【作者】 王桂玲；

【导师】 李丰；

【作者基本信息】 中国医科大学 ， 细胞生物学， 2007， 博士

【摘要】 目的最近的研究表明，肿瘤细胞信号传导通路的异常是导致肿瘤发生发展的主要原因。胃癌是我国最常见的恶性肿瘤，其发病率在我国近年来持续上升。因此胃癌发病机制及其诊断治疗的研究是我国目前肿瘤研究的一个重要课题。激酶在细胞信号转导过程中起到枢纽作用，这使它们在许多种肿瘤的治疗中成为有效的药物作用靶点。PAK1(P21-activated kinase1)是一保守的丝氨酸/苏氨酸蛋白激酶，为Rho家族小鸟苷三磷酸酶(Rho-GTPase)Cdc42和Rac1下游重要的靶蛋白。最近的研究表明它在癌症信号传导网络中起到关键的调节作用。PAKs可以被很多种细胞外刺激因子激活，参与许多重要的细胞活动如：细胞骨架重组、细胞周期演进、凋亡与存活、基因转录调节及癌细胞侵袭转移等。PAK1所具有的广泛生物学功能是由于其磷酸化下游靶蛋白引起的。到目前为止，已经发现了多种PAK1的底物及其相互作用蛋白。并且许多证据足以表明在肿瘤发生发展过程中存在PAK1信号转导途径。研究发现PAK1激酶在肿瘤细胞中存在广泛的过表达现象，并与细胞的恶性转化和癌细胞的侵袭性有着密切的联系，因此可以通过抑制PAK1的表达来抑制肿瘤生长。干扰PAK1信号转导途径有望成为肿瘤分子水平的干预靶点。因此对于PAK1及其下游分子的研究尤为重要。ZCW是我们利用酵母双杂交的方法，从乳腺上皮细胞cDNA文库中筛选了一个新的PAK1作用蛋白。生物信息学预测分析ZCW蛋白含有970个氨基酸(AA)，在其N-末端432-481AA区域含有一个新的CW(半胱氨酸-色氨酸)型锌指结构域，定位在细胞核内，可能作为一个转录因子发挥生物学作用。本研究在李丰教授前期工作基础上，采用BGC-823和SGC-7901等胃癌细胞系为研究对象，在体外和体内进一步证实PAK1蛋白与ZCW蛋白相互作用并确定磷酸化位点；用染色质免疫沉淀和启动子报告基因等分子细胞生物学技术研究PAK1调控ZCW转录因子的功能及其机制，探讨在胃癌发生发展中的作用，为胃癌治疗提供药物靶点。方法1、GST-pull down实验体外证实PAK1能与ZCW发生相互作用。克隆PAK1和ZCW基因的cDNA全长，构建不同质粒载体中；GST-pull down实验确定PAK1与ZCW相互作用区域。2、激酶分析实验验证PAK1磷酸化ZCW，并确立磷酸化位点。首先确定PAK1与ZCW磷酸化区域；在这个区域通过RXXS/T模序确立磷酸化位点，PCR方法进行定点突变(丝氨酸或苏氨酸→丙氨酸)并构建GST-ZCW融合载体，纯化ZCW野生型和ZCW突变体蛋白进行体外激酶实验。3、Northern Blot和Western Blot法筛选PAK1和ZCW高表达的胃癌细胞株，在ZCW低表达的SGC-7901胃癌细胞中转染pcDNA-3.1A-ZCW-wt，经G418筛选并检测建立ZCW稳定高表达的细胞株，同时用空载体pcDNA-3.1A做对照。4、细胞内共定位法证实PAK1蛋白与ZCW蛋白发生相互作用。在真核细胞中表达PAK1和ZCW，并进行亚细胞定位；在EGF或血清刺激条件下，共转染GFP-PAK1-wt和pcDNA3.1A-ZCW-wt，观察pcDNA3.1A-ZCWwt或内源性ZCW的定位变化及与PAK1的共定位。5、酵母交配实验证实PAK1蛋白与ZCW蛋白发生相互作用构建诱饵蛋白pGBKT7-PAK1(1-270)和pGBKT7-PAK1(270-545)载体；构建其作用蛋白pGADT7-ZCW和阳性对照蛋白pGADT7-Cdc42和pGADT7-Rac1的载体；酵母交配实验验证PAK1与ZCW蛋白相互作用。6、免疫共沉淀实验证实PAK1蛋白与ZCW蛋白发生相互作用。选取人胃癌7901-ZCW稳定细胞株，以7901-3.1A稳定细胞株为对照，在非变性条件下收集细胞，加1x IP buffer收集蛋白，超声破碎后离心去除不溶物。加入适量的PAK1多克隆抗体，4℃轻轻摇动混匀过夜，再加入30ul protein Aagarose Beads混合2小时，以1x IP buffer洗Besds 5次，Western Blot检测共沉淀的ZCW蛋白。进一步证实ZCW蛋白与PAK1在细胞内的相互结合。7、RNAi干扰技术分析ZCW的生物学功能RNAi阻断技术PAK1表达，Western Blot方法检测PAK1阻断的特异性和ZCW的表达；免疫共焦激光显微镜原位观察ZCW的定位变化。RNAi阻断ZCW表达，Western Blot法筛选三对ZCW RNAi阻断的特异性并检测下游靶基因p21(Waf1/Cip1)表达的影响。8、Northern Blot和RT-PCR方法检测ZCW对下游靶基因p21(Waf1/Cip1)表达的影响选取人胃癌7901-ZCW稳定细胞株，以7901-3.1A稳定细胞株为对照，在EGF干预的条件下，Northern Blot和RT-PCR法分别比较分析p21(Waf1/Cip1)在7901-ZCW稳定表达细胞株与对照组7901-3.1A稳定细胞株的表达情况。9、利用Dual-Luciferase Assay System分析ZCW对靶基因p21(Waf1/Cip1)启动子的调控构建pGL3-P21(Waf1/Cip1)-promoter(2.3kb)-Luciferase reporter载体，用pRL-TK载体做内对照，转染ZCW和PAK1质粒分析ZCW对其转录活性的调节，同时分析PAK1对ZCW转录活性的调控。10、染色质免疫沉淀技术(ChIP)分析ZCW蛋白作为转录因子与靶基因p21(Waf1/Cip1)的特异DNA结合选取人胃癌7901-ZCW稳定细胞株，以7901-3.1A稳定细胞株为对照。收集蛋白并进行处理，分别留出Input为对照，加入ZCW特异抗体孵育过夜，加入适量鲑鱼精DNA/蛋白A琼脂糖，进行样品处理和收集，分别以p21(Waf1/Cip1)启动子区的三对引物进行PCR，检测ZCW与p21(Waf1/Cip1)启动子区的DNA结合。结果1、体外GST-pull down证实PAK1能与ZCW发生相互作用已获得PAK1和ZCW基因的编码区全长，并构建不同实验用途的质粒载体中；GST-pull down实验确定PAK1与ZCW能相互作用。结果显示PAK1与ZCW的C-末端657-781AA区域发生作用，而ZCW与PAK1 N-末端的75-132AA区域相互作用。2、激酶分析实验验证ZCW是PAK1磷酸化的底物。激酶实验分析PAK1蛋白与ZCW蛋白C-末端657-781区域发生磷酸化。对ZCW四个磷酸化位点进行定点突变(Ser→Ala)，对突变体进行分析，结果第663、668和711丝氨酸都能被PAK1磷酸化；而第677丝氨酸突变为丙氨酸后，不能被PAK1磷酸化。因此PAK1与ZCW发生磷酸化位点在第677丝氨酸。