摘要
前言
PAK(p21 activated kinase)为一类保守的丝氨酸/苏氨酸蛋白激酶,通过其下游众多的激酶底物和结合蛋白参与众多的生物学功能。目前被认为是肿瘤细胞信号网络的关键调节因子。迄今鉴定的PAK家族成员包括六个成员:PAK1-PAK6,根据结构相似性分为两类,Ⅰ类包括PAK1-3,Ⅱ类包括PAK4-6。两类PAK在结构上包括N末端保守的PBD结构域(p21 binding domain)和C末端保守的激酶域(kinase domain,KD)。
PAK4是Ⅱ类PAK中最早被鉴定的,也是Ⅱ类PAK中最具有代表性的成员μ。与其他PAK家族成员一样,PAK4与Ⅰ类PAK不同的是N末端不含有自我抑制域(auto inhibitory domain,AID)因此两类PAK在活化方式和生物学功能上有所区别。PAK4在大多数组织中均有表达,在前列腺、睾丸及结肠中表达最高。重要的是PAK4在众多肿瘤细胞系中有过表达,并且其定位于19号染色体的基因在人结肠、卵巢及胰腺肿瘤中有扩增现象。在对人大肠癌340种丝氨酸/苏氨酸激酶突变筛查中发现了2个PAK4的基因突变。研究发现,激活型的PAK4可以导致肿瘤细胞的锚定非依赖性生长,而失活型的PAK4突变体(PAK4NE)则显著抑制了Ras引起的细胞转化。虽然PAK4是Ⅱ类PAK中研究的最为广泛的成员,但是目前发现的PAK4相互作用蛋白仍然十分有限,对PAK4的研究仍处于起步阶段。通过与Rho家族GEF-H1(guanine nucleotide exchange factor H1)相互作用,PAK4可引起细胞形态改变。PAK4通过与G蛋白偶联的Rho-GEF直接的相互作用,下调Rho活性,显著减少了Rho的GTP结合形式,进一步减少了血清或LPA刺激的应力纤维的形成。PAK4通过与整合素αvβ5的相互作用,言裥缘拇俳纤卅羦β5介导的细胞迁移。有报道证实LIMK1可以作为PAK1的磷酸化底物,最近研究证实,PAK4也可以磷酸化LIMK1。而活化的LIMK1可以磷酸化cofilin,有研究发现PAK4引起的细胞形状改变依赖cofilin的磷酸化,这表明对LIMK1活性的调节对于细胞移动是必须的,同时也说明PAK4,LIMK1和cofilin之间存在着功能上的关联。但对于PAK4引起的细胞迁移,仍然有很对机制等待研究。因此我们利用酵母双杂交技术从人胎脑文库中筛选出若干PAK4相互作用蛋白,我们选择了2个与肿瘤侵袭转移及细胞骨架相关的进行了研究,希望对PAK4促进肿瘤细胞骨架重组及肿瘤细胞迁移的作用及机制进行探讨。它们分别为DGCR6L和PKN1。
DGCR6L是我们利用酵母双杂交筛选出来的新的PAK4相互作用蛋白。GCR6L(DiGeorge critical region 6 like)基因定位于染色体22q11,存在两个高度同源的拷贝(DGCR6和DGCR6L),最初发现在velo-cardio-facial syndrome/DiGeorgesyndrome(VCFS/DGS)中缺失。两个基因高度同源,翻译的蛋白质均含有220个氨基酸残基,且保守度达97%。DGCR6L在乳腺癌中高表达,并于肿瘤转移相关,但具体机制不详。本研究中,我们将对PAK4与DGCR6L的相互作用进行验证,并探索DGCR6L在PAK4引起的胃癌细胞迁移中的作用和机制。
PKN1为RhoA和Rac1的下游靶蛋白,调节细胞骨架重组及细胞迁移。蛋白激酶C相关激酶1(PKC-related kinasel,PRK1)也称为PKN或PKN1。是脂质和RhoGTPase激活的丝氨酸/苏氨酸激酶,为PKC超家族一员,分子量为116KD。目前发现,该家族有三个成员,分别为PKN1、PKN2及PKN3,三者结构相似,其中PKN1与PKN2的同源性为58%。PKN1的N末端(aal～560)包含三个重复亮氨酸拉链样结构域,与蛋白质相互作用有关。C末端(aa561～942)有一个与蛋白激酶C(PKC)高度同源的催化结构域,与激酶活化有关。
PKN1的N末端含有两个同源重复序列,HR1和HR2域。HR1域内含有三个重复亮氨酸拉链样结构,呈反向平行卷曲排列(anti-parallel coiled-coil),也称ACC重复序列,分别为HR1a(ACC1),HR1b(ACC2)及HR1C(ACC3)。研究表明,RhoA能与PKN1的HR1a和HR1b结合,而不与HR1C结合。结合了GTP的RhoA即活化的RhoA只与HR1a(ACC1)结合,而结合了GTP的Rac1只与PKN1的HR1b区域(ACC2)结合。
一些实验证据表明,PRK1/PRK2作为RhoA和Rac1的下游靶蛋白或效应器,通过与RhoA和Rac1的相互作用在细胞骨架重组及细胞迁移中发挥作用。例如,RhoA和Rac1与PKN2结合并激活PKN2.在NIH3T3成纤维细胞中导入无激酶活性的PKN2突变体,会引起肌动蛋白应力纤维降解,说明PKN2激酶活性可以调节细胞骨架重组。PI3K下游靶蛋白PDK1能直接激活PKN1,介导胰岛素所致的肌动蛋白细胞骨架重组,应力纤维解聚,片状伪足形成。
