【作者】 邓欣；

【导师】 于秉治；

【作者基本信息】 中国医科大学 ， 细胞生物学， 2008， 博士

【摘要】 前言蛋白激酶B（PKB/AKT）是一种多功能丝氨酸/苏氨酸类蛋白激酶,在细胞的生长过程,如新陈代谢、细胞增殖、凋亡、转录以及细胞迁移中均起着重要的作用。在哺乳动物中,AKT1、AKT2和AKT3（又分别叫PKBα、PKBβ和PKBγ）有相似的结构域并且能够被大量的生长因子以PI3K依赖的方式被激活。AKT的N末端1～106位是它的PH结构域,这个区域在信号转导中被认为可以介导PKB/AKT与其它蛋白质的相互作用,PH域可以结合磷脂酰肌醇3,4,5-三磷酸盐（PtdIns（3,4,5）P₃）等磷脂,PH域与磷脂酰肌醇的结合使AKT定位于细胞膜,AKT在膜的聚集是其活化的标志,其Thr308位点的磷酸化可能促进C-末端疏水基序的构象发生改变,而其Ser473位点的磷酸化则是浆膜定位的一个必要条件。AKT在S期的起始和G₂/M转换过程中起了重要的作用,AKT1可以磷酸化p21的T145及p27的T157和T198,导致它们在细胞质中的易位与功能的尚失,而p21与AKT2结合后即不再被AKT1磷酸化。AKT也通过促进CDC25B的磷酸化调节G₂/M的转化,但是一直没有报道是哪一种亚型在G₂/M转化过程中发挥作用。脯氨酸丰富的肌醇多磷酸5磷酸酶（PIPP）,又名磷脂酰肌醇4,5-二磷酸盐5磷酸酶A（PIB5PA）,能去磷酸化PtdIns（3,4,5）P₃右旋第5位磷酸盐和PtdIns（4,5）P₂的肌醇环,分别形成磷脂酰肌醇3,4-二磷酸盐和磷脂酰肌醇4磷酸盐。PIPP在多种组织中表达,如脑、心、肾、胃、小肠和肺,但是它在这些组织中的特异性作用仍然需要探索。特别是,PIPP在1-细胞期胚胎中的作用,至今国内外没有任何报道。本研究以检测AKT的表达与活性为基础,探索PIPP和催化失活型的PIPP-H557A通过AKT及其下游底物对小鼠1-细胞期胚胎有丝分裂的作用,以期为研究PIPP在受精卵早期发育中的作用奠定基础。实验材料与方法一、动物来源中国医科大学实验动物部提供昆明系小白鼠。二、试剂pEFBOS-PIPP、pEFBOS vector和pEFBOS-PIPP-H557A由Mitchell教授惠赠;限制性内切酶MluI购自Fermentas公司;抗体购自Santa Cruz;其它试剂如果没有特指,均购于Sigma公司。三、小鼠卵母细胞以及受精卵的采集和培养取2～3周龄的昆明系雌性小鼠,腹腔注射5IU孕马血清PMSG,48h后颈椎脱臼法处死,取出卵巢,置于含有M2的培养液中,在显微镜下刺破大的有腔卵泡,释放含完整生发泡（GV）的天然裸卵母细胞,收集并贮存。根据Hogan的方法进行小鼠受精卵的超排和收集,取4～6周龄成熟雌性,腹腔注射PMSG 10 IU/只,48h后腹腔注射hCG 10 IU/只,当日与8周龄以上成熟雄性昆明系小鼠合笼过夜,次日清晨检查雌鼠阴栓,有阴栓者为交配成功,脱颈椎处死雌鼠,取双侧输卵管,剪下末端膨大置于M2培养液中,实体显微镜下撕开壶腹处,使受精卵细胞团自然流出,用M2培养液洗受精卵,把需要培养的受精卵移入M16培养液中,按不同的时间点收集G₁,S,G₂与M期的受精卵。四、RT-PCR将收集到的各期受精卵（200个卵/组）,用QuickPrep Micro mRNA Purification试剂盒按操作步骤提取RNA。用TaKaRa RNA PCR（AMV）试剂盒进行反转录和PCR。使用AMV（avian myeblastosia virus）反转录酶将mRNA合成cDNA的第一链,反转录实验按操作步骤说明。用Primer5软件设计引物在NCBI网站上BLAST确认之后,由上海生工生物技术有限公司合成。五、转染将受精卵接种于含100μl M16培养液的96孔板中,转染时受精卵处于G₁期（hCG注射后19～20h）。用脂质体Lipofectamine^TM2000转染质粒,按试剂盒按说明书,无菌条件下,首先,在含24.5μl的M16培养液的Eppendorf管中加入0.5μl的Lipofectamine^TM2000脂质体,同时在另一个含23.5μl的M16培养液中加入1.5μl（0.2μg）pEFBOS-PIPP、pEFBOS vector或pEFBOS-PIPP-H557A质粒,室温静置5min。然后,将两种液体混匀,室温静置20min。最后向含受精卵的培养液中加入脂质体与质粒DNA的复合物共50μl,置于37℃,5%CO₂培养箱中培养并按需要收集。六、间接免疫荧光法将待用的对照组及处理组受精卵移入新鲜配制的4%多聚甲醛液中,固定30min（37℃）,用含0.1%Triton-X100的PBS处理15min（37℃）,以增加细胞膜通透性,清洗液充分清洗后把卵移入封闭液滴中,37℃,1h。用兔多克隆抗pAKTSer473抗体室温孵育1h或4℃过夜。然后移入FITC标记的二抗,室温避光孵育30min后,用清洗液去除未结合的二抗,再用PI（用于激光共聚焦）或Hochest33258（用于荧光显微镜）染核。激光共聚焦或荧光显微镜下观察pAKTSer473蛋白定位的改变。七、AKT的抑制剂对小鼠受精卵卵裂率的影响将pEFBOS-PIPP和/或pEFBOS vector质粒转染的受精卵置于,M16培养液中,在37℃,5%CO₂饱和湿度的培养箱内培养,转染后10h,用相差显微镜观察并记录受精卵卵裂的情况,然后进行统计学分析,对照组比较,如果P＜0.05即具有统计学意义。八、Western Blot样品加入20μl蛋白提取缓冲液,反复冻融3次,加入SDS样品缓冲液2.0ul,100℃煮沸5min,12%SDS-PAGE电泳分离蛋白质,然后将蛋白转至硝酸纤维素膜上,再将滤膜与抗pAKT1/2/3 Ser473的抗体、抗CDC2 Tyr15抗体或抗β-actin抗体4℃温育过夜,经PBST洗涤后,用羊抗兔的IgG作为二抗室温孵育2h,经PBST洗涤膜后,化学发光法显影成像。