Abstract
The adaptor Nck links receptor signaling to cytoskeleton regulation. Here we found that Nck also controlled the phosphatidylinositol-3-OH kinase (PI(3)K)–kinase Akt pathway by recruiting the adaptor BCAP after activation of B cells. Nck bound directly to the B cell antigen receptor (BCR) via the non–immunoreceptor tyrosine-based activation motif (ITAM) phosphorylated tyrosine residue at position 204 in the tail of the immunoglobulin-α component. Genetic ablation of Nck resulted in defective BCR signaling, which led to hampered survival and proliferation of B cells in vivo. Indeed, antibody responses in Nck-deficient mice were also considerably impaired. Thus, we demonstrate a previously unknown adaptor function for Nck in recruiting BCAP to sites of BCR signaling and thereby modulating the PI(3)K-Akt pathway in B cells.
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Acknowledgements
We thank M. Way (Cancer Research UK) for constructs expressing His-Nck1 and His-Nck2; N. Engels (Georg-August-Universität Göttingen) for the CD8–immunoglobulin-α chimeric construct; M. Reth (Max Planck Institute of Immunobiology and Epigenetics) for the mouse expressing Cre from the promoter of the gene encoding MB-1 and the Syk-GFP-pcDNA3 plasmid; F. Bladt (Merck) and B. Arnold (German Cancer Research Centre and Institute of Molecular Biology) for mice with conditional deficiency in Nck1 or Nck1-Nck2; T. Kurosaki (Osaka University) for antibodies M1 and M4 (each anti-IgM); B. Montaner for help with breeding and maintenance of the mouse colony; S. Kjaer of the Protein Purification Facility (Cancer Research UK) for help with the expression of recombinant proteins; A. Weston and L. Collins of the Electron Microscopy Unit (Cancer Research UK) for assistance in the imaging of DT40 cells by electron microscopy; N. O'Reilly of the Peptide Synthesis Unit (Cancer Research UK) for help in the production of biotinylated peptides and peptide arrays; D. McCarthy of the Electron Microscopy Division (Cancer Research UK) for help in imaging naive B cells by electron microscopy; and all members of the Lymphocyte Interaction Laboratory (Cancer Research UK) for discussions and comments. Supported by Cancer Research UK and the Royal Society (F.D.B.).
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A.C. designed and analyzed peptide arrays, total internal reflection fluorescence and confocal microscopy, 'biolayer interferometry', ELISPOT assays, enzyme-linked immunosorbent assays (ELISAs) of basal antibodies, flow cytometry analysis of the development, activation, survival and proliferation mouse B cells and quantitative RT-PCR in vitro and in vivo and prepared the manuscript; M.G. contributed to the in vivo studies; M.E., J.T. and J.W. assisted in the design of the studies, generated the Nck-deficient DT40 cell line, designed and generated the DNA constructs, immunoprecipitation assays and Ca2+-mobilization measurements in DT40 cells and contributed to the preparation of the manuscript; T.O. did and analyzed SILAC–tandem mass spectrometry; K.-H.L., A.T. and T.P. provided the Nck-deficient mice; and F.D.B. conceived of the study and prepared the manuscript.
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Supplementary Figures 1–7 (PDF 3010 kb)
Nck is recruited at the sites of antigen clustering together with the kinase Syk
TIRF microscopy time-lapse imaging of wild-type DT40 cells expressing cit-Nck (green) together with Syk-cherry (blue) and spreading over M1-Alexa633 (red) containing planar lipid bilayers. (MOV 2535 kb)
Nck is recruited at the sites of antigen clustering in primary murine B cells
TIRF microscopy time-lapse imaging of purified primary murine B cells transduced with a retrovirus coding for a GFP-tagged version of Nck (green) and spreading over Alexa633-anti-κ chain-containing (red) planar lipid bilayers. (MOV 5441 kb)
WT and Nck1-2 KO B cells exhibit comparable migratory dynamics in lymph nodes under steady state
Multiphoton microscopy time-lapse imaging of SNARF-labeled wild-type (red) and CFSE-labeled Nck1-2 KO (green) B cells injected in a wild-type recipient animal and migrating in an explanted inguinal lymph node using. Migration of individual cells was tracked over time. (MOV 1483 kb)
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Castello, A., Gaya, M., Tucholski, J. et al. Nck-mediated recruitment of BCAP to the BCR regulates the PI(3)K-Akt pathway in B cells. Nat Immunol 14, 966–975 (2013). https://doi.org/10.1038/ni.2685
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DOI: https://doi.org/10.1038/ni.2685
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