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Quantitative proteomic analysis of B cell lipid rafts reveals that ezrin regulates antigen receptor–mediated lipid raft dynamics

Nature Immunology volume 7, pages 625–633 (2006)Cite this article

Abstract

Ligation of the B cell antigen receptor (BCR) with antigen induces lipid raft coalescence, a process that occurs after crosslinking of a variety of signaling receptors and is thought to potentiate cellular activation. To investigate lipid raft dynamics during BCR signaling, we quantitatively analyzed the B cell lipid raft proteome. BCR engagement induced dissociation of the adaptor protein ezrin from lipid rafts as well as threonine dephosphorylation of ezrin and its concomitant detachment from actin, indicating a transient uncoupling of lipid rafts from the actin cytoskeleton. Expression of constitutively active ezrin chimeras inhibited the BCR-induced coalescence of lipid rafts. Our data demonstrate that the release of ezrin from lipid rafts acts as a critical trigger that regulates lipid raft dynamics during BCR signaling.

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Figure 1: Isolation of lipid rafts for ICAT-based quantitative mass spectrometry.
Figure 2: BCR ligation induces changes in the subcellular localization of ezrin and its association with lipid rafts and the lipid raft–associated protein Cbp.
Figure 3: BCR ligation induces changes in ezrin Thr 567 phosphorylation and in the association of ezrin with actin.
Figure 4: Dominant positive mutants of ezrin.
Figure 5: Chimeric ezrin blocks BCR-induced lipid raft coalescence.

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Acknowledgements

We thank D. Barber (University of California at San Francisco) and members of the DeFranco lab for discussions, and C. MacArthur (Howard Hughes Medical Institute, University of California at San Francisco) for cell sorting. Supported by the National Institute of Diabetes and Digestive and Kidney Diseases (DK068292 to N.G.), the National Institutes of Health (AI20038 to A.L.D. and AI41109 to J.D.W.) and the National Heart, Lung, and Blood Institute (N01-HV-28179 to the Seattle Proteome Center at the Institute for Systems Biology).

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Author notes

  1. Ruedi Aebersold

    Present address: Institute for Molecular Systems Biology, Swiss Federal Institute of Technology, CH-8093, Zurich

Authors and Affiliations

  1. Department of Microbiology and Immunology, University of California, San Francisco, 94143, California, USA

    Neetu Gupta, Barbara Scheer & Anthony L DeFranco

  2. Institute for Systems Biology, Seattle, 98103, Washington, USA

    Bernd Wollscheid, Julian D Watts & Ruedi Aebersold

  3. Faculty of Natural Science, University of Zurich, CH-8057, Zurich, Switzerland

    Ruedi Aebersold

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Correspondence to Anthony L DeFranco.

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Supplementary information

Supplementary Fig. 1

Regulation of lipid raft coalescence by ezrin. (PDF 660 kb)

Supplementary Fig. 2

Blockade of lipid raft coalesence does not affect upstream signaling events. (PDF 163 kb)

Supplementary Fig. 3

Blockade of lipid raft coalesence does not affect CD69 upregulation. (PDF 276 kb)

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Gupta, N., Wollscheid, B., Watts, J. et al. Quantitative proteomic analysis of B cell lipid rafts reveals that ezrin regulates antigen receptor–mediated lipid raft dynamics. Nat Immunol 7, 625–633 (2006). https://doi.org/10.1038/ni1337

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