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STIM1, PKC-δ and RasGRP set a threshold for proapoptotic Erk signaling during B cell development

Nature Immunology volume 12, pages 425–433 (2011)Cite this article

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Abstract

Clonal deletion of autoreactive B cells is crucial for the prevention of autoimmunity, but the signaling mechanisms that regulate this checkpoint remain undefined. Here we characterize a previously unrecognized Ca2+-driven pathway for activation of the kinase Erk, which was proapoptotic and biochemically distinct from Erk activation induced by diacylglycerol (DAG). This pathway required protein kinase C-δ (PKC-δ) and the guanine nucleotide–exchange factor RasGRP and depended on the concentration of the Ca2+ sensor STIM1, which controls the magnitude of Ca2+ entry. Developmental regulation of these proteins was associated with selective activation of the pathway in B cells prone to negative selection. This checkpoint was impaired in PKC-δ-deficient mice, which developed B cell autoimmunity. Conversely, overexpression of STIM1 conferred a competitive disadvantage to developing B cells. Our findings establish Ca2+-dependent Erk signaling as a critical proapoptotic pathway that mediates the negative selection of B cells.

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Figure 1: Sensitization of B cells to antigen-induced apoptosis correlates with Ca2+-dependent activation of Erk.
Figure 2: BCR stimulation activates Erk via two distinct pathways activated mainly by either Ca2+ or DAG.
Figure 3: Ca2+-dependent Erk activation occurs selectively at stages of negative selection in primary bone marrow B cells.
Figure 4: STIM1 overexpression confers a competitive disadvantage to developing B cells.
Figure 5: Activation of Erk downstream of Ca2+ requires RasGRP.
Figure 6: Phosphorylation of RasGRP1 at Ser332 is required for the restoration of Ca2+-dependent Erk activation by RasGRP1 in RasGRP-deficient cells.
Figure 7: PKC-δ is required for Ca2+-dependent activation of Erk in developing bone marrow cells.
Figure 8: PKC-δ is required for STIM1-mediated sensitization of bone marrow B cells to negative selection.

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References

  1. Gay, D., Saunders, T., Camper, S. & Weigert, M. Receptor editing: an approach by autoreactive B cells to escape tolerance. J. Exp. Med. 177, 999–1008 (1993).

    Article  CAS  PubMed  Google Scholar 

  2. Goodnow, C.C. et al. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334, 676–682 (1988).

    Article  CAS  PubMed  Google Scholar 

  3. Nemazee, D.A. & Burki, K. Clonal deletion of B lymphocytes in a transgenic mouse bearing anti-MHC class I antibody genes. Nature 337, 562–566 (1989).

    Article  CAS  PubMed  Google Scholar 

  4. Tiegs, S.L., Russell, D.M. & Nemazee, D. Receptor editing in self-reactive bone marrow B cells. J. Exp. Med. 177, 1009–1020 (1993).

    Article  CAS  PubMed  Google Scholar 

  5. Wardemann, H. et al. Predominant autoantibody production by early human B cell precursors. Science 301, 1374–1377 (2003).

    CAS  PubMed  Google Scholar 

  6. Hertz, M. & Nemazee, D. BCR ligation induces receptor editing in IgM+IgD− bone marrow B cells in vitro. Immunity 6, 429–436 (1997).

    Article  CAS  PubMed  Google Scholar 

  7. Melamed, D., Benschop, R.J., Cambier, J.C. & Nemazee, D. Developmental regulation of B lymphocyte immune tolerance compartmentalizes clonal selection from receptor selection. Cell 92, 173–182 (1998).

    Article  CAS  PubMed  Google Scholar 

  8. Benschop, R.J., Brandl, E., Chan, A.C. & Cambier, J.C. Unique signaling properties of B cell antigen receptor in mature and immature B cells: implications for tolerance and activation. J. Immunol. 167, 4172–4179 (2001).

