【作者】 陈亮；

【导师】 曾宪录；

【作者基本信息】 东北师范大学 ， 细胞生物学， 2009， 硕士

【摘要】 血液中白细胞被血管内皮细胞捕获、沿着血管内皮细胞滚动、稳定粘附于血管内皮细胞上、跨血管内皮细胞迁移并最终到达炎症部位是一个受严格调控的复杂多步骤的过程。L-选择素组成性地表达在白细胞微绒毛顶端,是白细胞与血管内皮细胞相互作用过程中最先行使功能的分子。除了作为粘附分子发挥作用,L-选择素还作为信号分子在白细胞粘附过程中起始信号转导过程。L-选择素与单克隆抗体的结合能够引起肌动蛋白细胞骨架重排、细胞形态改变、氧呼吸爆发以及细胞内TNF-α和IL-8基因表达上调等一系列生理事件。但在白细胞起始粘附的过程中,L-选择素引起肌动蛋白细胞骨架重排的机制还缺乏研究。Lck激酶是非受体蛋白酪氨酸激酶Src家族的成员之一,特异性地在T淋巴细胞中表达,并且在T淋巴细胞的活化及发育过程中发挥着重要作用。Lck激酶包含一个N端膜定位结构域、一个独有结构域、一个SH3结构域、一个SH2结构域、一个酪氨酸激酶结构域和一个C端负调控结构域。在之前的研究中我们已经证明,Lck是L-选择素交联后最先活化的激酶。Vav1分子是一种分子量为95 kDa的蛋白,表达在所有血源性细胞中并且通过磷酸化而转变为活化形式。Vav1主要作为Rho家族GTP酶的鸟苷酸交换因子,因而可以参与多种和细胞骨架相关的信号通路。Vav1分子含有一个富含脯氨酸的序列和一个串联的SH3, SH2和SH3结构域,这也暗示着其可以在信号通路中充当接头蛋白。本论文主要探讨了在L-选择素的交联引发肌动蛋白细胞骨架重排的过程中,Lck激酶与Vav1分子所起到的作用。我们的工作证明,二者在L-选择素交联后会出现相互作用,并且都会发生磷酸化。进一步的研究表明Lck通过其SH2和SH3结构域与Vav1相互作用,而Vav1的SH2和两个SH3结构域都参与了与Lck的结合。实验结果表明,Lck激酶与Vav1分子都是L-选择素介导的信号通路中关键的信号分子。更多还原

【Abstract】 Leukocyte recruitment from the bloodstream to a site of inflammation entails a cascade of cellular adhesive events, including tethering, rolling, adhesion and transmigration. L-selectin constitutively expresses on the top of microvilli and appears to be the first receptor for the early kinetics of leukocyte-endothelial cell interaction. Besides, L-selectin contributes in leukocyte adhesion through its signaling properties. L-selectin engagement with antibody cross-linking could trigger actin cytoskeleton rearrangement, cell shape change, superoxide generation, increased gene expression of cytokine TNF-αand IL-8, and so on. But in initial adhesion of leukocytes, we still do not know what role L-selectin plays in actin cytoskeleton rearrangement.Lck kinase is a member of the Src family of non-receptor protein tyrosine kinases, which is expressed primarily in T lymphocytes and plays an important role in T cell activation and development. Lck contains an N-terminal membrane-targeting domain, a unique region, single Src SH3 and SH2 domains, and a tyrosine kinase domain followed by a C-terminal-negative regulatory domain. In our previous study, we have demonstrated that Lck was the first kinase activated in L-selectin signaling.Vav1 is a 95-kD protein expressed in all hemopoietic cells and it is activated following tyrosine phosphorylation.Vav1 acts as the guanine nucleotide exchange factor(GEF)for Rho-family GTPases and can mediate several cytoskeletal-associated cellular processes. Vav1 contains a proline-rich(PR) domain and an array of SH3, SH2 and SH3 domains, suggesting that Vav1 may have adapter functions.In this study, we focus on the function of Lck kinase and Vav1 in actin cytoskeleton rearrangement induced by L-selectin ligation. Our results demonstrated that Lck kinase could interact with Vav1 after the cross-linking of L-selectin in Jurkat T cells, and they were phosphorylated induced by L-selectin engagement. And then, we find that Lck can associate with Vav1 through its SH2 and SH3 domains. Finally, we suggested SH2 and both of SH3 domains in Vav1 could interact with Lck kinase. These results indicate that Lck kinase and Vav1 are all key signal transducers downstream of the L-selectin.更多还原