Study On The Biological Function Of ZCW Protein Interacted With P21-activated Kinase-1

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.