A Role for Protein Kinase CβI in the Regulation of Ca2+ Entry in Jurkat T Cells

Abstract

T cell activation leading to cytokine production and cellular proliferation involves a regulated increase and subsequent decrease in the intracellular concentration of Ca 2؉ ([Ca 2؉ ] i ). While much is understood about agonist-induced increases in [Ca 2؉ ] i , less is known about down-regulation of this pathway. Understanding the mechanism of this down-regulation is critical to the prevention of cell death that can be the consequence of a sustained elevation in [Ca 2؉ ] i . Protein kinase C (PKC), activated by the diacylglycerol produced as a consequence of T cell receptor engagement, has long been presumed to be involved in this down-regulation, although the precise mechanism is not wholly clear. In this report we demonstrate that activation of PKC by phorbol esters slightly decreases the rate of Ca 2؉ efflux from the cytosol of Jurkat T cells following stimulation through the T cell receptor or stimulation in a receptorindependent manner by thapsigargin. On the other hand, phorbol ester treatment dramatically reduces the rate of Ca 2؉ influx following stimulation. Phorbol ester treatment is without an effect on Ca 2؉ influx in a different T cell line, HSB. Down-regulation of PKC␤I expression by 18-h phorbol ester treatment is associated with a loss of the response to acute phorbol ester treatment in Jurkat cells, suggesting that PKC␤I may be the isozyme responsible for the effects on Ca 2؉ influx. Electroporation of an anti-PKC␤I antibody, but not antibodies against PKC␣ or PKC␥, led to an increase in the rate of Ca 2؉ influx following stimulation. Taken together, these data suggest that PKC␤I may be a component of the down-regulation of increases in [Ca 2؉ ] i associated with Jurkat T cell activation.Activation of T lymphocytes via stimulation through the T cell receptor for antigen (TCR) 1 leading to proliferation, cytokine production, or effector function requires a regulated increase and subsequent decrease in the intracellular concentration of Ca 2ϩ ([Ca 2ϩ ] i ). The biochemical events associated with activation of T lymphocytes and leading to Ca 2ϩ entry have been extensively studied (1). Engagement of the TCR leads to the activation of phospholipase C, which subsequently cleaves phosphatidylinositol bisphosphate into inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG). IP 3 , binding to an intracellular receptor, induces the release of Ca 2ϩ from an intracellular storage depot (2). In a receptor-independent manner, the Ca 2ϩ /ATPase inhibitor thapsigargin (3) causes an uncompensated leak of Ca 2ϩ from this internal storage pool. In T lymphocytes, as well as in most electrically nonexcitable cells, this depletion of the intracellular storage pool induces the opening of the plasma membrane Ca 2ϩ entry pathway and permits influx of extracellular Ca 2ϩ (1). Previously, we have provided evidence that the pathway between release of intracellular Ca 2ϩ and influx of extracellular Ca 2ϩ is mediated by Ca 2ϩ -activated calmodulin (4), which permits Ca 2ϩ entry carried by a current we have called I T...

Inhibition of the Calcium Release-activated Calcium (CRAC) Current in Jurkat T Cells by the HIV-1 Envelope Protein gp160

“…Indeed a short incubation of untreated Jurkat cells with the phorbolester TPA, a broad spectrum activator of classic and novel PKC isozymes, mimicked the effects of gp160 and reduced the CRAC current. This agrees with a study by Haverstick et al (39) showing that Jurkat cells incubated with PMA, another phorbol-ester PKC activator, had a reduced increase in [Ca 2ϩ ] i after activation by anti-CD3 antibody or TG.…”

“…We investigated the effects of Gö 6976, which is considered as inhibiting preferentially PKC␣ and PKC␤I (37)(38). This agent is interesting because PKC␣ is the predominant isoform in Jurkat cells, and PKC␤I has been implicated in I CRAC downmodulation (39). On gp160-treated cells, Gö 6976 (5 M) increased the time to reach the peak current after the addition of 10 mM CaCl 2 (Fig.…”

“…In particular, it does not inhibit PKC␦, -⑀, and - (38). This suggests the implication of PKC␣, an abundant PKC component, or PKC␤I, a less abundant isoform in Jurkat cells (39). Second, immunoblotting experiments indicate that gp160 had profound effects on both the protein amounts and the subcellular distribution of PKC␣ and -␤I, whereas PKC␦ and -were only weakly disturbed.…”

“…The (42), sphingosine and several ceramides in Jurkat T cells (43), and PKC in mast cells (44) and Jurkat T cells (39). A PKC-dependent process also seems to be involved in the reduction of I CRAC by gp160.…”

Redox modulation of intracellular free calcium concentration in thyroid FRTL-5 cells: evidence for an enhanced extrusion of calcium

“…Interestingly, thimerosal did not depolarize the membrane potential, although it potently stimulated [$H]phorbol 12,13-dibutyrate binding. However, in HEL cells and in Jurkat T-cells, activation of the PKC β isoenzyme specifically abrogates calcium entry [59,60]. At present we do not know which isoenzymes of PKC are activated by thimerosal in our cells.…”