Signal Transduction In Mast Cells

蛋白酪氨酸激酶林恩和Syk在高亲和力IgE受体（FceRI）诱导的信号转导中起重要作用。用Syk阴性细胞的实验表明，大多数受体诱导的酪氨酸磷酸化是Syk的下游，其中一些可能是这种激酶的实际底物。为了鉴定林恩和Syk的新底物，筛选RBL-2 H3细菌表达文库中被杆状病毒表达的林恩和Syk酪氨酸磷酸化的蛋白。鉴定了作为潜在林恩底物的5个克隆和作为Syk底物的8个克隆，包括已知底物如SLP-76、LAT和α-微管蛋白。鉴定的林恩的潜在底物是分子TOM 1 L1，其具有被认为对膜运输和蛋白质-蛋白质相互作用重要的几个结构域。由于TOM 1 L1的功能尚不清楚，因此分离大鼠TOM 1 L1全长cDNA并用于在COS-1和RBL-2 H3细胞中表达该蛋白。在COS-1细胞中，共转染TOM 1 L1和林恩，而不是Syk，导致TOM 1 L1的酪氨酸磷酸化。在RBL-2 H3肥大细胞中，内源性和过度表达的TOM 1 L1在非刺激细胞中被酪氨酸磷酸化，并且这种磷酸化通过IgE受体聚集而增加。通过亚细胞分级分离，野生型TOM 1 L1主要在细胞质中，有一小部分与膜组成性相关;这种相关性在缺失突变体中显着降低，缺乏几个蛋白质相互作用结构域。野生型TOM 1 L1的过表达，而不是缺失突变体，增强了IgE诱导的细胞因子的产生和释放。这些结果表明，TOM 1 L1是一种新的蛋白质，其参与FceRI信号转导以产生细胞因子。 免疫受体刺激导致细胞内钙增加，激活丝氨酸磷酸酶钙调磷酸酶，然后使活化T细胞的核因子（NFAT）去磷酸化。 去磷酸化的NFAT迅速易位到细胞核中并诱导各种细胞因子基因的转录。 NFAT在免疫细胞如T、B和肥大细胞的活化中起重要作用。因此，NFAT可用作免疫细胞活化的读出。将含有与增强型绿色荧光蛋白（GFP）的cDNA融合的三个串联NFAT结合位点的质粒转染到RBL-2 H3细胞中。然后将细胞克隆并再克隆以获得在高亲和力IgE受体刺激后几乎完全变成GFP+的细胞系。令人惊讶的是，使用这种灵敏的检测系统，在低至10 pg/ml的抗原浓度下存在IgE致敏细胞的活化，这比通过常规方法可以检测到的低至少一个对数。 在这种低抗原浓度下，没有可检测到的炎症介质释放，炎症介质是肥大细胞活化的标志。然而，在低抗原浓度下的NFAT活化似乎利用相同的信号传导途径;例如对钙调磷酸酶的需求。这些结果表明导致核基因表达的途径与诱导脱粒和释放炎症介质的途径之间存在差异。这些NFAT-GFP表达细胞也被用于筛选信号通路的调节剂，通过用RBL-2 H3 cDNA质粒文库瞬时转染它们，并测试受体诱导的GFP应答的抑制或增强。 蛋白质酪氨酸磷酸化是抗原受体活化后最早可检测到的事件之一。最近，我们观察到，在培养的胎肝前B细胞，前B细胞受体刺激诱导强烈的酪氨酸磷酸化的82 kDa的蛋白质，其身份尚不清楚。在抗原受体刺激的脾B细胞和肥大细胞中也观察到该蛋白的强磷酸化。 在肥大细胞中，这82 kDa的蛋白质的磷酸化需要Syk激酶活性，然而，这种磷酸化是独立的细胞内钙离子的上升。因此，该82 kDa蛋白的酪氨酸磷酸化与肥大细胞中的早期抗原信号传导事件直接相关。为了鉴定82 kDa磷酸化蛋白，用抗磷酸酪氨酸抗体免疫沉淀来自抗原刺激的RBL-2 H3细胞的裂解物，并对82 kDa磷酸化条带进行微测序。作为对照，还对已知含有Syk的磷酸化72 kDa条带进行微测序。82 kDa条带的主要组分是造血细胞特异性林恩底物（HS 1），而次要组分包括皮质素亚型B、肌醇多磷酸5磷酸酶（SHIP）、FYN结合蛋白、Ga B 1和蛋白激酶C-δ。72 kDa的条带，含有src同源2，含有76 kDa的蛋白（SLP-76）、HS 1、Syk、热休克蛋白70、Btk和SHIP。 在82和70 kDa条带中均检测到许多其他蛋白质的较低水平。 用对照和活化的RBL-2 H3细胞进行的免疫印迹和免疫沉淀实验证实了HS 1是82 kDa的磷蛋白。 因此，HS 1的酪氨酸磷酸化可以作为早期抗原信号通路功能状态的标志。

The protein tyrosine kinases Lyn and Syk play important roles in high affinity IgE receptor (FceRI)-induced signal transduction. Experiments with Syk negative cells indicate that most of the receptor-induced tyrosine phosphorylations are downstream of Syk and some of which may be actual substrates of this kinase. To identify novel substrates of Lyn and Syk, an RBL-2H3 bacterial expression library was screened for proteins that were tyrosine phosphorylated with baculoviral expressed Lyn and Syk. Five clones as potential Lyn substrates and eight clones as Syk substrates were identified including known substrates such as SLP-76, LAT, and alpha-tubulin. A potential substrate of Lyn identified was the molecule TOM1L1 which has several domains thought to be important for membrane trafficking and protein-protein interactions. Since the function of TOM1L1 is unclear, the rat TOM1L1 full-length cDNA was isolated and used to express the protein in COS-1 and RBL-2H3 cells. In COS-1 cells, the co-transfection of TOM1L1 and Lyn, but not Syk, resulted in the tyrosine phosphorylation of TOM1L1. In RBL-2H3 mast cells, both the endogenous and the over-expressed TOM1L1 were tyrosine phosphorylated in non-stimulated cells and this phosphorylation was increased by IgE receptor aggregation. By subcellular fractionation, wild-type TOM1L1 was mainly in the cytoplasm with a small fraction constitutively associated with the membrane; this association was markedly reduced in deletion mutants lacking several of the protein interaction domains. The over-expression of wild-type TOM1L1, but not the deletion mutants, enhanced the IgE-induced generation and release of cytokines. These results suggest that TOM1L1 is a novel protein involved in the FceRI signal transduction for the generation