Immunology Released by Calcium

Wei Wong

Science Signaling, AAAS, Washington, DC 20005, USA

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 CD3 and CD3 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 CD3 (CD3_CD) 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 CD3_CD 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 CD3_CD to shift from the membrane-bound state to the solution state. In vitro assays revealed that the tyrosine phosphorylation of CD3_CD 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 CD3 and CD3 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 CD3 and CD3 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 CD3_CD, 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]

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