Editors' ChoiceImmunology

Released by Calcium

Sci. Signal.  08 Jan 2013:
Vol. 6, Issue 257, pp. ec8
DOI: 10.1126/scisignal.2003941

Recognition of ligand by the T cell receptor (TCR) initiates signaling by triggering the tyrosine phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the cytoplasmic domains of associated CD3 subunits. In unstimulated T cells, the cytoplasmic domains of CD3ε and CD3ζ are inserted into the plasma membrane to render the ITAMs inaccessible to tyrosine kinases, such as Lck. Shi et al. noted the presence of Ca2+ microdomains around the TCR in stimulated T cells and investigated whether Ca2+ influx triggered by TCR engagement participated in the disassociation of CD3 ITAMs from the plasma membrane. A construct consisting of the cytoplasmic domain of CD3ε (CD3εCD) bound to vesicles composed of the anionic phospholipid POPG, an interaction that was attenuated by the addition of Ca2+. In Jurkat cells, the fluorescence resonance energy transfer (FRET) efficiency between fluorescently tagged CD3εCD and a membrane dye was decreased by ionomycin treatment (which induces Ca2+ influx) or by TCR cross-linking. Adding Ca2+ to POPG bicelles caused the amide resonance spectra of Tyr38 and Tyr49 in the ITAM of CD3εCD to shift from the membrane-bound state to the solution state. In vitro assays revealed that the tyrosine phosphorylation of CD3εCD by Lck was abrogated by the addition of POPG bicelles, an effect that was rescued by the addition of Ca2+. In Jurkat cells and primary mouse T cells, the tyrosine phosphorylation of CD3ε and CD3ζ in response to TCR ligation was enhanced by the presence of extracellular Ca2+ and decreased by intracellular chelation of Ca2+. The divalent cation Sr2+ can enter T cells in response to TCR ligation but not activate Ca2+-dependent signaling pathways, and it mimicked the increase in tyrosine phosphorylation of CD3ε and CD3ζ induced by extracellular Ca2+, suggesting the importance of the electrostatic charge of Ca2+ in promoting CD3 phosphorylation. Nuclear magnetic resonance spectroscopy showed that the phosphorus signal of POPG in lipid nanodiscs underwent similar chemical shift changes with the addition of Ca2+ or CD3εCD, suggesting that both bound to the same lipid phosphate region. Thus, Ca2+ that enters stimulated T cells disrupts the ionic interaction between the cytoplasmic domain of CD3 and the plasma membrane, thus making the ITAMs in CD3 accessible for tyrosine phosphorylation.

X. Shi, Y. Bi, W. Yang, X. Guo, Y. Jiang, C. Wan, L. Li, Y. Bai, J. Guo, Y. Wang, X. Chen, B. Wu, H. Sun, W. Liu, J. Wang, C. Xu, Ca2+ regulates T-cell receptor activation by modulating the charge property of lipids. Nature 493, 111–115 (2013). [PubMed]