c-Abl激酶在L-选择素介导的信号通路中的作用研究

发布时间：2018-08-19 06:01

【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。
[Abstract]:Leukocytes roll along the vascular endothelium, adhere steadily, and eventually reach the inflammation site. There are many molecules involved in this process. The selectin family molecules play an important role in the leukocyte rolling along the vascular endothelium. L-selectin is a member of the selectin family, which is constitutively expressed in leukocytes. L-selectin not only acts as an adhesion molecule, but also as a signal molecule in adhesion events. The binding of L-selectin with its monoclonal antibody can cause cell morphological changes, cytoskeleton rearrangement, oxygen respiration explosion, and intracellular IL-2. C-Abl kinases are a class of non-receptor tyrosine kinases with F-actin binding domains that can be activated by integrins and growth factor receptors, thereby regulating F-myocardium. We speculate that c-Abl kinase may also be involved in signal events that initiate L-selectin cross-linking. In this paper, we mainly discuss whether and how c-Abl molecules participate in signal events initiated by L-selectin cross-linking.
The activity of c-Abl kinase is strictly regulated by both intracellular and intramolecular mechanisms. Our study found that the activity of c-Abl kinase increases after L-selectin cross-linking. If cells are treated with c-Abl inhibitors before activation, the activity of c-Abl kinase decreases. In stationary cells, c-Abl kinases are uniformly distributed throughout the cell, while in L-selectin-crosslinked activated cells, c-Abl kinases are concentrated in F-actin and L-selectin-enriched sites. After treatment, the co-localization phenomenon disappeared, and the co-localization phenomenon was also weakened in the activated cells treated with CB, an inhibitor of F-actin polymerization. It was suggested that the changes of F-actin-dependent cytoskeleton induced by L-selectin cross-linking depended on the activity of c-Abl kinase.
C-Abl kinase also participates in the expression of colony-stimulating factor-1 (CSF-1) gene induced by L-selectin cross-linking. Overexpression of wild-type but not kinase mutant c-Abl enhances the up-regulation of CSF-1 gene induced by L-selectin cross-linking. Cells treated with c-Abl kinase inhibitors before activation decrease the C-Abl overexpression. The expression of SF-1 gene showed that the expression of CSF-1 gene was enhanced by L-selectin cross-linking, which was dependent on the activity of c-Abl kinase. ZAP70 kinase was one of the first molecules activated after T cell activation. Overexpression of wild type ZAP70 instead of kinase mutant or Y319F mutant ZAP70 also enhanced the expression of CSF-1 gene. Before activation, c-Abl and ZAP70 inhibitors were used to regulate the expression of CSF-1 gene by regulating the phosphorylation of ZAP70 Y319.
We found that L-selectin, c-Abl and ZAP70 bind together in L-selectin-activated T cells by protein immunoprecipitation and GST-pull down assay. By examining the binding relationship of L-selectin with different stimulation time, we found that c-Abl kinase and L-selectin bind together in composition with L-selection. With the increase of the cross linking time, more ZAP70 aggregates into the SH2 domain of c-Abl kinase.
Our results demonstrate that c-Abl kinase plays an important role in signal events induced by L-selectin crosslinking. It not only regulates F-actin-dependent cytoskeleton changes through changes in kinase activity, but also receives and transmits signal events induced by L-selectin crosslinking by binding to the tail of L-selectin cytoplasm. The upstream signal events are passed through the downstream substrate to regulate the expression of genes in the cell.
【学位授予单位】：东北师范大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R341

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