3、Northern Blot和Western Blot法选取了PAK1和ZCW高表达的胃癌BGC-823细胞系为实验对象，并选取SGC-7901胃癌细胞系建立ZCW稳定表达细胞系。4、细胞内共定位法证实PAK1蛋白与ZCW蛋白发生相互作用。间接免疫荧光显示内源性PAK1蛋白大部分定位在BGC-823胃癌细胞的胞浆中，少量定位在细胞核。而ZCW蛋白主要定位在细胞核。在EGF或血清刺激条件下，转染GFP-PAK1-wt或共转pcDNA-ZCW-wt分别观察内源性和外源性ZCW主要定位在细胞浆，少量在细胞核，并在细胞浆中与PAK1共定位。5、酵母双杂交实验证实PAK1蛋白与ZCW蛋白发生相互作用。成功构建诱饵蛋白pGBKT7-PAK1(1-270)和pGBKT7-PAK1(270-545)载体和其作用蛋白pGADT7-ZCW和阳性对照蛋白pGADT7-Cdc42和pGADT7-Rac1的载体，并进行酵母交配实验验证PAK1(1-270)区域与ZCW蛋白相互作用。6、免疫共沉淀实验证实PAK1蛋白与ZCW蛋白发生相互作用。选取人胃癌7901-ZCW稳定细胞株，以7901-3.1A稳定细胞株为对照，分别用ZCW和PAK1多克隆抗体进行免疫共沉淀，用ZCW和pcDNA3.1载体上的His标签抗体进行Western Blot，结果检测ZCW蛋白与PAK1在细胞内的相互结合。7、RNAi干扰技术研究ZCW生物学动能Western Blot方法结果检测Specific siRNA PAK1对PAK1具有特异性的阻断，而对高度同源的PAK2蛋白表达没有影响。同时检测对其作用蛋白ZCW的表达没有影响。通过设立PAK1 siRNA和PAK1 Control siRNA，免疫共焦激光显微镜原位观察PAK1 siRNA组，ZCW蛋白的定位主要在细胞核；而PAK1Control siRNA组，ZCW蛋白的定位主要在细胞浆。通过Western Blot法筛选出一对高阻断ZCW蛋白的ZCW RNAi。干扰ZCW后，Western Blot结果显示p21(Waf1/Cip1)蛋白表达与对照组相比明显增多；当ZCW高表达时，Western Blot结果显示p21(Waf1/Cip1)蛋白表达与对照组相比明显减少，说明ZCW下调p21(Waf1/Cip1)的表达。8、Northern Blot和RT-PCR方法检测ZCW对下游靶基因p21(Waf1/Cip1)表达的影响选取人胃癌7901-ZCW稳定细胞株，以7901-3.1A稳定细胞株为对照，在EGF干预的条件下，Northern Blot和RT-PCR检测在7901-ZCW稳定细胞株中低表达，而在对照组7901-3.1A稳定细胞株中p21(Waf1/Cip1)高表达。9、利用Dual-Luciferase Assay System分析ZCW对靶基因p21(Waf1/Cip1)启动子的调控利用pGL3-P21-promoter(2.3kb)-Luciferase reporter载体，荧光结果显示ZCW下调P21(Waf1/Cip1)-promoter的活性，同时显示PAK1能调控ZCW对P21(Waf1/Cip1)的抑制作用。10、染色质免疫沉淀技术(ChIP)分析ZCW蛋白作为转录因子与靶基因p21(Waf1/Cip1)的特异DNA结合选取人胃癌7901-ZCW稳定细胞株，以7901-3.1A稳定细胞株为对照，用ZCW特异抗体孵育，结果显示ZCW能与p21(Waf1/Cip1)启动子区的DNA结合。结论1、PAK1能与ZCW的C-末端657-781AA区域发生作用，并是通过磷酸化ZCW第677丝氨酸实现的。而ZCW是与PAK1N-末端的75-132AA区域相互作用。2、ZCW蛋白定位在细胞核，其定位的变化受PAK1和EGF的调节，可移位到细胞质内与PAK1共定位。3、干扰PAK1后，对细胞内ZCW总蛋白表达没有影响；同时也没有引起ZCW蛋白定位的变化。4、干扰ZCW后，能引起p21(Waf1/Cip1)蛋白高表达；在ZCW高表达的稳定细胞株中，p21(Waf1/Cip1)蛋白低表达。5、ChIP实验结果显示ZCW能与p21(Waf1/Cip1)启动子区的DNA结合；报告基因实验分析ZCW下调p21(Waf1/Cip1)启动子的活性，而PAK1对ZCW的转录抑制有调控作用。更多还原

【Abstract】 ObjectiveEvidence that cancer is a disease of deregulated signalling pathways has led to the development of signalling-based targeted therapies for various human tumour types. In recent years, Gastric cancer is the most common malignant tumor with increasing morbidity in China. So the research on pathogenesis,diagnosis and treatment of gastric cancer have become the focus of tumor research in our country. Further study on the mechanisms by which key proteins and its downstream target protein interact in cell signal transduction has revealed the regulation of tumor progression. Block of key target is also the key point for the therapy of tumor in this field.PAK1 have recently been found to be key regulators of cancer-cell signalling networks. The p21-activated kinases （PAKs） are serine/threonine protein kinases whose activity is stimulated by the binding of active Rac and Cdc42 GTPases.Our understanding of the regulation and biology of these important signaling proteins has increased tremendously. PAKs are activated by a variety of GTPase-dependent