Calcium-induced ERK activation in human T lymphocytes
Introduction
Increases in the level of cytosolic calcium play a key role in the activation of T lymphocytes. The intracellular concentration of calcium in resting T-lymphocytes is approximately 100 nM. This concentration is maintained against an extracellular concentration of approximately 1 mM. Following cross-linking of the T-cell receptor complex, by antibodies or mitogenic compounds, tyrosine phosphorylation of PLC-γ occurs. The increased activity of PLC-γ in turn results in the generation of inositol trisphosphate (IP3). IP3 diffuses from the cellular membrane and induces the release of calcium from intracellular stores sequestered in the endoplasmic reticulum. This release results in a rapid rise in cytoplasmic calcium levels. The depletion of the intracellular stores of calcium results in an influx of calcium across the cellular membrane giving rise to a prolonged increase in cytoplasmic calcium levels (Parekh et al., 1993, Randriamampita and Tsien, 1993, Randriamampita and Tsien, 1995).
The increases in intracellular calcium that follow T-cell receptor engagement clearly play a role in the activation of these cells. In T lymphocytes, calcium is clearly required for the production of interleukin-2 (Mills et al., 1985). When T lymphocytes are cultured in the presence of PMA they express the IL-2 receptor but fail to produce IL-2. For the production of IL-2 those studies demonstrated that a calcium flux is also required. A separate group of studies targeting the mechanism of action of the immunosuppressive compound cyclosporin A revealed one critical site on which calcium acts. Rises in intracellular calcium activate the cyclosporin A and FK506 inhibitable enzyme calcinurin. Calcinurin removes the phosphate group from the cytoplasmic element of the NF-AT allowing it to translocate to the nucleus. The nuclear form of NF-AT is then able to activate transcription of the IL-2 gene (Ho S et al., 1996, Schreiber and Crabtree, 1992).
While the activation of calcinurin is a critical event that follows increases in intracellular calcium within T lymphocytes, increases in intracellular calcium also activate kinases such as calmodulin and regulate the activity of phosphatases such as CD45 (Ostergaard and Trowbridge, 1991). Since it has previously been reported that calcium is capable of inducing the activation of the Extracellular Regulated Kinase (ERK) MAP kinases in human B-lymphocytes (Franklin et al., 1994), we determined if increases in intracellular calcium would induce the phosphorylation and activation of ERK in T lymphocytes.
Section snippets
Cells and reagents
The Jurkat cell line was obtained from ATCC (Rockville, MD). These cells were cultured in RPMI 1640 with 10% fetal calf serum, 2 mM glutamine, 100 U/ml, and 100 μg/ml streptomycin. In addition, these cells were also routinely cultured in 1% Anti-PPLO (Gibco; Grand Island, NY) to prevent mycoplasma contamination. Human peripheral blood lymphocytes were isolated from platelet-depleted blood by centrifugation over a Histopaque gradient. T lymphocytes were isolated as previously described by
Results
We previously reported that stimulation of normal resting B-lymphocytes with calcium ionophores induced the tyrosine phosphorylation and the enzymatic activation of ERK2 (Franklin et al., 1994). We have now determined that a similar response occurs in human T-lymphocytes. Stimulation of human T-lymphocytes with the calcium ionophores, ionomycin or A23187, induced the activation of both ERK1 and 2. Fig. 1 is an immunoblot of lysates from human primary T cells that were stimulated with either
Discussion
We have found that ionomycin induces the phosphorylation and activation of ERK1 and 2 in human T lymphocytes. This conclusion is based on: (1) Shifts in the mobility of ERK2 on SDS–PAGE gels; (2) The binding of an antibody that only recognizes the dual phosphorylated and hence activated form of the enzyme in immunoblots of lysates from ionomycin, but not DMSO, stimulated cells; (3) Increased MBP activity in lysates from ionomycin stimulated cells; and (4) Shifts in the mobility of p90rsk on
Acknowledgements
Supported by an institutional research grant from the American Cancer Society (IRG-97-149) and a faculty development award from East Carolina University School of Medicine awarded to R.A.F. P.A.A. was supported by a contribution from Glaxo Wellcome, Inc. MSN was a recipient of an East Carolina University School of Medicine doctoral student fellowship.
