肿瘤转移是恶性肿瘤生物学特征之一,也是临床治疗的难题,其中细胞迁移是肿瘤浸润和转移的必备条件.目前,对于调节细胞迁移和侵袭的相关机制仍知之甚少.细胞内细胞骨架蛋白的动态组装、细胞和细胞外基质之间的黏着变化、周围基质的重塑等以及这些反应在迁移侵袭过程中的协调都涉及复杂的信号调节.JWA(AF070523)基因是本课题组周建伟等发现并克隆的新基因,其编码蛋白是一种新的微管结合蛋白.那么,JWA基因是否涉及肿瘤细胞迁移和侵袭的相关分子机制呢?为此,本研究将致力于探讨JWA基因在人类肿瘤细胞迁移中的作用.   
目的   
    探讨JWA基因在肿瘤细胞迁移过程中的作用以及相关的分子机制,为进一步深入了解肿瘤的发生、发展和预后,为肿瘤的预防、诊断和治疗提供新的科学依据.    方法    HeLa(人宫颈癌细胞)、B16(小鼠黑色素瘤细胞)以及HCCLM3(高转移潜能的人肝癌细胞)都是体外研究肿瘤细胞迁移和侵袭的良好实验模型.本研究利用这些细胞在特定的促癌或抑癌剂诱导下细胞骨架肌动蛋白丝重组和迁移能力改变的特性,建立系列体外迁移模型.
细胞迁移能力从以下几方面进行评价：
(1)通过观察细胞肌动蛋白丝的重组来评价肿瘤细胞迁移能力的改变；
(2)通过划痕试验,检测细胞的不定向迁移来评价肿瘤细胞迁移能力；
(3)采用穿孔试验,检测细胞的定向迁移来评价肿瘤细胞迁移能力.使用凝胶酶谱和Western blot方法检测JWA及FAK、COX-2等细胞迁移相关基因的表达.并且利用JWA反义寡核苷酸技术下调JWA的表达,以进一步探讨JWA基因在促癌或抑癌剂诱导细胞迁移中的机制.使用免疫荧光技术,探讨JWA在促癌或抑癌剂诱导下调控肌动蛋白丝重组的功能.  
结果   
    (1) JWA是新的微管相关蛋白,为了解JWA是否类似于传统的MAP,具有肌动蛋白丝结合蛋白的功能,用JWA反义寡核苷酸处理细胞,经G418筛选后,得到稳定的JWA表达缺陷水平的HeLa细胞株(As-HeLa细胞),再观察对细胞肌动蛋白丝重组的影响.结果显示在稳定JWA表达缺陷的HeLa细胞株中,肌动蛋白丝开始重组,细胞的边缘随之伸出一些突触,提示JWA可能是微管和肌动蛋白丝结合蛋白,调节细胞骨架组装.   
    (2)在此基础上,进一步利用促癌剂佛波酯(phorbol ester,PMA)和抑癌剂三氧化二砷(arsenic trioxide,As_2O_3)分别诱导HeLa细胞株肌动蛋白丝重组的细胞模型：8 nM PMA分别处理细胞6,12和24h,免疫荧光显示,在稳定转染JWA空载体(HeLa-vector细胞)的细胞株中,PMA处理6h后,细胞边缘仍未发现突触的形成,肌动蛋白丝并未出现解聚,提示空载体细胞的肌动蛋白丝未发生明显的解聚聚合.而PMA处理12h后,观察到细胞学形态发生改变,肌动蛋白丝开始重组.同样的处理条件下,在稳定转染JWA表达缺陷载体的细胞株中,PMA处理6h后,细胞边缘伸出一定量的突触,提示肌动蛋白丝已经开始重组.PMA处理12h后,肌动蛋白丝发生更加明显的解聚聚合.与转染空载体的细胞相比,JWA表达缺陷的细胞肌动蛋白丝的重组明显加速.10μM As_2O_3处理细胞10,30和60min,免疫荧光同样观察到,JWA表达缺陷的细胞比空载体细胞提前出现肌动蛋白丝的解聚聚合,进一步提示JWA参与细胞肌动蛋白丝的重组过程,并且在促癌或抑癌剂诱导的细胞肌动蛋白丝的解聚聚合中起作用.  