九、AKT与MPF的活性测定将收集的鼠卵冻融3次,使细胞裂解,加入AKT或MPF反应液25μl,30℃水浴反应30min,取25μl点在Whatman P81强阳离子交换滤纸上,以75 mmol/L磷酸溶液终止反应,洗膜2次,每次2min。将滤纸置于含10ml蒸馏水的液闪瓶内,用Beckman液闪计数仪测定cpm值。十、数据处理实验结果应用SPSS 11.5统计学软件进行one-way ANOVA检验,所有的值用mean±SD表示,每组实验重复3次,P＜0.05被认为具有统计学意义。实验结果一、AKT在小鼠受精卵中的表达与活化RT-PCR的检测结果显示,AKT1的mRNA表达在小鼠GV期卵母细胞以及小鼠受精卵中的G₁,S,G₂和M期,而AKT2则仅表达在1-细胞胚胎中的G₁期。对p-AKT1/2/3 Ser473蛋白检测显示,Ser473位磷酸化的AKT在受精卵中表达的量远远大于它在GV期卵母细胞中的表达。免疫荧光分析的结果显示,结合Ser473位磷酸化AKT的绿色荧光FITC在GV期卵母细胞中均匀分布在胞浆中,而在S期的受精卵中绿色荧光分布于细胞膜,AKT的膜定位表明在S期的受精卵中它可能处于活化状态。当在G₁期的受精卵中加入AKT的抑制剂Wortmannin时,其定位从周边转向分布于整个细胞内,并以胞核为主,表明其活性受到抑制。结果建议,Ser473位磷酸化AKT的定位与其活性密切相关,可能是其活性的表现形式。对AKT活性的检测证明在小鼠受精卵中AKT的活性远远高于它在GV期卵母细胞中的活性,说明在小鼠受精卵中AKT可能处于充分的活化状态。二、PIPP在小鼠受精卵中对AKT及其信号途径的作用蛋白分析显示,PIPP明显降低了AKT Ser473位点磷酸化。建议,PIPP可能通过调节AKT的表达参与对AKT下游效应子的调节,并因此调控与AKT有关的小鼠受精卵的有丝分裂。结果显示,在PIPP转染的小鼠受精卵中,AKT的定位从细胞膜转向细胞核,受精卵卵裂率降低,MPF活性下降,CDC2 Tyr15的去磷酸化发生阻滞。三、PIPP-H557A在小鼠受精卵中对AKT及其信号途径的作用催化失活型PIPP-H557A对受精卵的作用与PIPP正好相反,说明,PIPP557位点的组氨酸对PIPP的催化活性具有决定意义,它的突变使PIPP失去了其5磷酸酶的催化活性。讨论作为一个维持有机体正常生命活动的重要调节子,AKT受许多生长因子的调控,如血小板源性生长因子PDGF、表皮生长因子（EGF）、胰岛素和神经生长因子（NGF）等。AKT的活化主要是通过PI3K/AKT调节,失活的、细胞质内的AKT转位到浆膜通过PH域去结合由PI3K活化产生的PtdIns（3,4,5）P₃和/或PtdIns（3,4）P₂,以此改变AKT的构象使其变得易于被上游激酶磷酸化,AKT的膜定位是其活化的标志。我们对小鼠受精卵中Ser473位磷酸化AKT定位的观察显示,AKT集中在S期小鼠受精卵的细胞膜,当使用PI3K/AKT途径的特殊抑制剂,Wortmannin等处理受精卵时,AKT的分布转位到小鼠受精卵的细胞核以及细胞质中,究其原因可能是由于PIPP使PtdIns（3,4,5）P₃生成下降并且转位到核区的结果。这些结果符合以前的对体细胞中AKT研究的报道,也应该为进一步研究小鼠受精卵发育过程中AKT的作用提供了重要的科学依据。我们检测到了AKT1在小鼠受精卵中的4个时期和不成熟卵母中的表达,说明AKT1在受精卵的发育过程中是必要的信号蛋白,但是令人惊异的是AKT2的mRNA仅仅表达在受精卵的G₁期。资料显示,AKT1是细胞增值所必需的蛋白,AKT1磷酸化p27可以损伤了p27的核输入序列,而AKT2则通过与p21结合促进细胞周期转变。结合实验结果,我们认为AKT2在G₁期的表达应该起到了一个负调控的作用,而AKT1与AKT2在表达时间上的不同说明它们在1-细胞胚胎分化的过程中起了不同的作用,并且应当具有不同的信号转导途径,当然,关于它们的具体作用需要进一步的研究。对Ser473位点磷酸化的AKT在蛋白表达水平的检测,我们发现在受精卵中Ser473位磷酸化AKT的表达量明显高于它在GV期卵母细胞中的表达,以G₂期为最高。此结果也与相应的AKT的活性分析一致,在受精卵中的活性明显高于其在GV期卵母细胞中的活性。在哺乳动物的有丝分裂中,M期促进因子MPF（M-Phase Promoting Factor）的活化在有丝分裂的G₂/M转换过程中起着关键的作用,它的活化主要由CDC2激活,CDC2 Thr14和Tyr15的磷酸化抑制CDC2活性,而它们的去磷酸化却激活了CDC2,同时CDC2的活化加速了M期的启动,在G₂/M转换期间,MPF的活性达到高峰。为探索AKT影响细胞周期的分子途径,我们研究了小鼠1-细胞胚胎中MFP的活性在AKT抑制剂作用下的改变。也检测了AKT的上游,肌醇多磷酸5-磷酸酶PIPP对AKT活性的调解,我们的结果与以前在体细胞中检测的报道一致,而且我们对PIPP mRNA在受精卵中表达的检测是首次报道,在对催化失活型PIPP-H557A的检测中,我们发现PIPP第557位的组氨酸突变为丙氨酸后其活性尚失并且失去了调节AKT活性的能力。根据实验结果,我们推论PIPP在小鼠受精卵中降低AKT Ser473位点的磷酸化,并且有可能通过此途径干涉细胞周期的进程,抑制其下游的MPF活性以及CDC2 Tyr15去磷酸化状态。总之,我们的实验揭示AKT Ser473位点的磷酸化在小鼠受精卵及初级卵母细胞中的定位、表达及其活性的变化。并且发现在小鼠受精卵中,PIPP能通过降低AKT Ser473位点的磷酸化调节1-细胞阶段胚胎的有丝分裂进程。我们的结果为哺乳动物受精卵的早期发育、细胞周期以及肿瘤的发生等研究提供了重要依据。结论1、在小鼠受精卵的中,AKT1与AKT2 mRNA的表达不同,AKT1在受精卵的发育过程中是必需的。2、在小鼠受精卵的S期中,AKT1定位于细胞膜。3、AKT1可能调控小鼠受精卵中G₂/M期的转换。4、在小鼠受精卵中,PIPP通过降低AKT的活性,调节G2/M期的转换,而催化失活型的PIPP-H557A不能降低AKT的活性。更多还原

