Dynamics of MAP kinase cascade in immune response

MAP激酶级联对于各种细胞外刺激向细胞核传递信号至关重要。通过抗原受体和其他刺激物的聚集刺激T细胞、B细胞和肥大细胞，导致MAP激酶的激活。在本研究中，我们利用黄色荧光蛋白（YFP）标记的MAPK （ERK2）和YFP标记的MAPKKK (Raf-1)，重点研究了肥大细胞中MAP激酶（MAPK）级联蛋白的动态。在这里，我们已经展示了MAP激酶信号在免疫应答中的许多方面。在野生型肥大细胞中，抗原刺激后，MAPKKK （Raf-1）的膜易位达到~ 3min，此后MAPK （ERK2）的核输入在5 ~ 7min达到最大，Raf-1不与脂质微域共定位。在leptomycin B（核输出抑制剂）存在的情况下，ERK2的输出变得更慢，尽管ERK2像未处理的细胞一样被输入到细胞核。我们研究了fc - riib对免疫细胞中fc - riib介导的MAP激酶核穿梭的影响。FcεRI和FcγRIIB的共聚增加了[Ca-<2+>]_i，诱导了ERK2的核输入。然而，钙的增加是短暂的，ERK2迅速输出到细胞质。我们研究了两种酪氨酸磷酸酶，HePTP（一种酪氨酸特异性磷酸酶）和VHR（一种双特异性蛋白-酪氨酸磷酸酶）对肥大细胞中fcε ri介导的ERK2核穿梭的影响。在heptp过表达的细胞中，ERK2在细胞核内的最大积累量远低于野生型细胞，尽管在相同的时间过程中，ERK2的细胞核输入量达到了最大值。在vhr过表达的细胞中，尽管ERK2像野生型细胞一样被导入细胞核，但输入的ERK2的数量减少得更快。因此，我们成功地揭示了MAP激酶级联蛋白在免疫应答中的分子机制。

The MAP kinase cascade is essential for signaling of various extracellular stimuli to the nucleus. Stimulation of T cells, B cells, and mast cells through aggregation of antigen receptors and other stimulants results in activation of MAP kinase. In this study, we focused on the dynamics of MAP kinase (MAPK) cascade protein in mast cells using yellow fluorescent protein (YFP)-tagged MAPK (ERK2) and YFP-tagged MAPKKK (Raf-1). Here, we have shown many aspects of MAP kinase signaling in immune response.1.In wild type mast cells, the membrane translocation of MAPKKK (Raf-1) reached 〜3 min after antigen stimulation, and thereafter the nuclear import of MAPK (ERK2) reached the maximum at 5〜7 min Raf-1 did not co-localize with lipid microdomain.2.In the presence of leptomycin B (inhibitor of nuclear export), the export of ERK2 became more slowly though ERK2 was imported to the nucleus as like as in non-treated cells.3.We have studied the effects of FcγRIIB on the FcεRI-mediated nuclear shuttling of MAP kinase in immune cells. The co-clustering of FcεRI and FcγRIIB increased the [Ca-<2+>]_i and induced the nuclear import of ERK2. However, the calcium increase was transient and ERK2 was rapidly exported to the cytoplasm.4.We have studied the effects of two tyrosine phosphatases, HePTP (a tyrosine-specific phosphatase) and VHR (a dual specificity protein-tyrosine phosphatase), on the FcεRI-mediated nuclear shuttling of ERK2 in the mast cells. In HePTP-overexpressing cells, the maximal amount of ERK2 accumulated in the nucleus was much less than that in wild type cells though the nuclear import of ERK2 reached the maximum in the similar time-course. In VHR-overexpressing cells, the amount of imported ERK2 decreased more rapidly though ERK2 was imported to the nucleus as like as in wild type cells.Thus we succeeded to show the molecular mechanism of MAP kinase cascade protein in immune response.