    (3)为了解JWA是否参与肿瘤细胞的迁移,HeLa细胞转染pEGFP-C1,pEGFP-C1-JWA或pEGFP-C1-AsJWA质粒,G418稳定筛选后,得到空载体,JWA高表达(HeLa-JWA细胞)和反义JWA稳定转染的细胞株,免疫印迹鉴定,建立划痕和细胞穿孔试验模型,观察JWA对细胞迁移能力的影响.在划痕模型中结果显示,JWA表达缺陷可促进细胞的伤口愈合,而JWA高表达则抑制伤口的愈合,表明JWA在细胞的非定向迁移中起作用.同样,细胞穿孔实验结果显示,JWA表达缺陷可促进细胞的迁移,而高表达的细胞则起抑制迁移的作用,表明JWA在细胞的定向迁移中起作用.我们用细胞穿孔模型检测了在HCCLM3和B16细胞株中JWA的作用,结果观察到类似的现象,进一步提示JWA在肿瘤细胞的迁移中起重要的作用,JWA可抑制细胞的迁移.   


     (4)为建立As_2O_3诱导细胞迁移的模型,首先用细胞增殖分析试剂盒分析As_2O_3的细胞毒性.结果显示,不同浓度的As_2O_3处理空载体和反义JWA稳定转染的HeLa细胞48h后,有血清处理的细胞均出现明显的细胞毒性,2μM As_2O_3处理的HeLa细胞大约50％出现生长抑制现象；但在无血清的情况下,As_2O_3浓度低于2μM诱导并未观察到抑制增殖和促进凋亡现象.提示在有血清的情况下,As_2O_3细胞毒性更大,所以选择无血清状态下As_2O_3(1μM)作为模型药物用于研究细胞迁移.   
    (5)不同浓度As_2O_3对细胞迁移作用有着不同的影响：用0.5,1和2μM As_2O_3处理空载体细胞0,1和2d后,划痕结果显示As_2O_3有显著抑制HeLa细胞伤口愈合的作用,且呈剂量依赖性关系.进一步研究发现,PMA(80 nM)能够显著增强As_2O_3抑制细胞迁移的作用,而对As_2O_3诱导的细胞凋亡和生长抑制没有显著影响.PMA(80 nM)与不同浓度的As_2O_3(0,0.5,1和2μM)序贯处理空载体细胞24h后,划痕试验结果显示As_2O_3呈剂量依赖性的抑制PMA诱导的HeLa细胞的伤口愈合.在相同的处理条件下,细胞穿孔试验也显示同样的结果.   
    (6)为探讨JWA参与细胞迁移的调控机制,考虑到药物诱导迁移的有效性及对细胞的毒性作用,我们选用PMA(80 nM)与1μM As_2O_3序贯处理空载体和反义JWA稳定转染的HeLa细胞.结果表明PMA能够显著增强HeLa细胞的迁移能力,而As_2O_3则明显抑制PMA诱导的HeLa细胞迁移,JWA基因表达下调后,能显著减弱As_2O_3对HeLa细胞迁移的抑制作用,同时促进PMA诱导的细胞迁移,这些结果进一步证明JWA可能作为信号分子参与细胞迁移的信号通路调控.   
    (7)在此基础上,为了解JWA是否为模型药物诱导的细胞迁移信号通路所必须,在上述药物诱导迁移的细胞模型中,用western blot检测丝裂原活化蛋白激酶信号通路(mitogen-activated protein kinase,MAPK)信号通路级联中各级信号分子,即Raf-MEK-ERK和其磷酸化的表达,结果显示在As_2O_3和PMA影响HeLa细胞迁移模型中,Raf-MEK-ERK信号途径被激活(即Raf、MEK、ERK磷酸化),JWA基因表达下调后,MEK和ERK不能被磷酸化,但Raf的磷酸化未受到影响,以上结果强烈提示JWA确有可能通过影响ERK信号通路而调控细胞迁移；其影响环节在于对MEK的调节；PKC可能作为JWA的上游调控因子之一,磷酸化JWA,影响ERK信号通路,进而调控细胞迁移.   