    Article  CAS  PubMed  Google Scholar 

  9. Gross, A.J., Lyandres, J.R., Panigrahi, A.K., Prak, E.T. & DeFranco, A.L. Developmental acquisition of the Lyn-CD22-SHP-1 inhibitory pathway promotes B cell tolerance. J. Immunol. 182, 5382–5392 (2009).

    Article  CAS  PubMed  Google Scholar 

  10. Hoek, K.L. et al. Transitional B cell fate is associated with developmental stage-specific regulation of diacylglycerol and calcium signaling upon B cell receptor engagement. J. Immunol. 177, 5405–5413 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. King, L.B., Norvell, A. & Monroe, J.G. Antigen receptor-induced signal transduction imbalances associated with the negative selection of immature B cells. J. Immunol. 162, 2655–2662 (1999).

    CAS  PubMed  Google Scholar 

  12. Parekh, A.B. & Putney, J.W. Jr. Store-operated calcium channels. Physiol. Rev. 85, 757–810 (2005).

    Article  CAS  PubMed  Google Scholar 

  13. Liou, J. et al. STIM is a Ca2+ sensor essential for Ca2+-store-depletion-triggered Ca2+ influx. Curr. Biol. 15, 1235–1241 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Roos, J. et al. STIM1, an essential and conserved component of store-operated Ca2+ channel function. J. Cell Biol. 169, 435–445 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Zhang, S.L. et al. STIM1 is a Ca2+ sensor that activates CRAC channels and migrates from the Ca2+ store to the plasma membrane. Nature 437, 902–905 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Liou, J., Fivaz, M., Inoue, T. & Meyer, T. Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca2+ store depletion. Proc. Natl. Acad. Sci. USA 104, 9301–9306 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Luik, R.M., Wu, M.M., Buchanan, J. & Lewis, R.S. The elementary unit of store-operated Ca2+ entry: local activation of CRAC channels by STIM1 at ER-plasma membrane junctions. J. Cell Biol. 174, 815–825 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Park, C.Y. et al. STIM1 clusters and activates CRAC channels via direct binding of a cytosolic domain to Orai1. Cell 136, 876–890 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Spassova, M.A. et al. STIM1 has a plasma membrane role in the activation of store-operated Ca2+ channels. Proc. Natl. Acad. Sci. USA 103, 4040–4045 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Wu, M.M., Buchanan, J., Luik, R.M. & Lewis, R.S. Ca2+ store depletion causes STIM1 to accumulate in ER regions closely associated with the plasma membrane. J. Cell Biol. 174, 803–813 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Mecklenbrauker, I., Saijo, K., Zheng, N.Y., Leitges, M. & Tarakhovsky, A. Protein kinase Cδ controls self-antigen-induced B-cell tolerance. Nature 416, 860–865 (2002).

    Article  PubMed  Google Scholar 

  22. Miyamoto, A. et al. Increased proliferation of B cells and auto-immunity in mice lacking protein kinase Cδ. Nature 416, 865–869 (2002).

    Article  CAS  PubMed  Google Scholar 

  23. Dower, N.A. et al. RasGRP is essential for mouse thymocyte differentiation and TCR signaling. Nat. Immunol. 1, 317–321 (2000).

    Article  CAS  PubMed  Google Scholar 

  24. Roose, J. & Weiss, A. T cells: getting a GRP on Ras. Nat. Immunol. 1, 275–276 (2000).

    Article  CAS  PubMed  Google Scholar 

  25. Roose, J.P., Mollenauer, M., Gupta, V.A., Stone, J. & Weiss, A. A diacylglycerol-protein kinase C-RasGRP1 pathway directs Ras activation upon antigen receptor stimulation of T cells. Mol. Cell. Biol. 25, 4426–4441 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Teixeira, C., Stang, S.L., Zheng, Y., Beswick, N.S. & Stone, J.C. Integration of DAG signaling systems mediated by PKC-dependent phosphorylation of RasGRP3. Blood 102, 1414–1420 (2003).

