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.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
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)
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)
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)
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)
Kim, C. et al. Basic amino-acid side chains regulate transmembrane integrin signalling. Nature 481, 209–213 (2012)
van den Bogaart, G. et al. Membrane protein sequestering by ionic protein–lipid interactions. Nature 479, 552–555 (2011)
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)
Kuhns, M. S. & Davis, M. M. The safety on the TCR trigger. Cell 135, 594–596 (2008)
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)
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)
Irvine, D. J., Purbhoo, M. A., Krogsgaard, M. & Davis, M. M. Direct observation of ligand recognition by T cells. Nature 419, 845–849 (2002)
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)
Smith-Garvin, J. E., Koretzky, G. A. & Jordan, M. S. T cell activation. Annu. Rev. Immunol. 27, 591–619 (2009)
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)
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)
Weiss, A. & Littman, D. R. Signal transduction by lymphocyte antigen receptors. Cell 76, 263–274 (1994)
van der Merwe, P. A. & Dushek, O. Mechanisms for T cell receptor triggering. Nature Rev. Immunol. 11, 47–55 (2011)
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)
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)
Leventis, P. A. & Grinstein, S. The distribution and function of phosphatidylserine in cellular membranes. Annu. Rev. Biophys. 39, 407–427 (2010)
Nika, K. et al. Constitutively active Lck kinase in T cells drives antigen receptor signal transduction. Immunity 32, 766–777 (2010)
Huse, M. et al. Spatial and temporal dynamics of T cell receptor signaling with a photoactivatable agonist. Immunity 27, 76–88 (2007)
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)
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)
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)
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)
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)
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)
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)
Zilly, F. E. et al. Ca2+ induces clustering of membrane proteins in the plasma membrane via electrostatic interactions. EMBO J. 30, 1209–1220 (2011)
Delaglio, F. et al. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293 (1995)
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).
The authors declare no competing financial interests.
This file contains Supplementary Figures 1-17, a Supplementary Discussion and additional references. (PDF 1986 kb)
Ca2+ microdomains were observed at the membrane proximal region. (MOV 34879 kb)
The result shows that Ca2+ co-localizes with TCR within the immunological synapse. (MOV 26593 kb)
The result shows Ca2+ co-localizes with TCR at the peripheral region (MOV 21990 kb)
About this article
Cite this article
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
Arachidonic acid-regulated calcium signaling in T cells from patients with rheumatoid arthritis promotes synovial inflammation
Nature Communications (2021)
Potentiating CD8+ T cell antitumor activity by inhibiting PCSK9 to promote LDLR-mediated TCR recycling and signaling
Protein & Cell (2021)
Cellular & Molecular Immunology (2020)
Cellular & Molecular Immunology (2020)
Nature Communications (2019)