Modulation of B cell tolerance checkpoints by distinct Ras/Erk Pathways

点击翻译按钮获取中文摘要

DESCRIPTION (provided by applicant): 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.