References (30)
- et al.
A potent synthetic inhibitor of the cAMP-dependent protein kinase
J. Biol. Chem.
(1986) - et al.
Activation of Ca2+/calmodulin-dependent protein kinase (CaM-kinase) IV by CaM-kinase kinase in Jurkat T lymphocytes
J. Biol. Chem.
(1995) - et al.
Mitogen-activated S6 kinase is stimulated via protein kinase C-dependent and independent pathways in swiss 3T3 cells
J. Biol. Chem.
(1987) - et al.
Degradation of a calcium influx factor (CIF) can be blocked by phosphatase inhibitors or chelation of Ca2+
J. Biol. Chem.
(1995) - et al.
The mechanism of action of cyclosporin A and FK506
Immunol. Today
(1992) - et al.
JNK is involved in signal integration during costimulation of T lymphocytes
Cell
(1994) - et al.
Involvement of p21ras in activation of extracellular signal-regulated kinase 2
Nature
(1992) - et al.
Anergic T cells are defective in both jun NH2-terminal kinase and mitogen-activated protein kinase signaling pathways
J. Exp. Med.
(1996) - et al.
Tyrosine phosphorylation of GAP and GAP-associated proteins in lymphoid and fibroblast cells expressing lck
Oncogene
(1991) - et al.
Regulation of mitogen-activated protein kinases by a calcium/calmodulin-dependent protein kinase cascade
Proc. Natl Acad. Sci. USA
(1996)
Tyrosyl phosphorylation and activation of MAP kinases by p56lck
Science
Blocked Ras activation in anergic CD4+ T cells
Science
Activation of MAP2-kinase in B lymphocytes by calcium ionophores
J. Immunol.
Evidence implicating utilization of different T cell receptor-associated signaling pathways by TH1 and TH2 clones
J. Immunol.
Translational control of interleukin 2 messenger RNA as a molecular mechanism of T cell anergy
J. Exp. Med.
Cited by (36)
ERβ regulated ovarian kisspeptin plays an important role in oocyte maturation
2021, Molecular and Cellular EndocrinologyChronic lymphocytic leukemia cells receive RAF-dependent survival signals in response to CXCL12 that are sensitive to inhibition by sorafenib
2011, BloodCitation Excerpt :We found that CXCL12 induced high levels of intracellular calcium flux in ZAP-70+ CLL cells, whereas most ZAP-70− CLL cells showed little or no intracellular calcium flux. Calcium-induced ERK activation has been described in B cells25 and T cells.26 We have previously shown that aberrant expression of ZAP-70 in CLL cells leads to increases in intracellular calcium flux in response to B-cell receptor engagement.27
Oxidative stress in the pathogenesis of psoriasis
2009, Free Radical Biology and MedicineInhibition of T cell MAPKs (Erk 1/2, p38) with thermal injury is related to down-regulation of Ca<sup>2+</sup> signaling
2005, Biochimica et Biophysica Acta - Molecular Basis of DiseaseCross-talk with Ca<sup>2+</sup> influx does not underlie the role of extracellular signal-regulated kinases in cytotoxic T lymphocyte lytic granule exocytosis
2004, Journal of Biological ChemistryCitation Excerpt :After transfer to nitrocellulose, the blot was cut in half and probed either with rabbit polyclonal antibodies against total ERK or against dually phosphorylated (i.e. active) ERK. In both cases, the blot was also probed with a rabbit polyclonal antibody specific for p90rsk to serve as a loading control (28, 29). Bands with molecular masses of ∼44 and 42 kDa were detected in the blot probed with the antibody recognizing total ERK (Fig. 1A).