    (8)为阐明JWA在模型药物激活的MEK-ERK信号通路中的作用,我们设计了JWA回复模型.JWA表达缺陷的HeLa细胞瞬转pEGFP-C1-JWA的质粒后得到JWA回复的HeLa细胞(As-HeLa-rescue细胞).回复模型中,PMA(80 nM)加或不加As_2O_3(1μM)处理空载体和JWA回复的细胞24h后,用western blot检测Raf-MEK-ERK的磷酸化.结果显示,JWA回复细胞中,As_2O_3和PMA诱导的MEK-ERK信号途径被激活,MEK和ERK的磷酸化被回复,但Raf的磷酸化回复并未受到影响,进一步证明JWA通过MEK-ERK级联影响细胞迁移.   
    (9) PMA和As_2O_3独立作用时均能激活MEK／ERK信号通路,但两者对HeLa细胞迁移的影响完全相反(PMA促进迁移,As_2O_3抑制迁移).为了解JWA是否参与激活了ERK不同的磷酸化位点,进而激活其下游不同的底物,我们用western bolt检测了迁移模型中As_2O_3和PMA诱导的ERK下游不同的底物黏着斑激酶(focal adhesion kinase,FAK)和环氧合酶2(cyclooxygenase-2,COX-2)的表达.结果显示As_2O_3可下调FAK的表达水平而PMA可上调COX-2的表达水平.进一步检测JWA在PMA调节COX-2的表达和As_2O_3调节FAK表达中的作用,结果显示,在As_2O_3和PMA影响HeLa细胞迁移模型中,用PMA(80 nM)处理JWA表达缺陷细胞株后,其相应的ERK下游底物COX-2的表达水平显著上调.而As_2O_3(1μM)处理则引起其相应的ERK下游底物FAK表达的上调.这些结果证明JWA与PMA／As_2O_3激活的MEK-ERK通路,分别调节其相应的ERK下游底物COX-2／FAK的表达,最终引起迁移的不同效应.   
    (10)用JWA回复模型进一步验证JWA在PMA调节COX-2的表达和As_2O_3调节FAK表达中的作用.用PMA(80 nM)加或不加As_2O_3(1μM)处理空载体和JWA回复的细胞24h后,用western blot检测COX-2和FAK的表达.结果显示,随着JWA表达水平的回复,COX-2和FAK的表达均被回复.以上结果提示JWA在PMA和As_2O_3诱导的MEK-ERK-下游底物通路中起重要作用.   


     (11)为探讨JWA是否在MAPK级联中起类似激酶分子的作用,我们设计了系列JWA磷酸化位点突变体.利用基因突变技术,构建了JWA编码区PKC磷酸化位点1、2以及两位点同时突变的三种pEGFP-N1载体,稳定转染,获得相应的三种HeLa细胞株,分别命名为As-HeLa-P1,As-HeLa-P2和As-HeLa-P12细胞株.PMA(80 nM)加或不加As_2O_3(1μM)序贯处理JWA空载体和磷酸化位点突变细胞24h后,用western blot检测MEK,ERK和其相应的下游分子FAK和COX-2的磷酸化水平.结果显示在As-HeLa-P1 HeLa细胞中,As_2O_3和PMA仍可诱导MEK和ERK的磷酸化.PMA诱导的COX-2和As_2O_3诱导的FAK表达被回复.但在As-HeLa-P2 HeLa细胞中,MEK和ERK的磷酸化水平下降.有趣的是,在As-HeLa-P12细胞中,JWA磷酸化位点突变后,As_2O_3或PMA刺激均不能引起MEK和ERK的磷酸化.甚至,PMA诱导的COX-2和As_2O_3诱导的FAK表达水平均不能回复.但JWA磷酸化位点突变并不影响Raf的磷酸化.总之,这些结果说明JWA在As_2O_3和PMA引起的MEK-ERK通路中起重要作用,提示JWA在MAPK通路中可能是介于Raf和MEK的磷酸化底物.   