and-independent mechanisms, which reflects the contributions of Pak function in many cellular signaling pathways and the need to carefully control Pak action in a highly localized manner. PAKs serve as important regulators of cytoskeletal dynamics and cell motility, transcription through MAP kinase cascades, death and survival signaling, and cell cycle progression. Pak1 is activated by growth-factor receptor tyroine kinase via Rac1 or Cdc42. Wide ranges of biological activities result from pak1 phosphorylation of downstream substrates. Consequently, PAKs have also been implicated in a number of pathological conditions and in cell transformation. Researches have found that overexpression of Pak exists in a number of tumor cells, which suggest a close relationship with malignant transformation and invasion. Therefore, blocking tumor growth may be induced by inhibitory Pak1 expression. Targeting developing Pak inhibitors will provide an important role in development of targeted anti-tumour agents. So a further study of PAK1 may contribute to clinical diagnosis and treatment.PAK1 was first identified as a member of the serine/threoine proten kinase that bind to Rac1 and Cdc42.It is increasingly recognized that downstream nuclear events can be triggered directly by pak1 relocalization to the nucleus and phosphorylation of target proteins.To date, Pak1-induced nuclear changes have centered on coactivator-mediated gene activation and phosphorylation of histone H3. To further understand the molecular mechanisms by which Pak1 regulates various signaling cascades, we carried out a yeast two-hybrid assay for screening Pak1-interacting proteins.A yeast two-hybrid system helped to identify new Pak1-interacting proteins in a human breast cDNA library. One positive clone was identical to ZCW which contain about 970 amino acids.The CW-type zinc finger domain in ZCW is located in the N-terminal 432-481 aminoacids. ZCW Predicted by bioinformatics is localized in nucleus and a transcriptional regulator. This study based on previous work that Professor Feng Li had identified ZCW interaction with Pak1 using glutathione S transferase （GST） assay. To further investigate the interaction between ZCW and Pak1 in vivo and as a transcriptional regulator in progression of gastric cancer,BGC-823 and SGC-7901 human gastric cancer cell lines was employed as a experiment model in the sequence studies. Methods1. ZCW interacts with PAK1 in vitroCloning the full-length PAK1 and ZCW and constructing different plasmids.To identity the interaction of ZCW with PAK1 in vitro using a GST pull down assay, we tested the ability of an in-vitro translated Pak1 protein binding to ZCW a fused to glutathione S-transferase （GST）.2. In vitro kinase assays to identify Pak1 phosphorylation of ZCW.Purified PAK1 enzyme readily phosphorlated GST-ZCW; To identify the specific site（s） of ZCW phosphorylation