Establishment of a proper B cell repertoire that is immunocompetent but not autoimmune depends on critical developmental checkpoints that edit, silence or delete autoreactive cells. The properties of B cells change as they progress through distinct stages in development, but the signaling mechanisms by which antigen drives the different selection checkpoints are incompletely defined. We have recently described a novel Ca2+- dependent Erk signaling pathway in developing B cells that is pro-apoptotic and mediates B cell negative selection. This pathway requires PKC? and RasGRP proteins and loss of this pathway in PKCg-deficient mice results in increased survival of B cells during negative selection and subsequent development of an SLE-like disease with lymphoproliferation and autoantibody production. RasGRP1-deficient mice have a substantial developmental block in T cell development that results in T cell lymphopenia, but as they age they also develop an SLE-like disease with B cell lymphoproliferation and autoantibody production, the etiology of which is not well understood. In addition, a recently identified RasGRP1Anaef allele, which carries a point mutation in the second EF-hand of RasGRP1, also causes an SLE-like disease with distinct effects on T cell development from those observed in the RasGRP1-deficient mice. The first goal of this proposal is to determine whether the SLE-like phenotype in these RasGRP1 mouse models is B cell intrinsic, and if it is due to loss of pro-apoptotic Erk signaling during B cell development. Secondly, because we have identified Serine 332 on RasGRP1 as a putative PKCg target phosphosite that is required for the activation of this novel Ca2+-Erk pathway, I will use in vitro biochemistry experiments to determine the effect of this phosphorylation on the function and specificity of RasGRP1. Finally, I will develop retroviruses encoding mutant S332 RasGRP1 to determine the relevance of this phospho-site in B cell development in vivo. Successful completion of these studies will greatly advance our understanding of Ras/Erk signaling in B cell development in normal and pathological settings. Such insight is essential to define the molecular mechanisms that confer functional specificity to different Ras/Erk pathways, which in turn may pinpoint events that can serve as therapeutic targets while having minor or no consequences on closely related but functionally distinct pathways.
Autoimmune diseases arise due to failure in the mechanisms that render immune cells tolerant to self-tissues. In B cells (the immune cells that produce antibodies) this tolerance to self is achieved through several checkpoints during their development that delete, edit or silence cells that can potentially elicit responses to self. This proposal aims to characterize the molecular mechanisms that mediate one of these checkpoints and their relevance in autoimmune disease development in mice. This checkpoint induces death of B cells with the potential to attack self and is mediated by a pathway that I recently discovered which involves the activation of molecules in the Ras/Erk pathway. Mutation of molecules in this pathway can induce lupus-like disease, and I will determine if this is due to loss of this checkpoint in B cell development. Because these molecules are also known to participate in survival pathways in other cellular contexts, understanding how these pathways are regulated to induce death or survival of immune cells is crucial to define strategies to target them therapeutically.
|Limnander, Andre; Zikherman, Julie; Lau, Tannia et al. (2014) Protein kinase C? promotes transitional B cell-negative selection and limits proximal B cell receptor signaling to enforce tolerance. Mol Cell Biol 34:1474-85|
|Daley, Stephen R; Coakley, Kristen M; Hu, Daniel Y et al. (2013) Rasgrp1 mutation increases naive T-cell CD44 expression and drives mTOR-dependent accumulation of Heliosýýý T cells and autoantibodies. Elife 2:e01020|
|Iwig, Jeffrey S; Vercoulen, Yvonne; Das, Rahul et al. (2013) Structural analysis of autoinhibition in the Ras-specific exchange factor RasGRP1. Elife 2:e00813|