    (12)为进一步证实JWA磷酸化在迁移中所起的作用,用PMA(80 nM)力口或不加As_2O_3(1μM)分别处理As-HeLa-P12和As-HeLa-rescue细胞后,细胞穿孔试验检测其迁移能力的改变.结果显示,在As-HeLa-rescue细胞中,As_2O_3和PMA诱导的迁移被回复,As-HeLa-P12细胞中结果则相反.因此,JWA蛋白磷酸化位点突变对调节细胞迁移起着重要的作用,但确切证据有待进一步研究提供.   
讨论
        JWA参与肿瘤细胞迁移的调节,是促癌或抑癌剂诱导细胞迁移信号转导通路中共同的信号分子,在MAPK信号通路以及肌动蛋白丝的重组过程中起重要的作用.JWA可能是细胞内控制肿瘤细胞转移新的分子标志物.JWA可以抑制HeLa,HCCLM3和B16等肿瘤细胞的迁移,可能在信号通路中同时调控微管和肌动蛋白丝两大细胞骨架系统,引起迁移.在HeLa细胞中,JWA通过调节细胞骨架肌动蛋白丝的重组抑制细胞的迁移；在抑癌剂As_2O_3和促癌剂PMA诱导的肌动蛋白丝的变化中,JWA均起重要的调节作用.JWA还参与MEK-ERK-下游底物的信号通路,可能通过激活ERK的不同磷酸化位点,使促癌或抑癌剂诱导的细胞迁移产生不同的终效应.JWA磷酸化在MAPK信号通路介导的细胞迁移中起重要作用,JWA表达与MEK和ERK的磷酸化有关,但不影响Raf,功能上类似于一个MAPKKK分子.对JWA基因功能深入、全面的探讨有助于我们进一步了解肿瘤的发生、发展和预后,为肿瘤的预防、诊断和治疗奠定理论基础.
    Cell migration is a process that is critical at many stages duringembryonic development, and essential for wound healing and tumor cellinvasion. Cell migration is regulated by coordinated changes in the actincytoskeleton and cell adhesion. Nevertheless, the molecular mechanismsparticipating in the regulation of cell migration remain largely unknown.JWA (AF070523) is a newly identified novel microtubule-associatedprotein (MAP) was essential for the rearrangement of F-actincytoskeleton and activation of MAPK cascades. To elucidate how cellmigration is regulated, we investigated the role of JWA in the migrationand invasion of human tumor cells.    
    Objectives To determine the influence of JWA expression on cellmotility and whether this affect was dependent upon the ability toremodel actin cytoskeleton and activate the MEK-ERK signalingpathway.      Methods Henrietta Lacks strain of cancer cells HeLa (human carcinomacervicis cell), B16 (melanoma cell) and HCCLM3 (human hepatoma carcinoma cell) were used for the models of migration and invasion invitro. The intracellular localization of JWA and F-actin was measured byimmunofluorescence microscopy. Tumor cell motility and invasion werenot only evaluated by means of wound-healing migration assay, butassayed by transwell. The involvement of expressions and theirphosphorylations of Raf, MEK and ERK were investigated duringPMA/As_2O_3-induced cell migration. Western blot was used to detect theprotein expression of JWA, FAK and COX-2. Antisense oligonucleotidewas applied to inhibit the expression of JWA and further explore thefunction of it. In order to determine the phosphorylations of MEK/ERKinduced by treatment of As_2O_3 and PMA are dependent on the status ofJWA phosphorylation at SDR or SLR sites, JWA protein PKCphosphorylation site mutation cell strains (ADR-SLR; SDR-ALR;ADR-ALR) were then constructed.   
Results   
     (1) Our previous studies showed that JWA is a novel microtubulebinding protein and affects microtubule assembly. This implies that JWAmay affect cytoskeleton rearrangement. Since the microtubule associatedproteins (MAPs) were shown to substitute for actin binding proteins inperipheral actin assemblies and induce acting gelatin in vitro, we firsttested hypothesis that JWA may exert an effect on actin assembly. Cellswere transfected with either pEGFP-C1 vector, pEGFP-C1-JWA (sense),or pEGFP-C1-AsJWA (anti-sense). After selection with G418, theresulting HeLa cells were verified and found to possess stable knockdownof JWA expression (knock-down HeLa cells, As-HeLa), stableover-expression of JWA (over-expression HeLa cells, HeLa-JWA) andvector control HeLa (HeLa-vector), respectively. We found that actinfilaments rearrangement was occurred followed by knockdown of JWA alone, the more protrusions were found at the leading edge of theAs-HeLa cells than vector control cells.   


