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


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.


  1. Xu, C. et al. Regulation of T cell receptor activation by dynamic membrane binding of the CD3epsilon cytoplasmic tyrosine-based motif. Cell 135, 702–713 (2008)

    Article  CAS  Google Scholar 

  2. Paddock, C. et al. Residues within a lipid-associated segment of the PECAM-1 cytoplasmic domain are susceptible to inducible, sequential phosphorylation. Blood 117, 6012–6023 (2011)

    Article  CAS  Google Scholar 

  3. Hansen, S. B., Tao, X. & MacKinnon, R. Structural basis of PIP2 activation of the classical inward rectifier K+ channel Kir2.2. Nature 477, 495–498 (2011)

    Article  ADS  CAS  Google Scholar 

  4. Whorton, M. R. & MacKinnon, R. Crystal structure of the mammalian GIRK2 K+ channel and gating regulation by G proteins, PIP2, and sodium. Cell 147, 199–208 (2011)

    Article  CAS  Google Scholar 

  5. Kim, C. et al. Basic amino-acid side chains regulate transmembrane integrin signalling. Nature 481, 209–213 (2012)

    Article  ADS  CAS  Google Scholar 

  6. van den Bogaart, G. et al. Membrane protein sequestering by ionic protein–lipid interactions. Nature 479, 552–555 (2011)

    Article  ADS  CAS  Google Scholar 

  7. Heo, W. D. et al. PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to the plasma membrane. Science 314, 1458–1461 (2006)

    Article  ADS  CAS  Google Scholar 

  8. Kuhns, M. S. & Davis, M. M. The safety on the TCR trigger. Cell 135, 594–596 (2008)

    Article  CAS  Google Scholar 

  9. DeFord-Watts, L. M. et al. The CD3ζ subunit contains a phosphoinositide-binding motif that is required for the stable accumulation of TCR–CD3 complex at the immunological synapse. J. Immunol. 186, 6839–6847 (2011)

    Article  CAS  Google Scholar 

  10. Aivazian, D. & Stern, L. J. Phosphorylation of T cell receptor ζ is regulated by a lipid dependent folding transition. Nature Struct. Biol. 7, 1023–1026 (2000)

    Article  CAS  Google Scholar 

  11. Irvine, D. J., Purbhoo, M. A., Krogsgaard, M. & Davis, M. M. Direct observation of ligand recognition by T cells. Nature 419, 845–849 (2002)

    Article  ADS  CAS  Google Scholar 

  12. Lioudyno, M. I. et al. Orai1 and STIM1 move to the immunological synapse and are up-regulated during T cell activation. Proc. Natl Acad. Sci. USA 105, 2011–2016 (2008)

    Article  ADS  CAS  Google Scholar 

  13. Smith-Garvin, J. E., Koretzky, G. A. & Jordan, M. S. T cell activation. Annu. Rev. Immunol. 27, 591–619 (2009)

    Article  CAS  Google Scholar 

  14. Palacios, E. H. & Weiss, A. Function of the Src-family kinases, Lck and Fyn, in T-cell development and activation. Oncogene 23, 7990–8000 (2004)

    Article  CAS  Google Scholar 

  15. Hogan, P. G., Lewis, R. S. & Rao, A. Molecular basis of calcium signaling in lymphocytes: STIM and ORAI. Annu. Rev. Immunol. 28, 491–533 (2010)

    Article  CAS  Google Scholar 

  16. Weiss, A. & Littman, D. R. Signal transduction by lymphocyte antigen receptors. Cell 76, 263–274 (1994)

    Article  CAS  Google Scholar 

  17. van der Merwe, P. A. & Dushek, O. Mechanisms for T cell receptor triggering. Nature Rev. Immunol. 11, 47–55 (2011)

    Article  CAS  Google Scholar 

  18. Purbhoo, M. A., Irvine, D. J., Huppa, J. B. & Davis, M. M. T cell killing does not require the formation of a stable mature immunological synapse. Nature Immunol. 5, 524–530 (2004)

    Article  CAS  Google Scholar 

  19. Sigalov, A. B., Aivazian, D. A., Uversky, V. N. & Stern, L. J. Lipid-binding activity of intrinsically unstructured cytoplasmic domains of multichain immune recognition receptor signaling subunits. Biochemistry 45, 15731–15739 (2006)

    Article  CAS  Google Scholar 

  20. Leventis, P. A. & Grinstein, S. The distribution and function of phosphatidylserine in cellular membranes. Annu. Rev. Biophys. 39, 407–427 (2010)

    Article  CAS  Google Scholar 

  21. Nika, K. et al. Constitutively active Lck kinase in T cells drives antigen receptor signal transduction. Immunity 32, 766–777 (2010)

    Article  CAS  Google Scholar 

  22. Huse, M. et al. Spatial and temporal dynamics of T cell receptor signaling with a photoactivatable agonist. Immunity 27, 76–88 (2007)

    Article  CAS  Google Scholar 

  23. Fanger, C. M., Hoth, M., Crabtree, G. R. & Lewis, R. S. Characterization of T cell mutants with defects in capacitative calcium entry: genetic evidence for the physiological roles of CRAC channels. J. Cell Biol. 131, 655–667 (1995)

    Article  CAS  Google Scholar 

  24. Park, C. Y., Shcheglovitov, A. & Dolmetsch, R. The CRAC channel activator STIM1 binds and inhibits L-type voltage-gated calcium channels. Science 330, 101–105 (2010)

    Article  ADS  CAS  Google Scholar 

  25. Yeromin, A. V. et al. Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai. Nature 443, 226–229 (2006)

    Article  ADS  CAS  Google Scholar 

  26. Denisov, I. G., Grinkova, Y. V., Lazarides, A. A. & Sligar, S. G. Directed self-assembly of monodisperse phospholipid bilayer nanodiscs with controlled size. J. Am. Chem. Soc. 126, 3477–3487 (2004)

    Article  CAS  Google Scholar 

  27. Quintana, A. et al. Calcium microdomains at the immunological synapse: how ORAI channels, mitochondria and calcium pumps generate local calcium signals for efficient T-cell activation. EMBO J. 30, 3895–3912 (2011)

    Article  CAS  Google Scholar 

  28. Varma, R., Campi, G., Yokosuka, T., Saito, T. & Dustin, M. L. T cell receptor-proximal signals are sustained in peripheral microclusters and terminated in the central supramolecular activation cluster. Immunity 25, 117–127 (2006)

    Article  CAS  Google Scholar 

  29. Huppa, J. B., Gleimer, M., Sumen, C. & Davis, M. M. Continuous T cell receptor signaling required for synapse maintenance and full effector potential. Nature Immunol. 4, 749–755 (2003)

    Article  CAS  Google Scholar 

  30. Zilly, F. E. et al. Ca2+ induces clustering of membrane proteins in the plasma membrane via electrostatic interactions. EMBO J. 30, 1209–1220 (2011)

    Article  CAS  Google Scholar 

  31. Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995)

    Article  CAS  Google Scholar 

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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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Authors and Affiliations



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.

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Correspondence to Junfeng Wang or Chenqi Xu.

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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).

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