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

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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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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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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