实验材料及方法
酵母配合实验(yeast mating assay)检测DGCR6L与PAK4的相互作用。DGCR6L全长cDNA克隆到pGADT7载体后转染至Y187酵母细胞,与转染pGBKT7-PAK4N/C的AH109酵母细胞进行配合实验,配合子接种于SD/-Leu/-Trp/-Ade/-His/x-α-gal培养板培养,生长并变蓝为阳性克隆。
蛋白质体外结合实验(GST pull-down assay)证实PAK4与DGCR6L或PKN1的相互作用,并寻找相互作用区域。pCDNA3.1C-PAKs进行体外转录翻译同时掺入~(35)S-methonine,得到~(35)S-PAKs;与体外纯化的GST-DGCR6/L或GST-PKN1s融合蛋白进行GST pull-down实验;通过构建相应蛋白的截短蛋白,利用GST-pulldown实验检测蛋白相互作用的区域。
免疫共沉淀(Immunoprecipitation)/免疫印迹(western blot)验证蛋白在哺乳动物细胞内的相互作用。免疫荧光检测蛋白在细胞内的定位或者是相互作用蛋白在细胞内的共定位。我们利用融合的荧光蛋白或者免疫荧光检测蛋白在细胞内的定位。GFP或DsRed融合的蛋白通过脂质体转染到细胞,然后利用激光共焦扫描显微镜检测蛋白在细胞内的定位。细胞内源性蛋白的定位通过免疫荧光的方式检测。细胞内的微丝和微管分别利用特异性的结合蛋白进行标记。利用激光共焦扫描显微镜检测蛋白在细胞内的定位。
DGCR6/L稳定表达细胞系建立用于细胞迁移实验。将pCDNA3.1C-DGCR6/L转染AGS细胞,利用G418进行筛选,得到DGCR6/L稳定表的的AGS~(DGCR6/L)细胞。
Transwell小室细胞迁移实验验证胃癌细胞的迁移能力改变。在PAK4和DGCR6L蛋白过表达或沉默表达条件下,检测细胞迁移能力变化。3次以上的实验结果进行统计学分析。
激酶反应检测PAK4对DGCR6L或PKN1的磷酸化。利用体外激酶反应,检测PAK4对DGCR6L、β-actin及PKN1的磷酸化;利用体外激酶反应检测PKN1对PAK4的磷酸化。
结果
1.利用酵母配合实验,DGCR6L与PAK4C发生相互作用;体外结合实验证实,PAK4特异性的与DGCR6L结合。
2.PAK4通过其466-572aa区域与DGCR6L结合,并且DGCR6L的基酸残基6G,14G,115L在结合中起重要作用。
3.PAK4与DGCR6L在胃癌细胞SGC-7901中相互作用并有共定位,但不直接结合。
4.PAK4在胃癌细胞SGC7901中与β-actin形成复合体,但经检测没有直接结合且PAK4不磷酸化β-actin;DGCR6L对于PAK4与β-actin形成复合体是必须的。
5.PAK4、DGCR6L和肌动蛋白微丝在SGC-7901细胞中两两共定位;DGCR6L可以剂量依赖性的促进细胞内LIMK1及cofilin的磷酸化水平。
6.稳定表达的DGCR6L促进胃癌细胞的迁移;沉默DGCR6L表达后细胞迁移能力下降,在此基础上沉默PAK4表达细胞的迁移能力进一步下降。
7.PAK4通过其激酶域与PKN1的HR2结构域结合。
8.PKN1磷酸化PAK4,磷酸化位点位于PAK4的1-118aa。
9.PKN1和PAK4在胃癌细胞SGC-7901内有共定位,且二者分别可以定位于高尔基复合体。
10.PKN1的表达使PAK4定位于细胞的肌动蛋白微丝,且三者有共定位。
11.PKN1和PAK4与细胞的微管有共定位。
结论
1.DGCR6L和PKN1分别是PAK4新的相互作用蛋白。
2.DGCR6L通过促进LIMK1磷酸化水平调节细胞肌动蛋白骨架促进肿瘤细胞迁移。
3.PKN1磷酸化PAK4的N-末端,且位点位于1-201aa区域。
4.PKN1结合并磷酸化PAK4可促进PAK4和微丝细胞骨架的结合。
5.PKN1和PAK4与细胞微管共定位。
Backgroud
p21 activated kinases(PAKs),serine/threonine kinases including two groups of members(groupⅠincludes PAK1-3 and groupⅡincludes PAK4-6),was recently found to be key regulators of cancer cell signaling networks.
