论文标题:APPC2与PRAK之间的相互作用
The Interaction between ARPC2 and PRAK
论文作者 龚小卫
论文导师 姜勇,论文学位 博士,论文专业 病理生理学
论文单位 第一军医大学,点击次数 86,论文页数 101页File Size5081k
2004-05-01论文网 http://www.lw23.com/lunwen_250646032/ p38调节/激活蛋白激酶,肌动蛋白相关蛋白复合体成份2,蛋白质-蛋白质相互作用,肌动蛋白相关蛋白2/3复合体,丝裂原激活蛋白激酶,p38,应激刺激,噬菌体展示,细胞骨架,免疫共沉淀,离体结合
p38-regulated/activated protein kinase, ARPC2, actin-related protein 2/3 complex, protein-protein interaction, stress stimulation,mitogen-activated protein kinase, p38, phage display, cytoskeleton,co-immunoprecipitation, in vitro binding
丝裂原激活蛋白激酶(MAPK)是真核细胞介导细胞外信号到细胞内反应的重要信号传导系统。p38是MAPK亚族之一，除了参与应激和生长因子诱导的基因表达及细胞增殖和凋亡的调节外，还在调节细胞骨架的重构中起重要作用。p38通过激活MAPKAPK2／3和PRAK，转而激活HSP27，从而介导细胞骨架重构，参与细胞应激反应。然而，尽管已经认识到PRAK与细胞骨架重构有关，但对其具体机制却知之甚少。为了研究PRAK在细胞内的确切功能，我们利用GST-PRAK作为诱饵，通过T7噬菌体展示系统进行了筛选。结果发现，PRAK能与一种在肌动蛋白细胞骨架装配中起重要作用的蛋白复合体—Arp2／3复合体中的一个亚单位ARPC2相互作用。为了验证两者之间是否确实存在这种相互作用，我们对这两者在体内和体外的结合情况都进行了研究，并对两者相互作用的影响因素进行了探讨。 在利用PRAK作为诱饵进行的噬菌体展示筛选中，我们共筛选到了13个编码蛋白的序列，其中就包括ARPC2。通过构建人肺cDNA文库，并在文库中利用特异性引物将ARPC2基因扩增出来，随后分别克隆到pET-14b和pcDNA3载体上。 在离体结合研究中，首先分别在大肠杆菌株BL21和DE3中对GST-PRAK和His-ARPC2进行了表达，并分别利用谷胱甘肽亲和树脂和Ni-NTA亲和树脂对这两种蛋白进行了纯化。在获得纯化蛋白的基础上，进行离体结合研究。结果表明，结合在Ni-NTA亲和树脂上的His-ARPC2能将GST-ARPC2共沉淀下来，而在相同条件下对单独的GST蛋白没有作用，说明ARPC2在离体条件下能与PRAK结合。 在在体结合研究中，将HA-PRAK和FLAG-ARPC2质粒共转染NIH／3T3细胞后，将细胞裂解并进行免疫共沉淀。利用抗FLAG抗体偶合琼脂微粒进行的免疫共沉淀结果表明，在未受刺激的情况下ARPC2与PRAK不结合，但在NaAsO_2的刺激下两者发生结合，且胰岛素的刺激也不能使两者结合。利用抗HA抗体偶合磁珠进行免疫共沉淀，结果发现PRAK也是在未受刺激时不与ARPC2结合，而NaAsO_2刺激时能将ARPC2共沉淀下来。在此基础上，利用GST-WA蛋白能与Arp2／3复合体结合的性质而将内源性ARPCZ沉淀下来，同时检测到内源性PRAK被共沉淀，进一步证实了ARPCZ能与PRAK相互作用，这也是两者在细胞内相互作用的最直接证据。对细胞进行不同时间刺激后，通过免疫共沉淀来了解ARPCZ与PRAK结合的时间过程。结果发现，两者在受到刺激后结合强度逐渐增加，至60~90而n时达最高峰，随后又逐渐下降。对多种不同的应激刺激是否能促进ARPCZ与PRAK的结合也进行了研究，结果表明各种应激刺激都能不同程度地促进两者结合，其中以N叭502、sorbitol和HZo:的作用较强。 利用p38通路抑制剂SB203580和ERK通路抑制剂PD98059预处理细胞后，观察对ARPCZ和PRAK结合的影响。结果发现，这两种不同的MAPK通路抑制剂对两者的结合都没有影响。利用PRAK的无活性突变体PRAK(182A)、活性突变体PRAK(182D)和失去激酶活性的突变体PRAK(KM)分别与FLAG一ARPCZ共转染细胞，发现PRAK(1 82A)和PRAK(KM)在未刺激的情况下，能与ARPCZ强烈结合，而在受到刺激后，结合反而显著减弱;PRAK(1 82D)与ARPCZ的结合模式则与野生型PRAK类似。在此基础上，对SB203580长时间处理细胞对野生型PRAK与ARPCZ结合的影响进行了研究，结果发现与PRAK(1 82A)和PRAK(KM)类似，也是未刺激时结合，刺激后结合减弱。利用微管解聚剂nocodazole、微管稳定剂taxol及微丝解聚剂细胞松弛素D等影响细胞骨架的药物预处理细胞后，观察这些药物对ARPcZ与PRAK结合的影响。结果表明，nocodazole及taxol对两者的结合没有影响，而细胞松弛素D则能显著抑制两者的结合。在认识到p38通路抑制剂对两者结合无影响，而微丝解聚剂则能抑制两者结合的基础上，设计了一个交叉实验来确认ARPCZ与PRAK的结合到底是由PRAK通路的激活，还是ARPCZ通路的激活，抑或是两者的同时激活所决定的。结果表明，单纯PRAK通路的激活不足以促使两者结合，而ARPCZ通路的激活才是导致两者结合的主要原因。 对ARPCZ和PRAK在细胞内的定位情况及受到刺激后的移位情况也进行了研究。结果发现，在外源性导入细胞的情况下，PRAK主要位于核内，刺激后逐渐出核，约90而n达最高峰，随后又逐渐恢复到静息状态的情况;而与此相反，ARPCZ主要位于胞质中，刺激后逐渐入核， 一6-约90而n达最高峰，随后又重新出核。 通过上述研究，可以得出以下结论:1.ARPCZ与PRAK在离体和在体情况下均能结合。2.ARPCZ与PRAK的结合具有刺激反应性，不同应激刺激都能促进两 者结合，提示两者的结合可能在细胞对应激刺激的反应中具有重要 作用。3.p38通路抑制剂SB203580、ERK通路抑制剂PD98059、微管解聚剂 nocodazole及微管稳定剂taxol都不能阻断ARPCZ与 PRAK的结合， 而微丝解聚剂细胞松弛素D可以显著抑制两者结合，提示微丝在两 者的结合中具有作用。4.PRAK的无活性突变体与失去激酶活性的突变体与SBZO358O长时间 作用于野生型PRAK的效应类似，都是未刺激时与ARPCZ强烈结合，
As key components of signal transduction systems in organisms, mitogen-activated protein kinases (MAPKs) are considered to be involved in many cellular processes and pathogenesis of many severe diseases. p38, a subfamily of MAPKs, is involved in the stress and growth factors-induced gene expression and the regulation of cell proliferation and cell apoptosis, as well as in the regulation of cytoskeleton remodeling. p38 can activate MAPKAP-K2/3 and PRAK, which in turn activate HSP27, leading to cytoskeleton remodeling. Although it is widely known that PRAK is involved in cytoskeleton remodeling, the exact mechanism is little elucidated. To investigate the