by PAK1, we first determined the region of ZCW that was phosphorylated by PAK1.Within this region are four RXXS consensus phosphorylation motifs,with amino acids 663,668,677 and 711 as potential phosphorylation sites.PCR-based point mutations thant resulted in Ser→Ala substitutions were made in the GST-ZCW vector.Purified wild-type or point-mutant ZCW proteions were phosphorylated by the PAK1 emzyme in vitro.3. To screen high expression of Pak1 and ZCW in Gastric cancer cell lines with Northern Blot and Western Blot assays.To obtain high expression of ZCW stable cell lines in SGC-7901 Gastric cancer cell lines by transfecting pcDNA-3.1A-ZCW-wt plasmid DNA and control pcDNA-3.1A plasmid DNA.4. The cellular localization of ZCW and PAK1 using indirect immunoflurescence to study ZCW interaction with PAK1 in vivoThe cellular localization of endogenous ZCW and endogenous Pak1 in BGC-823 cells. The cellular colocalization of ZCW and Pak1 when GFP-Pak1-wt and pcDNA3.1A-ZCW-wt were transiently transfected into BGC-823 cells with EGF stimulation.5. A yeast cotansformation assay to confirm in vivo Pak1-interacting ZCW Construct bait protein vector of pGBKT7-Pak1（1-270）and pGBKT7-Pak1（270-545）. construct grey protein vector of pGADT7-ZCW and positive control pGADT7-Cdc42, pGADT7-Racl. A yeast cotansformation assay to confirm in vivo PAK1-interacting ZCW6. Co-immunopecipitation assay to confirm the interaction between endogenous PAK1and endogenous ZCW or His-ZCW.Preparing Cell LysatesAspirate media. Treat 7901-ZCW stable cells by adding fresh media containing regulator for desired time.To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold PBS,Remove PBS and add 0.5 ml 1X ice-cold cell lysis buffer plus 1 mM PMSF to each plate （10 cm2） and incubate the plateon ice for 5 minutes. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice Sonicate four times for 5 seconds each on ice. Microcentrifuge for 10 minutes at 4℃, and transfer the supernatant to a new tube. The supernatant is the cell lysate.If necessary lysate can be stored at-80℃.ImmunoprecipitationTake 200μl cell lysate and add primary antibody; incubate with gentle rocking overnight at 4℃.Add protein A agarose beads （20μl of 50% bead slurry）. Incubate with gentle rocking for 1-3 hours at 4℃. Microcentrifuge for 30 seconds at 4℃. Wash pellet five times with 500μl of 1X cell lysis buffer. Keep on ice during washes. Resuspend the pellet with 20μl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 seconds. Heat the sample to 95-100℃for 2-5 minutes, Load the sample （15-30μl） on SDS-PAGE gel （12-15%）. Analyze sample by Western Blot.7. RNAi analysis ZCW biological functionWestern Blot detects the specific of Pak1 RNAi and ZCW expression. Examinetion the localization of endogenous ZCW when using Pak1-specific siRNA to block the expression of endogenous Pak1.The specific of ZCW RNAi and detects the protein expression of p21 （Waf1/Cip1） with Western Blot.8. Northern Blot and RT-PCR to detect the effect of ZCW on the p21 （Waf1/Cip1） gene expression. 