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Conformational states of the kinase Lck regulate clustering in early T cell signaling

Nature Immunology volume 14, pages 82–89 (2013)Cite this article

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Abstract

Phosphorylation of the T cell antigen receptor (TCR) by the tyrosine kinase Lck is an essential step in the activation of T cells. Because Lck is constitutively active, spatial organization may regulate TCR signaling. Here we found that Lck distributions on the molecular level were controlled by the conformational states of Lck, with the open, active conformation inducing clustering and the closed, inactive conformation preventing clustering. In contrast, association with lipid domains and protein networks were not sufficient or necessary for Lck clustering. Conformation-driven Lck clustering was highly dynamic, so that TCR triggering resulted in Lck clusters that contained phosphorylated TCRs but excluded the phosphatase CD45. Our data suggest that Lck conformational states represent an intrinsic mechanism for the intermolecular organization of early T cell signaling.

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Figure 1: TCR activation recruits Lck into pre-existing clusters.
Figure 2: Clustering of Lck10 and wild-type Lck.
Figure 3: Cluster analysis of Lck mutants in open and closed conformations.
Figure 4: Association with protein networks does not contribute to the clustering of Lck.
Figure 5: Live cell PALM imaging of wild-type Lck and Lck(Y505F).
Figure 6: Lck clusters segregate from CD45 clusters and localize together with phosphorylated TCRζ after activation of the TCR.

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Acknowledgements

We thank T. Harder (University of Oxford) for mammalian expression constructs encoding full-length wild-type human Lck and Lck(Y505F); O. Acuto (University of Oxford) for Lck(Y394F) and Lck(R273A); J. Wiedenmann (University of Southampton) for the mEos2 expression construct; and K. Nika and O. Acuto for discussions. Supported by the Australian Research Council, the National Health and Medical Research Council of Australia and the Human Frontier Science Program.

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Authors and Affiliations

  1. Centre for Vascular Research and Australian Centre for Nanomedicine, University of New South Wales, Sydney, Australia

    Jérémie Rossy, Dylan M Owen, David J Williamson, Zhengmin Yang & Katharina Gaus

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  1. Jérémie Rossy
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  2. Dylan M Owen
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  3. David J Williamson
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Contributions

J.R. conceived of and did PALM and dSTORM experiments and data analysis and prepared the manuscript; D.M.O. conceived of and analyzed PALM and dSTORM data; D.J.W. did and analyzed PALM experiments; Z.Y. contributed to molecular biology; and K.G. was responsible for conceptualization and manuscript preparation.

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Correspondence to Katharina Gaus.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 (PDF 4957 kb)

Supplementary Video 1

High clustering dynamics of wild-type Lck during TCR activation. (AVI 3524 kb)

Supplementary Video 2

Reorganization of a wild-type Lck cluster during TCR activation. (AVI 261 kb)

Supplementary Video 3

Clustering dynamics of Y505F Lck during TCR activation. (AVI 1908 kb)

Supplementary Video 4

Stability of a cluster of Y505F Lck during TCR activation. (AVI 565 kb)

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Rossy, J., Owen, D., Williamson, D. et al. Conformational states of the kinase Lck regulate clustering in early T cell signaling. Nat Immunol 14, 82–89 (2013). https://doi.org/10.1038/ni.2488

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