     (2) Both PMA and As_2O_3 were known to as effective model chemicalsto enhance or inhibit cell motility via the activation of MAPK signalcascades and the rearrangement of F-actin cytoskeleton, respectively. Inthis study, cell culture models were established to observe whether JWAaffects PMA or As_2O_3 induced cellular F-actin rearrangement. The resultsshowed that vector only transfected HeLa cells stretched out evenly, andno protrusions were found at the leading edge of the cells after incubationwith PMA for 6 h. The actin filaments were well organized, indicatingthat the HeLa vector cells do not undergo a significant rearrangement ofactin filaments. Upon stimulation with PMA for 12 h, however, thecellular morphology was changed, and the actin filaments wereremodeled. In As-HeLa cells, there were some actin protrusion structuresat the leading edge of the cells upon PMA stimulation within 6 h,indicating that the actin filaments were reorganized. After the stimulationwith PMA for 12 h, the actin filaments underwent a more profoundreorganization. Compared to the vector controls, actin reorganization wasobviously accelerated in these JWA deficient As-HeLa cells. In this study,As_2O_3-induced rearrangements of the actin cytoskeleton were similar tothose induced by PMA. The data above suggest that knock down JWAalone contributes to induce rearrangement of actin filaments, and alsoenhances PMA and As_2O_3 induced actin rearrangement in HeLa cells.   
     (3) Rearrangement of actin filament was usually linked with changedcell motility. To characterize the potential effect of JWA on cell motility,we first examined whether JWA affects the migration of HeLa cells. Theselected cell populations were employed to test for their migrationabilities. Data shown that knockdown of JWA alone significantlypromoted cellular migration when compared with cells carrying control vector. In contrast, over-expression of JWA inhibited cell migration. Thereduced directional persistence suggested that JWA rendered cells unableto maintain a stable direction during targeted migration. Therefore, theseHeLa derivatives were assessed for wound healing migration. Aspredicted, over-expression of JWA produced cells with a marked delay inwound closure, whereas, JWA deficient cells showed accelerated woundclosure. The result from transwell assay was also supported that JWAover expressed HeLa cells reduced the ability to path through the filter,whereas JWA knock-down HeLa cells significantly enhanced themobility. To confirm the biological function of JWA on cell motility, wefurther designed transwell assays in another two kinds cancer cell linesHCCLM3 and B16 cells. As a result, similar phenomena were found,suggesting that the role of JWA on cell migration might exist in multiplecancer cell lines.
    (4) Since As_2O_3 is also a well-established as anti-proliferative orpro-apoptotic metal, investigations were thus undertaken to determine ifthe anti cell-migration effects of As_2O_3 were due to its anti-proliferationroles. The effect of As_2O_3 on cell cytotoxicity was determined. Theresults showed that significant cytotoxic effects were observed in theserum-supplemented media following the As_2O_3 treatment for 48 h.Exposure of HeLa cells to 2μM As_2O_3 induced approximately 50%growth inhibition. However, in serum-free culture condition, As_2O_3 didnot inhibit cellular proliferation or induce cell apoptosis whenconcentrations under 2μM. However, As_2O_3 was found to exertpronounced cytotoxic effects on HeLa cells in the presence of serum; Thedata in this study was also showed that knock-down JWA was not affectcell viability in HeLa cells after exposed to As_2O_3 within indicatedconcentrations for 48 h when compared to the vector control. To excludethe role of relative inhibition of cell migration in cytotoxicity, the influence of lower concentration As_2O_3 (≤1μM) with serum-free cultureconditions were examined in this study.   
