【作者】 程春明；

【导师】 顾建新；

【作者基本信息】 复旦大学 ， 疾病蛋白组学， 2009， 博士

【摘要】 Rac1/Cdc42的效应子PAK被不同的信号级联反应活化,包括酪氨酸激酶受体和integrins受体,并且它也调控着大量的生物学过程,如细胞增殖和细胞迁移等。PAK的活化曾被报道对RAF-MEK-MAPK信号通路的最大活化是必须的,可能是因为PAK能够同时活化RAF和MEK的缘故。在本论文中,我们证实了起初被鉴定为A-RAF激酶特异负调节子的TH1在细胞中也和PAK1有相互作用。荧光共聚焦显微镜分析显示TH1和PAK1在细胞核和胞浆中都有共定位,这种现象和我们目前的结果相一致。GST pull-down和免疫共沉淀实验证实了TH1和PAK1能直接相互作用,并且选择性的结合到PAK1 C端的激酶域上。TH1和PAK1结合的能力随着PAK1的活化而增强。和PAK1的这种结合形式暗示了他们间的相互作用部分是被PAK1激酶活性所调控的。正如体外激酶活性分析和Western Blot检测所显示的,TH1抑制了PAK1激酶活性并且负调节了MAPK信号的传导。有趣的是,TH1在体内和体外都和MEK1/ERK结合,但并不直接抑制他们的激酶活性。此外,我们还观察到TH1定位于迁移细胞前沿的粘附斑和纤维伪足上,并且通过影响肌动蛋白和粘附动力学变化干扰了细胞的迁移。基于这些实验数据,我们提出了一个模型:TH1和PAK1相互作用,并通过MAPK通路的上游特异地限制了MAPK模块的活化,从而影响了细胞的迁移。更多还原

【Abstract】 The Rac1/Cdc42 effector,p21-activated kinase,is activated by various signaling cascades,including receptor-tyrosine kinasesand integrins,and regulates a number of processes such as cell proliferation and motility.PAK activity has been shown to be required for maximal activation of the canonical RAF-MEK-MAPK signaling cascade,possibly because of PAK co-activation of RAF and MEK.Here we have shown that trihydrophobin 1(TH1),originally identified as a negative regulator of A-RAF kinase,also interacted with PAK1 in cultured cells.Confocal microscopy assay indicated that TH1 colocalized with PAK1 in both the cytoplasm and nucleus,which is consistent with our previous results.GST pull-down and coimmunoprecipitation experiments demonstrated that TH1 interacted directly with PAK1 and bound selectively to the carboxyl-terminal kinase domain of PAK1,and the ability of the binding was enhanced along with activation of PAK1.The binding pattern of PAK1 implies that this interaction was mediated in part by PAK1 kinase activity. As indicated by in vitro kinase activity assays and Western blot detections, TH1 inhibited PAK1 kinase activity and negatively regulated MAPK signal transduction.Interestingly,TH1 bound with MEK1/ERK in cells and in vitro without directly suppressing their kinase activity.Furthermore,we observed that TH1 localized to focal adhesions and filopodia in the leading edge of cells,where TH1 reduced cell migration through affecting actin and adhesion dynamics.Based on these observations,we propose a model in which TH1 interacts with PAK1 and specifically restricts the activation of MAPK modules through the upstream of the MAPK pathway,thereby influencing cell migration.更多还原