TY - JOUR
T1 - Differential roles of PKC-θ in the regulation of intracellular calcium concentration in primary T cells
AU - Manicassamy, Santhakumar
AU - Sadim, Maureen
AU - Ye, Richard D.
AU - Sun, Zuoming
N1 - Funding Information:
We thank Drs Amnon Altman and Xin Lin for providing expression plasmids of various isoforms of PKCs, Dr Reuven Agami for the pSuper plasmid, Drs Prasad Kanteti and Bellur Prabhakar for critically reading the manuscript and helpful discussion. This work was supported by grants from American Cancer Society of Illinois Division, Schweppe Foundation, UIC Cancer center and UIC IRB and NIH R01-AI053147-01.
PY - 2006/1/20
Y1 - 2006/1/20
N2 - Activation of T lymphocytes requires protein kinase C theta (PKC-θ) and an appropriately elevated free intracellular Ca2+ concentration ([Ca2+]i). Here, we show that phorbol 12 myristate 13-acetate (PMA) inhibited Ca2+ influx in wild-type but not PKC-θ-/- T cells, suggesting that PKC-θ plays a role in PMA-mediated inhibition of Ca2+ influx. In contrast, T cell receptor (TCR) crosslinking in the same PKC-θ-/- T cells did result in significantly decreased [Ca2+]i compared to wild-type T cells, suggesting a positive role for PKC-θ in TCR-mediated Ca 2+ mobilization. In PKC-θ-/- mice, peripheral mature T cells, but not developing thymocytes, displayed significantly decreased TCR-induced Ca2+ influx and nuclear factor of activated T cells (NFAT) translocation upon sub-optimal TCR crosslinking. The decreased intracellular free Ca2+ was due to changes in Ca2+ influx but not efflux, as observed in extracellular and intracellular Ca2+ mobilization studies. However, these differences in Ca2+ influx and nuclear factor of activated T cells (NFAT) translocation disappeared with increasing intensity of TCR crosslinking. The enhancing effect of PKC-θ on Ca2+ influx is not only dependent on the strength of TCR crosslinking but also on the developmental stage of T cells. The underlying mechanism involved phospholipase Cγ1 activation and inositol triphosphate production. Furthermore, knockdown of endogenous PKC-θ expression in Jurkat cells resulted in significant inhibition of TCR-induced activation of NFAT, as evidenced from NFAT reporter studies. Forced expression of a constitutively active form of calcineurin in PKC-θ-/- Jurkat cells could readily overcome the above inhibition. Thus, PKC-θ can both positively and negatively regulate the Ca2+ influx that is critical for NFAT activity.
AB - Activation of T lymphocytes requires protein kinase C theta (PKC-θ) and an appropriately elevated free intracellular Ca2+ concentration ([Ca2+]i). Here, we show that phorbol 12 myristate 13-acetate (PMA) inhibited Ca2+ influx in wild-type but not PKC-θ-/- T cells, suggesting that PKC-θ plays a role in PMA-mediated inhibition of Ca2+ influx. In contrast, T cell receptor (TCR) crosslinking in the same PKC-θ-/- T cells did result in significantly decreased [Ca2+]i compared to wild-type T cells, suggesting a positive role for PKC-θ in TCR-mediated Ca 2+ mobilization. In PKC-θ-/- mice, peripheral mature T cells, but not developing thymocytes, displayed significantly decreased TCR-induced Ca2+ influx and nuclear factor of activated T cells (NFAT) translocation upon sub-optimal TCR crosslinking. The decreased intracellular free Ca2+ was due to changes in Ca2+ influx but not efflux, as observed in extracellular and intracellular Ca2+ mobilization studies. However, these differences in Ca2+ influx and nuclear factor of activated T cells (NFAT) translocation disappeared with increasing intensity of TCR crosslinking. The enhancing effect of PKC-θ on Ca2+ influx is not only dependent on the strength of TCR crosslinking but also on the developmental stage of T cells. The underlying mechanism involved phospholipase Cγ1 activation and inositol triphosphate production. Furthermore, knockdown of endogenous PKC-θ expression in Jurkat cells resulted in significant inhibition of TCR-induced activation of NFAT, as evidenced from NFAT reporter studies. Forced expression of a constitutively active form of calcineurin in PKC-θ-/- Jurkat cells could readily overcome the above inhibition. Thus, PKC-θ can both positively and negatively regulate the Ca2+ influx that is critical for NFAT activity.
KW - Ca influx
KW - NFAT
KW - PKC-theta
KW - T cells
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U2 - 10.1016/j.jmb.2005.10.043
DO - 10.1016/j.jmb.2005.10.043
M3 - Article
C2 - 16309697
AN - SCOPUS:28944445460
SN - 0022-2836
VL - 355
SP - 347
EP - 359
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 3
ER -