    (5) As_2O_3 treatment alone showed a significant inhibitory effect onHeLa cell migration and in a concentration-dependent manner. In order toevaluate the effects of As_2O_3 in response to PMA accelerated cellmigration behaviors, confluent monolayer of HeLa cells were scrapedwith a pipette tip to remove a section of the monolayer, and the cells werecultured for 24 h with PMA (80 nM) in the presence or absence of As_2O_3.The cell migration rates were assessed by counting the number of cellsthat migrated into the pre-defined denuded area of the cell monolayer.The results showed that As_2O_3 concentration-dependently inhibitedPMA-stimulated HeLa cells migration. Transwell assays also showedsimilar results, in which the ability of the cells to pass through the filterswas measured.   
     (6) The role of JWA on chemicals induced migration of HeLa cellswere also determined by using transwell assays. Data showed thattreatment of the cells with As_2O_3 markedly inhibited PMA-stimulated cellmigration and in a concentration-dependent manner. However, thisinhibitory effect of As_2O_3 was significantly blocked in JWA deficientHeLa cells. In contrast, JWA deficiency further enhancedPMA-stimulated cell migration. Suggesting that intracellular expressionof JWA is important for the effects of As_2O_3 on PMA induced cellmigration.   


    (7) It is well known that both As_2O_3 and PMA treatment inducedactivation of MAPK cascade. JWA was also proposed as a putativeMAPK activated protein. Therefore, it was hypothesized that JWA mightaffect cell migration via MAPK cascade. To test this hypothesis, weconducted western blot analysis for MEK-ERK expressions and theirphosphorylation status in cell migration models. The effects of JWA transfection on RAF-MEK-ERK phosphorylation in HeLa cells were firstinvestigated. Data showed that, without PMA or As_2O_3 treatment, thecells with vector control, knockdown JWA expression or followed rescuetransfection were no significant phosphorylation can be detected for thesesignal molecules of MAPK cascades. The phosphorylations ofRaf-MEK-ERK occurred in vector only transfected cells after treatmentwith either PMA or As_2O_3 or the both, however, the phosphorylations ofMEK-ERK but not Raf were completely blocked in JWA deficient HeLacells. These data provide evidence that knockdown of JWA enhanced cellmigration might be mediated by MEK-ERK associated signal pathway.   
   (8) To further confirm whether JWA was essential for thephosphorylation of MEK-ERK induced by As_2O_3/PMA, we designedJWA rescue cell culture models. JWA-deficient HeLa cells weretransiently  transfected with pEGFP-C1-JWA to produce JWA-rescuedHeLa cells which recovered JWA expression. In the serum-free cellmigration models, JWA-rescued HeLa cells and vector-control HeLacells were treated with PMA (80 nM) for 12 h in the presence or absenceof As_2O_3 (1μM) for a further 12 h, and subsequently the phosphorylationstatus of Raf-MEK-ERK was determined. We found that As_2O_3 or PMAtreatment triggered MEK and ERK phosphorylations were completelyrecovered in the cells presence of rescued JWA expression. Interestingly,JWA rescue did not influence the activation of Raf.   
   (9) Both As_2O_3 and PMA induced similar activation patterns of theMEK-ERK cascade and played an essential role in cell migration. Basedon these experimental facts, the questions arose as to how similarMEK-ERK phosphorylation patterns mediate reversal effects on cellmigration? A reasonable explanation might be that As_2O_3 and PMAactivating different downstream substrates of ERK, separately, whichmay involve different phosphorylation sites of these MAPK cascades molecules. To test our hypothesis, we selected COX-2 and FAK as thedifferential downstream target responsive to PMA and As_2O_3,respectively, and determined their expressions in As_2O_3 or PMA-inducedHeLa cell migration model. Data showed that, after PMA (80 nM)stimulation, the expression of COX-2 was markedly up-regulated in JWAknockdown HeLa cells accompanied by no changes in vector-controlcells and no influence on FAK. In contrast, As_2O_3 (1μM) treatment onlyled to up-regulation of FAK not COX-2 expressions. These data suggestthat JWA plays a key role in PMA/As_2O_3 via MEK-ERK-dependantpathway which regulates COX-2/FAK separately and linked to thecontrasting effects on cell migration.      