【Abstract】 ObjectivesProtein Kinase B（PKB/AKT）is a multifunctional serine-threonine protein kinase, which paly a key role in the process of cell growth,such as metabolism,reproducation, apoptosis,transcription and migration.In mammalian,AKT1,AKT2 and AKT3（also termed PKBα,PKBβand PKBγrespectively）have similar domain and can be activated by a large part of growth factors in a PI3K-dependent manner.AKT N-terminal（1～106） is its PH domain.The PH domain may mediate the interaction of PKB/AKT with other proteins.It can bind certain phospholipids such as PtdIns（3,4,5）P₃ and/or PtdIns （3,4）P₂ and meanwhile target AKT to plasma membrane.Plasma membrane localization cooperates with basal AKT activity to activate Ser473 phosphorylation of AKT C-termiinal.Therefore membrane recruitment of AKT is a hallmark of activation. Thr308 phosphorylation of AKT might promote a conformational change of a C-terminal hydrophobic motif（HM）.And membrane localization of AKT is very critical for AKT activity and Ser 473 phosphorylation.Previous reports have indicated that AKT play an important role in the initiation of S and G₂/M transition.AKT1 can phosphorylate p21 T145 and p27 T157 and result in their cytoplasmic translocation and loss of function.However,when p21 binds with AKT2,it can no longer be phosphorylated by AKT1.AKT can promote CDC25B phosphorylation and regualte G₂/M transition.Proline-rich inositol polyphosphate 5-phosphatases（PIPP）,also called PIB5PA,can dephosphorylate dextrorotation 5-position phosphorylation of PtsIns（3,4,5）P₃ and/or PtdIns（4,5）P₂ to form PtsIns （3,4）P₂ and/or PtdIns（4）P.PIPP expresses in multiple tissues such as brain,heart, kidney,stomach,small intestine and lung.Its special function remains to be established, especially there is still no report about the role of PIPP in mouse one-cell embryos.In this study,based on the expression and activity of AKT,we explore the effect of PIPP on AKT and its downstream substrates in mitotic division of mouse one-cell embryos in order to lay a important foundation to study the effect of PIPP on early development of mouse one-cell embryos.Materials and Methods1.AnimalKunming strain mice were obtained from the Department of Laboratory Animals in China Medical University.2.ReagentsThe pEFBOS-PIPP,pEFBOS-PIPP-H557A and pEFBOS vector plasmids were provided by Prof.Mitchell;Restriction endonuclease MluI were purchased from MBI. Antibodies were purchased from Santa-Cruz.Other