    Article  CAS  PubMed  Google Scholar 

  27. Downward, J., Graves, J.D., Warne, P.H., Rayter, S. & Cantrell, D.A. Stimulation of p21ras upon T-cell activation. Nature 346, 719–723 (1990).

    Article  CAS  PubMed  Google Scholar 

  28. Zhong, X.P. et al. Enhanced T cell responses due to diacylglycerol kinase zeta deficiency. Nat. Immunol. 4, 882–890 (2003).

    Article  CAS  PubMed  Google Scholar 

  29. Das, J. et al. Digital signaling and hysteresis characterize ras activation in lymphoid cells. Cell 136, 337–351 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Ebinu, J.O. et al. RasGRP, a Ras guanyl nucleotide-releasing protein with calcium- and diacylglycerol-binding motifs. Science 280, 1082–1086 (1998).

    Article  CAS  PubMed  Google Scholar 

  31. Ebinu, J.O. et al. RasGRP links T-cell receptor signaling to Ras. Blood 95, 3199–3203 (2000).

    CAS  PubMed  Google Scholar 

  32. Oh-hora, M., Johmura, S., Hashimoto, A., Hikida, M. & Kurosaki, T. Requirement for Ras guanine nucleotide releasing protein 3 in coupling phospholipase C-γ2 to Ras in B cell receptor signaling. J. Exp. Med. 198, 1841–1851 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Roose, J.P., Mollenauer, M., Ho, M., Kurosaki, T. & Weiss, A. Unusual interplay of two types of Ras activators, RasGRP and SOS, establishes sensitive and robust Ras activation in lymphocytes. Mol. Cell. Biol. 27, 2732–2745 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Guilbault, B. & Kay, R.J. RasGRP1 sensitizes an immature B cell line to antigen receptor-induced apoptosis. J. Biol. Chem. 279, 19523–19530 (2004).

    Article  CAS  PubMed  Google Scholar 

  35. Stang, S.L. et al. A proapoptotic signaling pathway involving RasGRP, Erk, and Bim in B cells. Exp. Hematol. 37, 122–134 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Aiba, Y. et al. Activation of RasGRP3 by phosphorylation of Thr-133 is required for B cell receptor-mediated Ras activation. Proc. Natl. Acad. Sci. USA 101, 16612–16617 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Zheng, Y. et al. Phosphorylation of RasGRP3 on threonine 133 provides a mechanistic link between PKC and Ras signaling systems in B cells. Blood 105, 3648–3654 (2005).

    Article  CAS  PubMed  Google Scholar 

  38. Martiny-Baron, G. et al. Selective inhibition of protein kinase C isozymes by the indolocarbazole Go 6976. J. Biol. Chem. 268, 9194–9197 (1993).

    CAS  PubMed  Google Scholar 

  39. Blom, N., Gammeltoft, S. & Brunak, S. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J. Mol. Biol. 294, 1351–1362 (1999).

    Article  CAS  PubMed  Google Scholar 

  40. Eswar, N. et al. Tools for comparative protein structure modeling and analysis. Nucleic Acids Res. 31, 3375–3380 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Freedman, T.S. et al. A Ras-induced conformational switch in the Ras activator Son of sevenless. Proc. Natl. Acad. Sci. USA 103, 16692–16697 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Mecklenbrauker, I., Kalled, S.L., Leitges, M., Mackay, F. & Tarakhovsky, A. Regulation of B-cell survival by BAFF-dependent PKCδ-mediated nuclear signalling. Nature 431, 456–461 (2004).

    Article  PubMed  Google Scholar 

  43. Steinberg, S.F. Distinctive activation mechanisms and functions for protein kinase Cδ. Biochem. J. 384, 449–459 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Yoshida, K. PKCδ signaling: mechanisms of DNA damage response and apoptosis. Cell. Signal. 19, 892–901 (2007).

    Article  CAS  PubMed  Google Scholar 

  45. Hartley, S.B. et al. Elimination of self-reactive B lymphocytes proceeds in two stages: arrested development and cell death. Cell 72, 325–335 (1993).

