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Ca2+ regulates T-cell receptor activation by modulating the charge property of lipids

Nature volume 493, pages 111–115 (2013)Cite this article

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Abstract

Ionic protein–lipid interactions are critical for the structure and function of membrane receptors, ion channels, integrins and many other proteins1,2,3,4,5,6,7. However, the regulatory mechanism of these interactions is largely unknown. Here we show that Ca2+ can bind directly to anionic phospholipids and thus modulate membrane protein function. The activation of T-cell antigen receptor–CD3 complex (TCR), a key membrane receptor for adaptive immunity, is regulated by ionic interactions between positively charged CD3ε/ζ cytoplasmic domains (CD3CD) and negatively charged phospholipids in the plasma membrane1,8,9,10. Crucial tyrosines are buried in the membrane and are largely protected from phosphorylation in resting T cells. It is not clear how CD3CD dissociates from the membrane in antigen-stimulated T cells. The antigen engagement of even a single TCR triggers a Ca2+ influx11 and TCR-proximal Ca2+ concentration is higher than the average cytosolic Ca2+ concentration12. Our biochemical, live-cell fluorescence resonance energy transfer and NMR experiments showed that an increase in Ca2+ concentration induced the dissociation of CD3CD from the membrane and the solvent exposure of tyrosine residues. As a consequence, CD3 tyrosine phosphorylation was significantly enhanced by Ca2+ influx. Moreover, when compared with wild-type cells, Ca2+ channel-deficient T cells had substantially lower levels of CD3 phosphorylation after stimulation. The effect of Ca2+ on facilitating CD3 phosphorylation is primarily due to the charge of this ion, as demonstrated by the fact that replacing Ca2+ with the non-physiological ion Sr2+ resulted in the same feedback effect. Finally, 31P NMR spectroscopy showed that Ca2+ bound to the phosphate group in anionic phospholipids at physiological concentrations, thus neutralizing the negative charge of phospholipids. Rather than initiating CD3 phosphorylation, this regulatory pathway of Ca2+ has a positive feedback effect on amplifying and sustaining CD3 phosphorylation and should enhance T-cell sensitivity to foreign antigens. Our study thus provides a new regulatory mechanism of Ca2+ to T-cell activation involving direct lipid manipulation.

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Figure 1: Ca 2+ induced the dissociation of CD3ε CD from the membrane bilayer.
Figure 2: Ca 2+ induced the solvent exposure of tyrosine residues in CD3ε CD ITAM.
Figure 3: Ca 2+ facilitated CD3 phosphorylation.
Figure 4: Ca 2+ bound to the phosphate group of anionic phospholipids at physiological concentrations.

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Acknowledgements

We would like to thank R. Lewis for his gift of CJ-1 Jurkat cell line; S.-c. Sun, A. Lin, D. Li, J. J. Chou, H. Gu and M. Lei for discussions. C.X. is funded by the National Basic Research Program of China (973 Program, no. 2011CB910901 and no. 2012CB910804), the National Science Foundation of China (no. 31070738), the Chinese Academy of Sciences (Hundred Talents Program and no. KSCX2-EW-J-11), the Shanghai Municipal Commission for Science and Technology (10PJ1411500) and the Young Talent Program of Shanghai Institutes for Biological Sciences, CAS (no. 2010KIP101). J.W. is funded by the National Basic Research Program of China (973 Program, no. 2012CB917202) and the Chinese Academy of Sciences (Hundred Talents Program).

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Author notes

  1. Xiaoshan Shi, Yunchen Bi and Wei Yang: These authors contributed equally to this work.

Authors and Affiliations

  1. State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China ,

    Xiaoshan Shi, Wei Yang, Xingdong Guo, Yan Jiang, Lunyi Li, Yibing Bai, Jun Guo & Chenqi Xu

  2. National Center for Protein Science Shanghai, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 333 Haike Road, Shanghai 201203, China ,

    Xiaoshan Shi, Wei Yang, Xingdong Guo, Yan Jiang, Lunyi Li, Yibing Bai, Jun Guo & Chenqi Xu

  3. High Magnetic Field Laboratory, Hefei Institutes of Physical Science, Chinese Academy of Sciences, 350 Shushanhu Road, Hefei 230031, Anhui Province, China ,

    Yunchen Bi, Chanjuan Wan, Yujuan Wang, Bo Wu, Hongbin Sun & Junfeng Wang

  4. School of Life Sciences, Tsinghua University, Beijing, 100084, China

    Xiangjun Chen & Wanli Liu

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Contributions

C.X. and J.W. conceived the project. X.S. and W.Y. performed the biochemical and T-cell activation experiments. Y.Bi and X.S. performed NMR, circular dichroism and Fourier transform infrared experiments. X.G. and Y.J. performed the FRET experiment. W.L. and C.X. supervised the TIRFM and spinning-disk confocal microscopy experiments. X.G. performed these imaging experiments and X.C. participated in the initial part of these imaging experiments. L.L., Y.Bai and J.G. helped on protein sample preparation. C.W., Y.W., B.W. and H.S. helped on nanodisc sample preparation. C.X. wrote the manuscript. J.W. and other authors revised the manuscript.

Corresponding authors

Correspondence to Junfeng Wang or Chenqi Xu.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1-17, a Supplementary Discussion and additional references. (PDF 1986 kb)

Real time cytosolic Ca2+ images in a Jurkat T cell encountering immobilized CD3 antibodies

Ca2+ microdomains were observed at the membrane proximal region. (MOV 34879 kb)

Real time TCR proximal Ca2+ images in the immunological synapse

The result shows that Ca2+ co-localizes with TCR within the immunological synapse. (MOV 26593 kb)

Real time TCR proximal Ca2+ images in the peripheral region outside of the immunological synapse

The result shows Ca2+ co-localizes with TCR at the peripheral region (MOV 21990 kb)

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Shi, X., Bi, Y., Yang, W. et al. Ca2+ regulates T-cell receptor activation by modulating the charge property of lipids. Nature 493, 111–115 (2013). https://doi.org/10.1038/nature11699

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