of cytokines. Immune receptor stimulation results in an increase in intracellular calcium that activates the serine phosphatase calcineurin, which then dephosphorylates the nuclear factor of activated T cells (NFAT). The dephosphorylated NFAT rapidly translocates into the nucleus and induces the transcription of various cytokine genes. NFAT plays an important role in the activation of immune cells such as T, B and mast cells. Therefore, NFAT can be used as readout for immune cell activation. A plasmid containing three tandem NFATbinding sites fused to the cDNA of enhanced green fluorescent protein (GFP) was transfected into the RBL-2H3 cells. The cells were then cloned and recloned to obtain a cell line that became almost totally GFP+ upon high affinity IgE receptor stimulation. Surprisingly with this sensitive detection system, there is activation of IgE sensitized cells at concentrations of antigen as low as 10 pg/ml, which is at least a log lower than can be detected by conventional methods. At this low antigen concentration there is no detectable release of inflammatory mediators which are the hallmarks of mast cell activation. However, NFAT activation at low antigen concentrations appears to utilize the same signaling pathways; for example the requirement for calcineurin. These results suggest differences between the pathways that result in nuclear gene expression from those that induce degranulation and the release of inflammatory mediators. These NFAT-GFP expressing cells are also being used to screen for regulators of the signaling pathway by transiently transfecting them with an RBL-2H3 cDNA plasmid library and testing for either inhibition or enhancement of the receptor-induced GFP response. Protein tyrosine phosphorylation is one of the earliest detectable events after antigen receptor activation. Recently, we observed that in cultured fetal liver pre-B cells, pre-B cell receptor stimulation induced the strong tyrosine phosphorylation of an 82 kDa protein, whose identity is unclear. The strong phosphorylation of this protein was also observed in antigen-receptor stimulated spleen B cells and mast cells. In mast cells, the phosphorylation of this 82 kDa protein required Syk kinase activity; however, this phosphorylation was independent of the rise of intracellular calcium. Therefore, the tyrosine phosphorylation of this 82 kDa protein directly correlated with early antigen signaling events in mast cells. To identify the 82 kDa phosphorylated protein, lysates from antigen stimulated RBL-2H3 cells were immunoprecipitated with anti-phospho-tyrosine antibody and the 82 kDa phosphorylated band was micro-sequenced. As a control, the phosphorylated 72 kDa band, that is known to contain Syk, was also micro-sequenced. The major component of the 82 kDa band was the hematopoietic cell specific Lyn substrate (HS1), while minor components included cortactin isoform B, inositol polyphosphate 5 phosphatase (SHIP), FYN binding protein, Gab 1, and protein kinase C-delta. The 72 kDa band, contained src homology 2 containing protein of 76 kDa (SLP-76), HS1, Syk, heat shock 70 protein, Btk and SHIP. There were a number of other proteins detected at lower levels in both the 82 and 70 kDa bands. Immunoblotting and immunoprecipitation experiments with control and activated RBL-2H3 cells confirmed that HS1 was the 82 kDa phospho-protein. Therefore, tyrosine phosphorylation of HS1 can serve as a marker for the functional status of early antigen signaling pathways.