca2+ Mobilization and Erk Activation in Jurkat T Cells in Response to T-Cell Antigen Receptor Ligation

2001 ◽  
Vol 21 (21) ◽  
pp. 7137-7149 ◽  
Author(s):  
Xiaochuan Shan ◽  
Richard Balakir ◽  
Gabriel Criado ◽  
Jason S. Wood ◽  
Maria-Cristina Seminario ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Tyrosine Phosphorylation ◽  
Antigen Receptor ◽  
Jurkat Cells ◽  
Jurkat T Cells ◽  
T Cell Antigen Receptor ◽  
Erk Activation ◽  
Cell Antigen Receptor ◽  
Cell Antigen

ABSTRACT The tyrosine kinase ZAP-70 has been implicated as a critical intermediary between T-cell antigen receptor (TCR) stimulation and Erk activation on the basis of the ability of dominant negative ZAP-70 to inhibit TCR-stimulated Erk activation, and the reported inability of anti-CD3 antibodies to activate Erk in ZAP-70-negative Jurkat cells. However, Erk is activated in T cells receiving a partial agonist signal, despite failing to activate ZAP-70. This discrepancy led us to reanalyze the ZAP-70-negative Jurkat T-cell line P116 for its ability to support Erk activation in response to TCR/CD3 stimulation. Erk was activated by CD3 cross-linking in P116 cells. However, this response required a higher concentration of anti-CD3 antibody and was delayed and transient compared to that in Jurkat T cells. Activation of Raf-1 and MEK-1 was coincident with Erk activation. Remarkably, the time course of Ras activation was comparable in the two cell lines, despite proceeding in the absence of LAT tyrosine phosphorylation in the P116 cells. CD3 stimulation of P116 cells also induced tyrosine phosphorylation of phospholipase C-γ1 (PLCγ1) and increased the intracellular Ca2+ concentration. Protein kinase C (PKC) inhibitors blocked CD3-stimulated Erk activation in P116 cells, while parental Jurkat cells were refractory to PKC inhibition. The physiologic relevance of these signaling events is further supported by the finding of PLCγ1 tyrosine phosphorylation, Erk activation, and CD69 upregulation in P116 cells on stimulation with superantigen and antigen-presenting cells. These results demonstrate the existence of two pathways leading to TCR-stimulated Erk activation in Jurkat T cells: a ZAP-70-independent pathway requiring PKC and a ZAP-70-dependent pathway that is PKC independent.

scholarly journals The T-cell antigen receptor regulates sustained increases in cytoplasmic free Ca2+ through extracellular Ca2+ influx and ongoing intracellular Ca2+ mobilization

1987 ◽  
Vol 247 (3) ◽  
pp. 695-700 ◽  
Author(s):  
J B Imboden ◽  
A Weiss
Keyword(s):  
T Cell ◽  
Antigen Receptor ◽  
Jurkat Cells ◽  
Elevated Concentration ◽  
T Cell Antigen Receptor ◽  
Cell Antigen Receptor ◽  
Cell Antigen ◽  
Human T Cell ◽  
Inositol 1,4,5 Trisphosphate ◽  
Inositol Tetrakisphosphate

Signal transduction by the T-cell antigen receptor involves the turnover of polyphosphoinositides and an increase in the concentration of cytoplasmic free Ca2+ ([Ca2+]i). This increase in [Ca2+]i is due initially to the release of Ca2+ from intracellular stores, but is sustained by the influx of extracellular Ca2+. To examine the regulation of sustained antigen-receptor-mediated increases in [Ca2+]i, we studied the relationships between extracellular Ca2+ influx, the mobilization of Ca2+ from intracellular stores, and the contents of inositol polyphosphates after stimulation of the antigen receptor on a human T-cell line, Jurkat. We demonstrate that sustained antigen-receptor-mediated increases in [Ca2+]i are associated with ongoing depletion of intracellular Ca2+ stores. When antigen-receptor-ligand interactions are disrupted, [Ca2+]i and inositol 1,4,5-trisphosphate return to basal values over 3 min. Under these conditions, intracellular Ca2+ stores are repleted if extracellular Ca2+ is present. There is a tight temporal relationship between the fall in [Ca2+]i, the return of inositol 1,4,5-trisphosphate to basal values, and the repletion of intracellular Ca2+ stores. Reversal of the increase in [Ca2+]i preceeds any fall in inositol tetrakisphosphate by 2 min. These studies suggest that sustained antigen-receptor-induced increases in [Ca2+]i, although dependent on extracellular Ca2+ influx, are also regulated by ongoing inositol 1,4,5-trisphosphate-mediated intracellular Ca2+ mobilization. In addition, an elevated concentration of inositol tetrakisphosphate in itself is insufficient to sustain an increase in [Ca2+]i within Jurkat cells.