reagents,unless otherwise specified, were purchased from Sigma（St.Louis,MO）.3.Collection and Culture of Mouse fertilized Eggs and GV intact OocytesMouse one-cell embryos were collected from female mice with the method described by Hogan and Constantini.These eggs were then collected according to different cell cycles needed（G₁ phase:11～20 hr after hCG injection;S phase:21～26 hr after hCG injection;G₂ phase:27～29 hr after hCG injection;M phase:30～32 hr after hCa injection）.As control group,mouse oocytes were collected and cultured from 20- to 28-day-old Kunming strain female mice according to the method described by Schultz and Jaroslav.The oocytes were matured in vitro for 0,24 h to reach the germinal vesicle（GV）and metaphaseⅡ（MⅡ）stage respectively.4.RT-PCRTotal mRNA was extracted from mouse GV-intact oocytes and mouse fertilized eggs（200 eggs for each sample）at different phases by use of the QuickPrep^TM MicromRNA Purification Kit（Amersham Bioscience Co.）according to the manufacturers’s instruction.RT-PCR reaction was carried out by RNA PCR Kit（AMV） Ver2.1（Takara Bio.,Inc.,Japan）according to its instruction.The sequences of the primers were designed by Primer 5.0 software and synthesized by Shanghai Sangon Biological Engineering Technology & Services Co.,Ltd.5.Transfection of FLAG-PIPP,FLAG-PIPP-H557A and FLAG vectorMouse one-cell embryos were transfected with 0.2μg DNA（FLAG-PIPP, FLAG-PIPP-H557A,or FLAG vector,respectively）and 0.5μl Lipofectamine 2000 （Invitrogen,Carlsbad,CA）according to the manufacturer’s instructions.After transfection,these eggs were cultured in M16 medium at 37℃24 hr and collected at required time.The supernatant was removed and then stored in -70℃until use.6.Immunoflueresence to analyse the distribution of phosphorylated AKT Ser473 in mouse fertilized eggsCollected mouse oocytes with GV and one-cell embryos at S phase,fixed in 4% polyformaldehyde,perforate with 0.1%Triton-X100;samples were incubated with the first antibody and the FITC conjugated second antibody,stain the DNA with PI; observed under the fluorescene microscope or confocal microscope.7.Effect of AKT inhibitors on division rate of mouse fertilized eggsEggs treated with AKT inhibitors were cultured in M16 medium,after 10 h,the division rate of eggs was observed,recorded and analyzed with SPSS 11.5 software. Compared with control group,it is significant for statistics if P＜0.05.8.Western BlotProtein extracts of mouse eggs were prepared with the similar method as previous reports.The primary antibodies against