    Article  CAS  PubMed  Google Scholar 

  46. Hartley, S.B. et al. Elimination from peripheral lymphoid tissues of self-reactive B lymphocytes recognizing membrane-bound antigens. Nature 353, 765–769 (1991).

    Article  CAS  PubMed  Google Scholar 

  47. Freedman, T.S. et al. Differences in flexibility underlie functional differences in the Ras activators son of sevenless and Ras guanine nucleotide releasing factor 1. Structure 17, 41–53 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Kolch, W. Coordinating ERK/MAPK signalling through scaffolds and inhibitors. Nat. Rev. Mol. Cell Biol. 6, 827–837 (2005).

    Article  CAS  PubMed  Google Scholar 

  49. Murphy, L.O. & Blenis, J. MAPK signal specificity: the right place at the right time. Trends Biochem. Sci. 31, 268–275 (2006).

    Article  CAS  PubMed  Google Scholar 

  50. Philips, M.R. Compartmentalized signalling of Ras. Biochem. Soc. Trans. 33, 657–661 (2005).

    Article  CAS  PubMed  Google Scholar 

  51. Daniels, M.A. et al. Thymic selection threshold defined by compartmentalization of Ras/MAPK signalling. Nature 444, 724–729 (2006).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank the Sandler-Moore Mass Spectrometry Core Facility at the University of California at San Francisco (funded by the Sandler Family Foundation, the Gordon and Betty Moore Foundation and the National Cancer Institute of the US National Institutes of Health (P30 CA82103)) for assistance in protein identification; the Flow Cytometry Core Facility at the Department of Pathology and Diabetes Center of the University of California at San Francisco for assistance; G. Koretzky (University of Pennsylvania) for the pEF-Flag-hDGK-ζ plasmid; A. Roque for assistance with animal husbandry; H. Phee and M. Hermiston for critical reading of the manuscript and suggestions; B. Au-Yeung and H. Wang for help with tail-vein injections; and members of the Weiss laboratory for discussions. Supported by the Howard Hughes Medical Institute and the Sidney Kimmel Foundation (J.P.R.).

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Authors and Affiliations

  1. Department of Medicine, Howard Hughes Medical Institute, Rosalind Russell Medical Research Center for Arthritis, University of California at San Francisco, San Francisco, California, USA

    Andre Limnander, Tanya S Freedman & Arthur Weiss

  2. Department of Anatomy, University of California at San Francisco, San Francisco, California, USA

    Philippe Depeille & Jeroen P Roose

  3. Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA

    Jen Liou

  4. Division of Nephrology, Department of Medicine, Hannover Medical School, Hannover, Germany

    Michael Leitges

  5. The Biotechnology Centre of Oslo, University of Oslo, Oslo, Norway

    Michael Leitges

  6. Laboratory for Lymphocyte Differentiation, World Premier International Immunology Frontier Research Center, Osaka University, Osaka, Japan

    Tomohiro Kurosaki

  7. Laboratory for Lymphocyte Differentiation, RIKEN Research Center for Allergy and Immunology, Yokohama, Japan

    Tomohiro Kurosaki

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Contributions

A.L. designed and did experiments, analyzed the data and wrote the manuscript; P.D. collaborated with A.L. in determining expression of various proteins on sorted bone marrow B cell populations and in collecting reconstituted mice; T.S.F. did homology modeling of RasGRP1; J.L. and T.K. provided reagents; M.L. generated the Prkcd−/− mice; J.P.R. designed experiments and provided reagents; and A.W. designed experiments, supervised the research, revised the manuscript and provided support.

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Correspondence to Arthur Weiss.

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Limnander, A., Depeille, P., Freedman, T. et al. STIM1, PKC-δ and RasGRP set a threshold for proapoptotic Erk signaling during B cell development. Nat Immunol 12, 425–433 (2011). https://doi.org/10.1038/ni.2016

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