function of PRAK in vivo, a GST fusion protein of PRAK was used as a bait and screened through T7 phage display system. It was found that PRAK may interact with ARPC2 ? a subunit of Arp2/3 complex, which is an important protein complex in the actin cytoskeleton assembly. Then, in order to validate the interaction between these two proteins, in vitro binding and in vivo binding were executed, and the influential factors of the binding were investigated.In the T7 phage display screening in which PRAK as the bait, 13 sequences of coded protein were found, and ARPC2 was one of them. Then, human lung cDNA library was constructed and ARPC2 gene was amplified by specific primers from the library, and was cloned into pET-14b and pcDNA3 vectors, respectively.In the in vitro binding experiment, GST-PRAK and His-ARPC2 were expressed in E. coli strain BL21 and DE3, then purified with GST seprose and Ni-NTA resin, respetively. When these two purified protein were accessible, the in vitro binding was performed. The results showed that GST-PRAK could be co-precipitated by His-ARPC2, which pre-bound to the Ni-NTA resin, and the GST protein alone could not in the same condition, which suggested that ARPC2 could bind to PRAK in vitro.In the in vivo binding experiment, HA-PRAK and FLAG-ARPC2plasmids were co-transfected into NIH/3T3 cells, then the cells were lysed and the supernatant was used in co-immunoprecipitation. The co-immunoprecipitated results through anti-FLAG antibody beads showed that ARPC2 and PRAK could not bind to each other without stimulation or with the stimulation of insulin, but could bind to each other with the stimulation of arsenite. The co-immunoprecipitated results through anti-HA antibody magnetic beads showed the same results. Because of the ability of binding to Arp2/3 complex, GST-WA fusion protein was used to immunoprecipitate endogenous ARPC2, and co-immunoprecipitated endogenous PRAK was detected, which further confirmed that interaction did exist between ARPC2 and PRAK. It was also the most direct evidence of these two proteins interacted with each other. After NIH/3T3 cells were stimulated with different time, the time course of the binding between ARPC2 and PRAK was studied. The results showed that the binding intensity of these two proteins gradually increased upon stimulation, and reached the peak when stimulated 60~90 min approximately, then descended gradually. Whether different stress stimuli could also improve the binding between ARPC2 and PRAK was investigated, and the results indicated that different stress could lead to interaction between these two proteins in a variant degree, and the effect of arsenite, sorbitol and H2C2 were relatively strong among them.The influential issues on the binding between ARPC2 and PRAK were then studied. Firstly, NIH/3T3 cells were pretreated with SB203580, the specific inhibitor of p38 pathway, and PD98059, the specific inhibitor of ERK pathway, and it is shown that these two inhibitors had no effect on the binding. Then, PRAK(182A), the dominant negative mutant of PRAK, PRAK(182D), the dominant positive mutant of PRAK, and PRAK(KM), the loss-of-activity mutant of PRAK, were co-transfected with FLAG-ARPC2 respectively. The responsive pattern of PRAK(182A) was similar to that of wild type PRAK. But to our surprise, PRAK(182A) and PRAK(KM) were found to bind to ARPC2 intensively, but the bindings wer
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