【作者】 陈翠霞；

【导师】 郝水； 曾宪录；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2007， 博士

【摘要】 白细胞沿着血管内皮滚动、稳定粘附,最终到达炎症部位是一个复杂的,多步骤的过程。在这个过程中有很多分子参与。选择素家族分子对于白细胞沿着血管内皮的滚动起重要作用。L-选择素是选择素家族的一员,组成性的表达在白细胞微绒毛顶端,对白细胞与血管内皮的起始粘附起主要作用。L-选择素不仅可以作为一个粘附分子,还能作为一个信号分子在粘附事件中发挥作用。L-选择素与其单克隆抗体的结合,能引起细胞形态变化,细胞骨架重排,氧呼吸爆发,细胞内IL-8以及TNFα基因的表达上调等。在L-选择素起始的信号事件中,有激酶分子的参与,但具体的分子机制还很不清楚。c-Abl激酶是一类非受体酪氨酸激酶,具有F-肌动蛋白结合结构域,能够被整合素以及生长因子受体结合而活化,从而调节F-肌动蛋白依赖的细胞骨架的变化。我们推测,c-Abl激酶也可能参与了L-选择素交联起始的信号事件。本文主要探讨了c-Abl分子是否以及如何参与了由L-选择素交联起始的信号事件。c-Abl的激酶活性在细胞内受到分子间和分子内双重的严格调控。我们的研究发现,当L-选择素交联以后,c-Abl激酶活性增强。如果在活化细胞前用c-Abl的抑制剂处理细胞,则c-Abl的激酶活性降低。c-Abl的激酶活性变化和L-选择素交联引起的F-肌动蛋白的变化相一致。在活化的细胞内,c-Abl的定位也发生变化。在静止细胞中,c-Abl激酶均匀的分布在整个细胞中,而L-选择素交联活化的细胞内,c-Abl激酶聚集在F-肌动蛋白以及L-选择素富集的部位。在细胞活化前用c-Abl的激酶抑制剂处理,三者的共定位现象消失。用抑制F-肌动蛋白聚合的抑制剂CB处理活化前的细胞,三者的共定位现象也减弱。说明L-选择素交联引起的F-肌动蛋白依赖的细胞骨架的变化依赖于c-Abl激酶的活性。c-Abl激酶还参与了由L-选择素交联引起的集落刺激因子-1(CSF-1)基因的表达。过表达野生型而不是激酶突变型的c-Abl能够增强由L-选择素交联引起的CSF-1基因的表达上调。用c-Abl激酶抑制剂处理活化前的细胞则降低了由c-Abl过表达引起的CSF-1基因的表达。说明L-选择素交联引起的CSF-1基因的表达增强依赖于c-Abl的激酶活性。ZAP70激酶是T细胞活化后最先被活化的分子之一。过表达野生型而不是激酶突变型或者Y319F突变型的ZAP70也能增强CSF-1基因的表达增强。在L-选择素交联活化以前,用c-Abl以及ZAP70抑制剂处理发现c-Abl激酶通过调节ZAP70 Y319的磷酸化来调节CSF-1基因的表达。通过蛋白质免疫共沉淀以及GST-pull down实验,我们发现在L-选择素活化的T细胞内,L-选择素、c-Abl以及ZAP70三者结合在一起。通过L-选择素不同刺激时间来检测三者的结合关系,我们发现c-Abl激酶和L-选择素组成性的结合在一起,随着L-选择素交联时间的增加,更多的ZAP70聚集到c-Abl激酶的SH2结构域。我们的结果证明了L-选择素交联引起的信号事件中,c-Abl激酶起到了很重要的作用。它不仅通过激酶活性的变化来调节F-肌动蛋白依赖的细胞骨架的变化,还能通过和L-选择素胞浆尾部结合,接受并传递由L-选择素交联引起的信号事件。将上游的信号事件通过与下游的底物结合来传递从而调控细胞内基因的表达。更多还原

【Abstract】 Lymphocyte recruitment onto inflamed tissues requires cells tethering to and rolling on vascular surfaces under flow. Recruitment of leukocytes is controlled by a variety of adhesion molecules expressed on the surface of the endothelium and circulating leukocytes. The members of selectin family play an important role during the lymphocytes rolling on the vessel. L-selectin, a member of selectin family and constitutively expressed on leukocytes, plays a fundamental role in the lymphocyte tethering on the vessel. Except for its role in adhesion, L-selectin can function as a signal transduction molecule. Crosslinking of human L-selectin with its antibody leads to cell shape change, cytoskeleton reorganization, superoxide generation, increased cytokine gene transcription, and activation of intracellular protein pathways. Though many kinases are involved in the signaling transduction mediated by L-selectin crosslinking, the regulation of them is unclear. c-Abl, a nonreceptor protein tyrosine kinase, contains the F-actin binding domain. It has been shown to be very important for cell signal transduction triggered by integrin and PDGF engagement. In this study, we focus on the c-Abl kinase function in the signaling transduction induced by L-selectin ligation.It has been reported that the kinase activity of the cytoplasmic c-Abl is tightly regulated by inter- and intra- molecular regulation. In our present work, we found that c-Abl kinase activity was increased following L-selectin crosslinking. STI571 preincubation would reduce the c-Abl kinase activity. The regulation of c-Abl kinase activity is closely related to the F-actin assembly triggered by L-selectin crosslinking, and the localization of c-Abl changes in the L-selectin stimulated neutrophils. c-Abl was faint and obscure in resting cells, while in majority of stimulated neutrophils c-Abl redistributed to the region where L-selectin and F-actin polarized. The colocalization of c-Abl and F-actin and L-selectin was reduced by cytochalasin B and STI571 preincubation. These results suggested that F-actin alteration triggered by L-selectin crosslinking was regulated by c-Abl kinase.c-Abl kinase was also involved in the CSF-1 gene transcription induced by L-selectin engagement. Our results demonstrated that overexpression of WT c-Abl kinase increased CSF-1 gene transcriptional activity. Alternatively, CSF-1 transcription was blockaded by STI571 incubation as well as by overexpression of the KD mutant of c-Abl, suggesting that c-Abl kinase regulates CSF-1 gene transcription dependenting on its kinase activity. ZAP70 is one of the earliest activated molecules in TCR signaling. Preincubation with STI571 reduced the phosphorylation of ZAP70Y319, while the increase of c-Abl kinase activity triggered by L-selectin ligation was not inhibited by pretreating the cells with piceatannol. Overexpression of WT ZAP70 other than KD ZAP70 or Y319F ZAP70 enhanced CSF-1 gene transcription which was greatly reduced in the presence of STI571. These data suggested that c-Abl kinase could regulate ZAP70 Y319 phosphorylation in the L-selectin ligation stimulated cells and ZAP70 kinase acted as a substrate of the c-Abl kinase in the CSF-1 transcriptional events triggered by L-selectin ligation.We found that L-selectin, c-Abl and ZAP70 was connected closely in the L-selectin engagement stimulated T cells by using the co-IP and GST-pull down assays. In the rest cells, the cytoplamic domain of L-selectin was connected with c-Abl SH2 domain constitutively. After L-selectin ligation, more ZAP70 would recruit to the c-Abl SH2 domain and made a complex with L-selectin and c-Abl kinase.In summary, the results we obtained provide the proofs for understanding the key role of c-Abl kinase in the signaling transduction triggered by L-selectin ligation. c-Abl kinase can regulate cytoskelecton reorganization in a kinase activity dependent manner. It also can associate with the cytoplasmic domain of L-selectin to accept the L-selectin intiated signaling and connect with the substrate proteins to regulate gene transcription.更多还原