PAK4 was found to be expressed in most tissues examined,with levels highest in the prostate,testis,and colon.More important,PAK4 was found to be overexpressed in cell lines that were derived from many cancer types and is localized to a region of chromosome 19 that is also commol/Lonly amplified in several human colon,ovarian and pancreatic tumors.Two PAK4 genetic mutations were also found among 340 serine/threonine kinases in human colorectal cancers.In addition,an activated allele of PAK4 leads to anchorage-independent cancer-cell growth,whereas a dominant-negative PAK4 mutant(PAK4NE) effectively inhibits Ras-induced transformation.Since PAK4 is newly identified,there are far less reported substrates than PAK1,but PAK4 is the most extensively studied member of the GroupⅡPAKs. PAK4 mediates morphological changes through its association with the Rho-family guanine nucleotide exchange factor(GEF),GEF-H1.PAK4 can negatively regulate the activation of Rho through a direct protein-protein interaction with G protein-linked Rho GEFs,dramatically decrease Rho-GTP loading in vivo and the formation of actin stress fibers in response to serum or LPA stimulation.PAK4 interacts with integrinαvβ5 and selectively promotes integrinαvβ5-mediated cell migration.LIMK1 was identified as a substrate for PAK1 and recently PAK4.
LIMK1 phosphorylates cofilin,and PAK4-induced cell rounding is dependent on cofilin phosphorylation.This suggests that regulation of LIMK1 activity is required for cell migration,indicating that there is a functional linkage between PAK4,LIMK and cofilin.However,there are still uncovered mechanisms for cancer cell migration induced by PAK4.
The DiGeorge critical region 6(DGCR6) gene exists in two highly homologous copies(DGCR6 and DGCR6L) on chromosome 22q11 and is first found deleted in patients with velo-cardio-facial syndrome/DiGeorge syndrome(VCFS/DGS).Both genes share highly conserved intron/exon structures and the deduced proteins,each 220 amino acids in length,are 97%identical at the amino-acid level.DGCR6L was highly expressed in mammary carcinomas and relates to the metastasis,its biochemical activity remained unclear.