scholarly journals Two independently regulated Ca2+ entry mechanisms coexist in Jurkat T cells during T cell receptor antigen activation

1993 ◽  
Vol 293 (2) ◽  
pp. 395-398 ◽  
Author(s):  
S C Chow ◽  
G E Kass ◽  
S Orrenius
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Antigen Receptor ◽  
Cell Receptor ◽  
Jurkat Cells ◽  
Receptor Complex ◽  
T Cell Antigen Receptor ◽  
Bivalent Cation ◽  
Cell Antigen Receptor ◽  
Cell Antigen

Receptor-mediated Ca2+ influx was studied in the human leukaemic T cell line, Jurkat. Stimulation of these cells through the T cell antigen-receptor complex with OKT3 (an antibody against the CD3 molecules of the T cell antigen-receptor complex), or inhibition of the endoplasmic reticular Ca(2+)-ATPase with thapsigargin, resulted in Ca2+ mobilization from intracellular stores and the activation of Ca2+ and Mn2+ entry. The rates of thapsigargin-induced Ca2+ and Mn2+ entry in Jurkat cells were 76% and 64% respectively of those observed after treatment of these cells with OKT3. The combined addition of thapsigargin plus OKT3 to Jurkat cells produced an enhanced effect on the sustained increase in the cytosolic free Ca2+ concentration that was greater than that obtained by addition of thapsigargin or OKT3 alone. The rates of Ca2+ and Mn2+ entry were increased to 119% and 112% respectively of the OKT3-induced rates. Taken together, these results suggest that the inositol 1,4,5-trisphosphate-sensitive Ca(2+)-pool-dependent bivalent cation entry only accounts for 57% and 52% respectively of the total OKT3-dependent Ca2+ and Mn2+ entry, and that the rest is mediated by second messenger(s). Thus two separate pathways coexist in regulating Ca2+ entry in Jurkat cells during activation mediated through the T cell receptor.

scholarly journals Transmembrane signalling by the T cell antigen receptor. Perturbation of the T3-antigen receptor complex generates inositol phosphates and releases calcium ions from intracellular stores.

1985 ◽  
Vol 161 (3) ◽  
pp. 446-456 ◽  
Author(s):  
J B Imboden ◽  
J D Stobo
Keyword(s):  
T Cell ◽  
Calcium Ions ◽  
Inositol Phosphates ◽  
Inositol Trisphosphate ◽  
Antigen Receptor ◽  
Jurkat Cells ◽  
Receptor Complex ◽  
T Cell Antigen Receptor ◽  
Cell Antigen Receptor ◽  
Cell Antigen

Antibodies against the T3-antigen receptor complex can activate the human T cell line, Jurkat, to produce interleukin 2 (2-5). This activation is initiated by a receptor-mediated increase in the concentration of free cytoplasmic calcium ions [Ca2+]i (3, 4). In this communication, we investigate the mechanism by which the receptor complex increases [Ca2+ )i in Jurkat cells. The initial receptor-mediated change in [Ca2+]i can occur when extracellular Ca2+ is depleted by EGTA. Perturbation of the T cell antigen receptor, therefore, generates a signal which mobilizes Ca2+ from intracellular stores. As inositol trisphosphate appears to function as such a signal for certain hormone receptors, we measured the levels of inositol trisphosphate and of the other inositol phosphate compounds in Jurkat. Antibodies to either the antigen receptor heterodimer or T3 determinants result in marked elevations of all three inositol phosphates. These changes in inositol phosphates are not secondary to the receptor-mediated increases in [Ca2+]i as demonstrated by the inability of the Ca2+ ionophore, ionomycin, to affect the levels of any of these compounds. In concentrations between 0.1 and 1 microM, purified inositol trisphosphate releases Ca2+ from permeabilized Jurkat cells. Taken together, these data indicate that, during activation, perturbation of the T3-antigen receptor complex generates inositol trisphosphate. This compound functions as an intracellular signal to release Ca2+ from intracellular stores, leading to increases in [Ca2+]i.

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