9. To study ZCW regulation of pGL3-p21 （Waf1/Cip1）-promoter （2.3kb） with Dual-Luciferase Assay System10. Chromatin immunoprecipitation （ChIP） assay to determine whether ZCW binding to p21 （Waf1/Cip1）-promoter DNA.7901-ZCW and control 7901-3.1 A stable cells （～10⁶） treated as described were subjected to crosslinking of proteins to DNA.ChIP assay was done as Upstate described. After immunoprecipitation with ZCW or His-tagged antibody, eluted DNA was amplified by PCR using the p21 promoter primers for the upstream region （₂264 to₁971） were:5’ primer: 5’-ttgagctctggcatagaaga-3’;3’ primer: 5’-tacccagacacactctaagg-3’.The primers for element region （₁357 to₁063） were:5’ primer: 5’-agactctgagcagcctgag-3’;3’ primer: 5’-aaccctcatttgcagatggt-3’.The primers for the downstream core promoter region （₁94 to+81） were:5’ primer: 5’-accggctggcctgctggaact-3’;3’ primer: 5’-tctgccgccgctctctcacct-3’.Results1. In vitro transcription and translation of the Pak1 and ZCW proteins were carried out by using the TNT transcription-translation system, ZCW-GST but not GST efficiently interacted with 35S-labelled full-length Pak1.Conversely, in-vitro-translated ZCW protein specifically interacted with GST-Pak1. We found that the N-terminal region of Pak1 containing residues 75-132 can interact with ZCW and that the CRIB domain of Pak1 containing residues 52-132 can efficiently interact with the C terminal 657-781 domain of ZCW.2. To show that ZCW is a direct substrate of Pak1, we carried out an in vitro kinase assay by using purified ZCW, pak1 enzyme was purified from Escherichia coli. As a result, Pak1 phosphorylated full-length ZCW, these results suggested that the Pak1 phosphorylation sites were localized in the region of ZCW containing residues 657-781. Accordingly, mutation of Ser 677 to Ala completely abolished the phosphorylationof ZCW by Pak1.Mutation of Ser 663 to alanine had no effect on Pak1-mediated phosphorylation, whereas the mutation of Ser 668 and Ser711 to alanine reduced the magnitude of Pak1 phosphorylation of ZCW in vitro. Ser 677 also seemed to be important for Pak1-mediated phosphorylation in vivo.3. To be showed high expression of Pak1 and ZCW in the BGC-823 Gastric cancer cell and low expression of ZCW in the SGC-7901 with Northern Blot and Western Blot assays. To obtain high expression of ZCW stable cell lines in SGC-7901 Gastric cancer cell lines.4. Colocalization of Pak1 and ZCW is induced by physiological signalsThe cellular localization of endogenous Pak1 in BGC-823 cells is in the cytoplasm while endogenous ZCW in the nucleus, when GFP-Pak1-wt and pcDNA-ZCW wt was transiently transfected into BGC-823 cells, PAK1 is in the cytoplasm while endogenous ZCW in the nucleus. Colocalization of ZCW and PAK1 when GFP-Pak1-wt and pcDNA-ZCW-wt