    (10) To further elucidate the role of JWA in these consequent events,we employed JWA rescue cell culture models. Treatment ofJWA-rescued HeLa cells and HeLa-vector cells with PMA (80 nM) for12 h was conducted in the presence or absence of As_2O_3 (1μM) forfurther 12 h; subsequently, the expressions of COX-2 and FAK weredetermined. We found that recovered JWA expression in JWA-deficientHeLa cells significantly reversed expression patterns of COX-2 by PMAand FAK by As_2O_3 treatments. These results supported the postulationthat JWA plays a critical role in activation of the MEK-ERK downstreamas a consequence of As_2O_3 and PMA stimulation.   


    (11) In order to determine whether JWA serves as a kinase-likemolecule in MAPK cascade, we constructed series JWA expressionvectors with PKA/PKC phosphorylation sites mutants which were thentransiently transfected into JWA knockdown HeLa cell lines, respectively.Therefore, three JWA protein PKC phosphorylation site mutation cellstrains (ADR-SLR; SDR-ALR; ADR-ALR) were then constructed,expressed as As-HeLa-P1, As-HeLa-P2 and As-HeLa-P12, respectively.These conditional cell lines were used for determine if the phosphorylations of MEK/ERK induced by treatment of As_2O_3 and PMAare dependent on the status of JWA phosphorylation at SDR or SLR sites.JWA vector control and phosphorylation site-mutated HeLa cells weretreated with PMA (80 nM) for 12 h in the presence or absence of As_2O_3(1μM) for further 12 h, the phosphorylations of MEK or ERK and itsdownstream effectors FAK and COX-2 were determined. Data showedthat, in As-HeLa-P1 HeLa cells, As_2O_3 and PMA treatments were stillable to induce activation of MEK and ERK. Further, the expression ofCOX-2 by PMA and FAK by As_2O_3 was almost rescued. However,As-HeLa-P2 cells showed a reduced level of activation of MEK and ERK.It is of interest that, in As-HeLa-P12 cells, As_2O_3 or PMA treatment wascompletely unable to induce activation of MEK and ERK. Moreover,PMA-induced up-regulation of COX-2 and As_2O_3-induced down-regulation of FAK was also not reversible in As-HeLa-P12 cells.However, JWA phosphorylation site mutation did not influence theactivation of Raf. Taken together, these studies indicate that thephosphorylation motif of JWA is critical for activation of the MEK-ERKpathway induced by either As_2O_3 or PMA.    
    (12) To further investigate the role of JWA phosphorylation on cellmigration, As-HeLa cells were transfected with a series pEGFP-N1-JWAPKC phosphorylation sites mutation vectors, or pEGFP-C1-JWA,separately. After selection with G418, pools of cells that phosphorylationsite mutation JWA (As-HeLa-P12) and JWA rescue proteins (As-HeLa-rescue) were generated. Transwell assays showed that in As-HeLa-rescued cells, the facilitative effect of JWA deficiency on cell migrationby PMA and As_2O_3 was significantly rescued. Interestingly, the cells withJWA mutants (As-HeLa-P12) inversely stimulated the migration either byAs_2O_3 or PMA. Therefore, the existence of SDR-SLR motifs in JWAprotein is critical for regulating cell migration. Conclusion JWA, a newly identified novel microtubule-associatedprotein (MAP) was essential for the rearrangement of F-actincytoskeleton and activation of MAPK cascades induced by arsenictrioxide (As_2O_3) and phorbol ester (PMA).    
    All the data suggest that JWA inhibits random and directed tumor cellmigration. JWA may initiate the regulatory pathways, such as regulationof MT and actin cytoskeleton, leading to intensive migration.    In HeLa cells, knock down JWA alone contributes to inducerearrangement of actin filaments, and also enhances PMA and As_2O_3induced actin rearrangement. JWA plays a critical role in activation of theMEK-ERK downstream as a consequence of As_2O_3 and PMA stimulationand links to the contrasting effects on cell migration. JWA may befunctional as a kinase-like molecule in MAPK cascade; the SDR-SLRphosphorylation motifs play a critical role in MAPK pathway mediatedtumor cells migration.    Taken together, JWA, works as a potential critical molecule, associatedwith migration and invasion of human tumor cells, and may be a novelmolecular marker for it. A full understanding of JWA function mayprovide significant insights into the pathogenesis, development andprognostic of tumor.