In this study,we found that the C-terminal kinase domain of PAK4 binds specifically with DGCR6L,which promotes the migration of gastric cancer cells.These findings suggest the existence of a novel biochemical route by which the binding of PAK4 with DGCR6L promotes migration of gastric cancer cells by increasing phosphorylation level of LIMK1.
Methods
Yeast mating assay for identifying interaction between PAK4 with DGCR6L.
DGCR6L full length cDNA was subcloned into pGADT7 vector and using yeast strain Y187 as a host.PAK4N/C was transformed into yeast strain AH109.Then yeast mating performed.
GST pull-down assay for binding of PAK4 with DGCR6L or PKN1.In vitro transcription and translation of PAK proteins were performed by using the TNT-coupled transcription and translation system.Using a T7-TNT kit,we translated 1μg of pCDNA3.1 vector in the presents of ~(35)S-methonine.The GST pull-down assay were performed by incubating equal amounts of GST,GST-fusion proteins immol/Lobilized by GST Sepharose Beads with in vitro translated ~(35)S-labeled PAK proteins.Bound proteins were isolated by SDS-PAGE.The bound proteins were then visualized by Fluorography.GST-fused proteins were used to map the regions of two binding proteins.
Immol/Lunoprecipitation and western hot were used to identify the interaction of two proteins in mammol/Lalian cells.We determined the cellular localization of proteins by indirect immol/Lunofluorescence.Cells grown on glass coverslips were transfected with GFP-fusion or DsRed-fusion proteins if necessary, then immol/Lnofluorescenced with specific antibodies.Confocal scanning analysis was performed by using a Zeiss Laser Scanning Confocal Microscope in accordance with established methods.
Establishment of DGCR6/L stable expression AGS cell lines. pCDNA3.1C-DGCR6/L,which containing selectable marker G418 to facilitate selection of stable transfected cells,were transfected into AGS cells which has a lower DGCR6L protein level.G418 was added and protein expression was detected by western blot after clones were picked.Transwell migration assay was used to assess cell migration in vitro.
Kinase assay was used to detect the kinase activity of PKN1 and PAK4.The phosphorylations of PAK4 on DGCR6L,β-actin or PKN1 were detected by in vitro kinase assay;the phosphorylation of PKN1 on PAK4 was detected by in vitro kinase assay.
Results
1.DGCR6L interacted with PAK4C specifically by yeast mating assay and GST-pull down assay.
2.PAK4 interacted with DGCR6L by its 466-572aa region,and the 6G,14G and 115L were critical amino acids for the binding.
3.PAK4 interacted and colocalized with DGCR6L in SGC-7901 cells,but direct binding was not found,PAK4 did not phosphorylate DGCR6L.
4.PAK4 formed complex,but did not bind,withβ-actin in SGC-7901 cells,and DGCR6L was necessary for this complex.PAK4 didn't phosphorylateβ-actin.
5.PAK4,DGCR6L and F-actin colocalized each two in SGC-7901 cells,and DGCR6L promote the phosphorylation level of LIMK1 and cofilin in a dose dependent manner.
6.Stable expressed DGCR6L increased the migration of gastric cancer cells,when DGCR6L was silenced,the potential of migration decreased.
7.PAK4 bind to the HR2 domain of PKN1 through its kinase domain.
8.PKN1 phosphorylated PAK4,phosphorylation sites was within the region of 1-201aa of PAK4.
9.PKN1colocalized with PAK4 in SGC-7901cells and both them colocalized at Golgi apparas.
10.The expression of PKN1 induced the localization of PAK4 on F-actin.
11.PKN1,PAK4 colocalized with MT in SGC-7901.
Conclusions
1.DGCR6L and PKN1 are both novel binding protein of PAK4.
2.DGCR6L promote PAK4-induced reorgnazition and migration of gastric cancer cells via LIMK1.
3.PKN1 phosphorylates PAK4.
4.Expression of PKN1 mediates colocalization of both PKN1 and PAK4 with MF.
5.PKN1,PAK4 colocalized with MT in gastric cancer cells.
引文
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