pAKT1/2/3（Ser 473）,β-actin（Santa Cruz,CA, USA）,pCDC2（Tyr15）（New England Biolabs）or CDC2（Neomarkers）were used at 1:500 dilution.HRP-conjugated secondary antibody was diluted 1:2000.The proteins were detected using an ECL detection system（Pierce Biotechnology Inc.,USA）9.Assays of AKT and MPF activitiesAKT or MPF kinase activity was measured by histone H2B kinase or histone H1 kinase assay.The frozen eggs were treated with the same method as we previously reported.The radioactivity on the filter paper was counted with a BECKMAN scintillation counter.10.Statisitical AnalysisMeasurements were expressed as mean±SD and statistical analysis was performed by one-way ANOVA test with SPSS 11.5 Software.P＜0.05 was regarded as a significant difference.Each experiment was repeated three times,and the value of three samples averaged was compared among groups.Results1.Expression and Activity of AKT in mouse fertilized eggsAKT1 transcripts showed detectable level in each phase of mouse one-cell embryos and GV-intact oocytes.However,AKT2 transcript was detected only in G₁ phase of mouse one cell embryos.And the protein levels of AKT（Ser473） phosphorylation were much higher in mouse one cell embryos than those in immature oocytes.AKT activity measurement indicated that the activity of AKT in one-cell embryos was higher than that in GV intact oocytes,suggesting that AKT may play a key role in mouse one cell embryos.Localization of AKT（Ser473）phosphorylation was regulated in an activity-dependent manner.2.Effect of PIPP on AKT and its G₂/M transition in mouse fertlilized eggsMouse fertilized eggs transfected with FLAG-PIPP were found decrease of AKT activity,compared with these eggs transfected with FLAG vector（P＜0.001）. Meanwhile,eggs transfected with FLAG-PIPP or treated with LY294002 showed the change of AKT membrane localization,the decrease of cleavage rate,MPF activity and CDC2 Tyr15 dephosphorylation.3.Effect of PIPP-H557A on G2/M transition of mouse fertlilized eggsMouse fertilized eggs transfected with FLAG-PIPP-H557A showed that there were little effect on MPF activity and division rates and dephosphorylation of CDC2 Tyr15 in mouse fertilized eggs.DiscussionAs an important regulator to maintain the normal vital movement of organism, AKT is activated by the stimulation of many growth factors.AKT activity is regulated mainly by PI3K/AKT signaling pathway.Pleckstrin homology（PH）domain of AKT binds to PtdIns（3,4,5）P₃ and/or PtdIns（3,4）P₂ that is generated in response to PI3K activation.This binding alters AKT’s