Differential roles of PKC-theta in the regulation of intracellular calcium concentration in primary T cells.
Activation of T lymphocytes requires protein kinase C theta (PKC-theta) 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-theta-/- T cells, suggesting that PKC-theta plays a role in PMA-mediated inhibition of Ca2+ influx. In contrast, T cell receptor (TCR) crosslinking in the same PKC-theta-/- T cells did result in significantly decreased [Ca2+]i compared to wild-type T cells, suggesting a positive role for PKC-theta in TCR-mediated Ca2+ mobilization. In PKC-theta-/- 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-theta 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 Cgamma1 activation and inositol triphosphate production. Furthermore, knockdown of endogenous PKC-theta 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-theta-/- Jurkat cells could readily overcome the above inhibition. Thus, PKC-theta can both positively and negatively regulate the Ca2+ influx that is critical for NFAT activity.
Manicassamy
Santhakumar
S
Department of Microbiology and Immunology, College of Medicine, University of Illinois, Chicago, IL 60612, USA.
Sadim
Maureen
M
Ye
Richard D
RD
Sun
Zuoming
Z
D000818:Animals; D019703:Calcineurin / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002454:Cell Differentiation; D003432:Cross-Linking Reagents / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D066298:In Vitro Techniques; D007295:Inositol Phosphates / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics / Q000502:physiology*; D019169:Jurkat Cells; D051379:Mice; D018345:Mice, Knockout; D016328:NF-kappa B / Q000378:metabolism; D050778:NFATC Transcription Factors / Q000378:metabolism* / Q000502:physiology; D051966:Phospholipase C gamma / Q000378:metabolism; D011493:Protein Kinase C / Q000235:genetics / Q000502:physiology*; D000074121:Protein Kinase C-theta; D021381:Protein Transport; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism
D003432:Cross-Linking Reagents; D007295:Inositol Phosphates; D007527:Isoenzymes; D016328:NF-kappa B; D050778:NFATC Transcription Factors; D011948:Receptors, Antigen, T-Cell; D018808:Transcription Factor AP-1; C051853:inositol 1,3-bisphosphate; C076233:PRKCQ protein, human; C489593:Prkcq protein, mouse; D011493:Protein Kinase C; D000074121:Protein Kinase C-theta; D019703:Calcineurin; D051966:Phospholipase C gamma; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium