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Nck-mediated recruitment of BCAP to the BCR regulates the PI(3)K-Akt pathway in B cells

Nature Immunology volume 14, pages 966–975 (2013)Cite this article

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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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Figure 1: Nck is recruited to the BCR signalosome and is necessary for efficient signal transduction.
Figure 2: Nck binds directly to the phosphorylated immunoglobulin α-chain.
Figure 3: Nck deficiency does not affect B cell development, except for the B-1 compartment.
Figure 4: The activation of B cells is impaired in the absence of Nck.
Figure 5: Nck deficiency compromises T cell–independent and early T cell–dependent immune responses.
Figure 6: Nck is critical for phosphorylation of BCAP and recruitment of BCAP to the BCR.
Figure 7: Nck is required for efficient BCR-induced activation of Akt.

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References

  1. Burkhardt, A.L., Brunswick, M., Bolen, J.B. & Mond, J.J. Anti-immunoglobulin stimulation of B lymphocytes activates src-related protein-tyrosine kinases. Proc. Natl. Acad. Sci. USA 88, 7410–7414 (1991).

    CAS  PubMed  Google Scholar 

  2. Hutchcroft, J.E., Harrison, M.L. & Geahlen, R.L. B lymphocyte activation is accompanied by phosphorylation of a 72-kDa protein-tyrosine kinase. J. Biol. Chem. 266, 14846–14849 (1991).

    CAS  PubMed  Google Scholar 

  3. Hutchcroft, J.E., Harrison, M.L. & Geahlen, R.L. Association of the 72-kDa protein-tyrosine kinase PTK72 with the B cell antigen receptor. J. Biol. Chem. 267, 8613–8619 (1992).

    CAS  PubMed  Google Scholar 

  4. Chen, M. et al. Identification of Nck family genes, chromosomal localization, expression, and signaling specificity. J. Biol. Chem. 273, 25171–25178 (1998).

    CAS  PubMed  Google Scholar 

  5. Antón, I.M., Lu, W., Mayer, B.J., Ramesh, N. & Geha, R.S. The Wiskott-Aldrich syndrome protein-interacting protein (WIP) binds to the adaptor protein Nck. J. Biol. Chem. 273, 20992–20995 (1998).

    PubMed  Google Scholar 

  6. Rivero-Lezcano, O.M., Marcilla, A., Sameshima, J.H. & Robbins, K.C. Wiskott-Aldrich syndrome protein physically associates with Nck through Src homology 3 domains. Mol. Cell Biol. 15, 5725–5731 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Barda-Saad, M. et al. Dynamic molecular interactions linking the T cell antigen receptor to the actin cytoskeleton. Nat. Immunol. 6, 80–89 (2005).

    CAS  PubMed  Google Scholar 

  8. Gil, D., Schamel, W.W.A., Montoya, M., Sánchez-Madrid, F. & Alarcón, B. Recruitment of Nck by CD3ɛ reveals a ligand-induced conformational change essential for T cell receptor signaling and synapse formation. Cell 109, 901–912 (2002).

    CAS  PubMed  Google Scholar 

  9. Roy, E. et al. Fine tuning of the threshold of T cell selection by the Nck adapters. J. Immunol. 185, 7518–7526 (2010).

    CAS  PubMed  Google Scholar 

  10. Roy, E. et al. Nck adaptors are positive regulators of the size and sensitivity of the T-cell repertoire. Proc. Natl. Acad. Sci. USA 107, 15529–15534 (2010).

    CAS  PubMed  Google Scholar 

  11. Fu, C., Turck, C.W., Kurosaki, T. & Chan, A.C. BLNK: a central linker protein in B cell activation. Immunity 9, 93–103 (1998).

    CAS  PubMed  Google Scholar 

  12. Oellerich, T. et al. The B-cell antigen receptor signals through a preformed transducer module of SLP65 and CIN85. EMBO J. 30, 3620–3634 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Garrity, P.A. et al. Drosophila photoreceptor axon guidance and targeting requires the dreadlocks SH2/SH3 adapter protein. Cell 85, 639–650 (1996).

    CAS  PubMed  Google Scholar 

  14. Weber, M. et al. Phospholipase C-{gamma} 2 and Vav cooperate within signaling microclusters to propagate B cell spreading in response to membrane-bound antigen. J. Exp. Med. 4, 853–868 (2008).

    Google Scholar 

  15. Fleire, S. et al. B cell ligand discrimination through a spreading and contraction response. Science 312, 738–741 (2006).

    CAS  PubMed  Google Scholar 

  16. Takata, M. et al. Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways. EMBO J. 13, 1341–1349 (1994).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Ishiai, M. et al. BLNK required for coupling Syk to PLCγ2 and Rac1-JNK in B cells. Immunity 10, 117–125 (1999).

    CAS  PubMed  Google Scholar 

  18. Chiu, C.W., Dalton, M., Ishiai, M., Kurosaki, T. & Chan, A.C. BLNK: molecular scaffolding through 'cis'-mediated organization of signaling proteins. EMBO J. 21, 6461–6472 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Cambier, J.C., Pleiman, C.M. & Clark, M.R. Signal transduction by the B cell antigen receptor and its coreceptors. Annu. Rev. Immunol. 12, 457–486 (1994).

    CAS  PubMed  Google Scholar 

  20. Engels, N., Wollscheid, B. & Wienands, J. Association of SLP-65/BLNK with the B cell antigen receptor through a non-ITAM tyrosine of Ig-α. Eur. J. Immunol. 31, 2126–2134 (2001).

    CAS  PubMed  Google Scholar 

  21. Kabak, S. et al. The direct recruitment of BLNK to immunoglobulin α couples the B-cell antigen receptor to distal signaling pathways. Mol. Cell Biol. 22, 2524–2535 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  22. Siemasko, K. et al. Receptor-facilitated antigen presentation requires the recruitment of B cell linker protein to Igα. J. Immunol. 168, 2127–2138 (2002).

    CAS  PubMed  Google Scholar 

  23. Bladt, F. et al. The murine Nck SH2/SH3 adaptors are important for the development of mesoderm-derived embryonic structures and for regulating the cellular actin network. Mol. Cell Biol. 23, 4586–4597 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Jones, N. et al. Nck adaptor proteins link nephrin to the actin cytoskeleton of kidney podocytes. Nature 440, 818–823 (2006).

    CAS  PubMed  Google Scholar 

  25. Hobeika, E. et al. Testing gene function early in the B cell lineage in mb1-cre mice. Proc. Natl. Acad. Sci. USA 103, 13789–13794 (2006).

    CAS  PubMed  Google Scholar 

  26. Hardy, R.R., Carmack, C.E., Shinton, S.A., Kemp, J.D. & Hayakawa, K. Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow. J. Exp. Med. 173, 1213–1225 (1991).

    CAS  PubMed  Google Scholar 

  27. Okada, T., Maeda, A., Iwamatsu, A., Gotoh, K. & Kurosaki, T. BCAP: the tyrosine kinase substrate that connects B cell receptor to phosphoinositide 3-kinase activation. Immunity 13, 817–827 (2000).

    CAS  PubMed  Google Scholar 

  28. Yamazaki, T. et al. Essential immunoregulatory role for BCAP in B cell development and function. J. Exp. Med. 195, 535–545 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Aiba, Y., Kameyama, M., Yamazaki, T., Tedder, T.F. & Kurosaki, T. Regulation of B-cell development by BCAP and CD19 through their binding to phosphoinositide 3-kinase. Blood 111, 1497–1503 (2007).

    PubMed  Google Scholar 

  30. Frese, S. et al. The phosphotyrosine peptide binding specificity of Nck1 and Nck2 Src homology 2 domains. J. Biol. Chem. 281, 18236–18245 (2006).

    CAS  PubMed  Google Scholar 

  31. Patterson, H.C.K., Kraus, M., Kim, Y.-M., Ploegh, H. & Rajewsky, K. The B cell receptor promotes B cell activation and proliferation through a non-ITAM tyrosine in the Igalpha cytoplasmic domain. Immunity 25, 55–65 (2006).

    CAS  PubMed  Google Scholar 

  32. Omori, S.A. et al. Regulation of class-switch recombination and plasma cell differentiation by phosphatidylinositol 3-kinase signaling. Immunity 25, 545–557 (2006).

    CAS  PubMed  Google Scholar 

  33. Srinivasan, L. et al. PI3 kinase signals BCR-dependent mature B cell survival. Cell 139, 573–586 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. Yusuf, I. Optimal B-cell proliferation requires phosphoinositide 3-kinase-dependent inactivation of FOXO transcription factors. Blood 104, 784–787 (2004).

    CAS  PubMed  Google Scholar 

  35. Hinman, R.M., Bushanam, J.N., Nichols, W.A. & Satterthwaite, A.B. B cell receptor signaling down-regulates forkhead box transcription factor class O 1 mRNA expression via phosphatidylinositol 3-kinase and Bruton's tyrosine kinase. J. Immunol. 178, 740–747 (2007).

    CAS  PubMed  Google Scholar 

  36. Dengler, H.S. et al. Distinct functions for the transcription factor Foxo1 at various stages of B cell differentiation. Nat. Immunol. 9, 1388–1398 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. Suzuki, A. et al. Critical roles of Pten in B cell homeostasis and immunoglobulin class switch recombination. J. Exp. Med. 197, 657–667 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Carrasco, Y.R., Fleire, S.J., Cameron, T., Dustin, M.L. & Batista, F.D. LFA-1/ICAM-1 interaction lowers the threshold of B cell activation by facilitating B cell adhesion and synapse formation. Immunity 20, 589–599 (2004).

    CAS  PubMed  Google Scholar 

  39. Stork, B. et al. Grb2 and the non-T cell activation linker NTAL constitute a Ca2+-regulating signal circuit in B lymphocytes. Immunity 21, 681–691 (2004).

    CAS  PubMed  Google Scholar 

  40. Stork, B. et al. Subcellular localization of Grb2 by the adaptor protein Dok-3 restricts the intensity of Ca2+ signaling in B cells. EMBO J. 26, 1140–1149 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Scaplehorn, N. et al. Grb2 and Nck act cooperatively to promote actin-based motility of vaccinia virus. Curr. Biol. 12, 740–745 (2002).

    CAS  PubMed  Google Scholar 

  42. Donnelly, S.K., Weisswange, I., Zettl, M. & Way, M. WIP provides an essential link between Nck and N-WASP during Arp2/3-dependent actin polymerization. Curr. Biol. 11, 999–1006 (2013).

    Google Scholar 

  43. Carrasco, Y.R. & Batista, F.D. B cells acquire particulate antigen in a macrophage-rich area at the boundary between the follicle and the subcapsular sinus of the lymph node. Immunity 27, 160–171 (2007).

    CAS  PubMed  Google Scholar 

  44. Barral, P. et al. CD169+ macrophages present lipid antigens to mediate early activation of iNKT cells in lymph nodes. Nat. Immunol. 11, 303–312 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. Weber, C., Schreiber, T.B. & Daub, H. Dual phosphoproteomics and chemical proteomics analysis of erlotinib and gefitinib interference in acute myeloid leukemia cells. J. Proteomics 75, 1343–1356 (2012).

    CAS  PubMed  Google Scholar 

  46. Barnden, M.J., Allison, J., Heath, W.R. & Carbone, F.R. Defective TCR expression in transgenic mice constructed using cDNA-based α- and β-chain genes under the control of heterologous regulatory elements. Immunol. Cell Biol. 76, 34–40 (1998).

    CAS  PubMed  Google Scholar 

  47. Eckl-Dorna, J. & Batista, F.D. BCR-mediated uptake of antigen linked to TLR9 ligand stimulates B-cell proliferation and antigen-specific plasma cell formation. Blood 113, 3969–3977 (2009).

    CAS  Google Scholar 

  48. Barral, P. et al. B cell receptor-mediated uptake of CD1d-restricted antigen augments antibody responses by recruiting invariant NKT cell help in vivo. Proc. Natl. Acad. Sci. USA 105, 8345–8350 (2008).

    CAS  Google Scholar 

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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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Authors and Affiliations

  1. Lymphocyte Interaction Laboratory, London Research Institute–Cancer Research UK, London, UK

    Angelo Castello, Mauro Gaya & Facundo D Batista

  2. Institute of Cellular and Molecular Immunology, Georg August University of Göttingen, Göttingen, Germany

    Johannes Tucholski, Jürgen Wienands & Michael Engelke

  3. Department of Medicine II, Hematology/Oncology, Goethe University, Frankfurt, Germany

    Thomas Oellerich

  4. Department of Molecular Immunology, German Cancer Research Centre, Heidelberg, Germany

    Kun-Hui Lu & Anna Tafuri

  5. Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada

    Tony Pawson

  6. Department of Molecular Genetics, University of Toronto, Toronto, Canada

    Tony Pawson

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  1. Angelo Castello
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Contributions

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.

Corresponding authors

Correspondence to Angelo Castello, Michael Engelke or Facundo D Batista.

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Supplementary information

Supplementary Text and Figures

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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