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

描述（由申请人提供）：建立具有免疫能力但非自身免疫性的适当B细胞库依赖于编辑、沉默或删除自身反应性细胞的关键发育检查点。B细胞的特性随着它们在不同发育阶段的进展而改变，但抗原驱动不同选择检查点的信号机制尚不完全明确。我们最近描述了一种新的Ca2+依赖性Erk信号通路，它在发育中的B细胞是促凋亡的，并介导B细胞的负选择。这一途径需要PKC¿和RasGRP蛋白，在pkcg缺失的小鼠中，这一途径的缺失导致B细胞在阴性选择过程中的存活增加，随后发展为具有淋巴细胞增殖和自身抗体产生的slea样疾病。rasgrp1缺陷小鼠在T细胞发育中具有实质性的发育阻滞，导致T细胞淋巴细胞减少，但随着年龄的增长，它们也会发展为具有B细胞淋巴细胞增殖和自身抗体产生的slea样疾病，其病因尚不清楚。此外，最近发现的一个RasGRP1Anaef等位基因，在RasGRP1的第二个EF-hand中携带一个点突变，也会导致一种sle样疾病，其对T细胞发育的影响与在RasGRP1缺陷小鼠中观察到的不同。本提案的第一个目标是确定这些RasGRP1小鼠模型中的sle样表型是否是B细胞固有的，以及是否由于B细胞发育过程中促凋亡Erk信号的丢失。其次，由于我们已经确定RasGRP1上的丝氨酸332是激活这种新型Ca2+-Erk途径所需的PKCg靶磷酸化，我将使用体外生物化学实验来确定这种磷酸化对RasGRP1的功能和特异性的影响。最后，我将开发编码突变体S332 RasGRP1的逆转录病毒，以确定该磷酸化位点在体内B细胞发育中的相关性。这些研究的成功完成将极大地促进我们对正常和病理条件下B细胞发育中Ras/Erk信号传导的理解。这种见解对于定义赋予不同Ras/Erk通路功能特异性的分子机制至关重要，这反过来又可以确定可以作为治疗靶点的事件，同时对密切相关但功能不同的通路产生轻微或没有影响。

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.