was transiently transfected into BGC-823 cells with EGF stimulation resulted in the intracellular colocalization of endogenous PAK1and ZCW both in the cytoplasm and in the nucleus.5. A yeast cotansformation assay to confirm in vivo PAK1-interacting ZCWThe specificity of the Pak1-ZCW interactions was verified by one-on-one transformation. Cotransfection of ZCW and Pak1 constructs conferred on transformed colonies the ability to grow in medium lacking adenosine, histidine, tryptophan and leucine and to turn blue in aα-galactosidase assay, whereas cotransfection of ZCW or Pak1 with control vectors did not.In this yeast two-hybrid assay, full-length ZCW interacted specifically with the N-terminal regulatory domain of Pak1. 6. Co-immunopecipitation assay to confirm the interaction between endogenous PAK1 and endogenous ZCW or His-ZCW. When His-tagged ZCW was stablely transfected in SGC-7901 human gastric cancer cells, endogenous PAK1 interacted with His-ZCW.7. We used Pak1-specific siRNA to block the expression of the endogenous Pak1 gene product and examin the localization of endogenous ZCW.The control siRNA had no effect on normal ZCW distribution. However, Pak1 siRNA blocked Pak1 protein expression, and resulted in exclusively nuclear localization of ZCW.8. Northern Blot and RT-PCR assay to detect the effect of ZCW on the p21（Waf1/Cip1） gene expression: high expression in the ZCW high stable cell lines compared with that of control.9. To study ZCW regulation of pGL3-P21 （2.3kb） with Dual-Luciferase Assay System, we found ZCW repress the p21（Waf1/Cip1）-promoter activity.PAK1 regulation of ZCW repressor functions.10. When using chromatin immunoprecipitation （ChIP） with anti-ZCW antibody and PCR primers designed against the p21 （Waf1/Cip1）-promoter（2264 to₁971,₁357 to 1063 and₁94 to +81 base pairs）,we found ZCW can bind the p21 （Waf1/Cip1）-promoter DNA target.Conclusions1. We found that the N-terminal region of Pak1 containing residues 75-132 can interact with ZCW and that the CRIB domain of Pak1 containing residues 52-132 can interact with the C-terminal 657-781 domain of ZCW.To show that ZCW is a direct substrate of Pak1, these results suggested that the Pak1 phosphorylation sites were localized in the region of ZCW containing residues 657-781. Ser 677 also seemed to be important for Pak1-mediated in vivo. 2. The cellular localization of endogenous PAK1 in BGC-823cells is in the cytoplasm while endogenous ZCW in the nucleus.Colocalization of ZCW and PAK1 when GFP-Pak1-wt and pcDNA-ZCW-wt was transiently transfected into BGC-823 cells with EGF stimulation resulted in the intracellular colocalization of endogenous PAK1and ZCW both in the cytoplasm and in the nucleus.3. ZCW may be a transcriptional repressor and PAK1 regulation of ZCW repressor functions.The mechanism by which ZCW repress the p21（Waf1/Cip1）-promoter activity is currently under investigation.更多还原