conformation such that it becomes accessible to phosphorylation by its upstream kinases,such as phosphoinositide-dependent kinase 1. The Localization of AKT（Ser473）phosphorylation in mouse fertilized eggs of S and G₂ phase indicated that AKT was activated and translocated in an activity-dependent manner.During the mitosis of fertilized eggs,mRNA expression is distinct.It requires genome reprogramming and proper regulation of different gene expression patterns. Our data showed that AKT2 transcript fluctuates in mitotic cycle of mouse fertilized eggs,whereas AKT1 transcript level is unchanged,suggesting that AKT1 and AKT2 gene patterns might be properly regulated in mitotic cycle of mouse fertilized eggs.The apparent differences with respect to their expression suggest that their signal transduction pathways may differ,particularly with regard to their downstream targets or substrates.Our data would contribute to an understanding of relationship between embryogenesis and tumorigenesis.In dividing eukaryotic cells,entry into mitosis is governed by the MPF.Its activation is achieved mainly by phosphorylation of CDC2.Clearly,AKT activity is complex, exhibiting a series of spatial restrictions that limit phosphorylation by upstream activiators.It is very significant to explore the effect of AKT and PIPP on mitotic division of mouse fertilized eggs.Our data are rather compatible with previous reports. Potential inhibitors of AKT activity,Wortmannin or PIPP,distinctly decreased AKT activity.Moreover,the dramatic downregulation of AKT1 activity blocked the procedure of mitotic division of mouse fertilized eggs,such as decrease of MPF activity and increase of CDC2 Tyr15 phosphorylation.Meanwhile catalytic inactive PIPP-H557A was detected to loss the activity of PIPP and have little effect on AKT and its substrates.These data should be of great benefit for our understanding of AKT1 and PIPP sensitive signal cascades in mitotic cycle of mouse fertilized eggs.Taken together,our study indicated that AKT1 in mouse one-cell embryos may be located in cytomembrane and activated sufficiently.AKT1 activity may promote mitotic division of mouse fertilized eggs.However,PIPP paly a negative role to AKT1 and different with catalytic inactive PIPP-H557A.And whether AKT2 expression provides physiologic significance,or is predictive of response to a particular signaling pathway,needs a further study.Conclusion1.AKT1 and AKT2 mRNA expression were different in mitotic cycle of mouse fertilized eggs.AKT1 is the necessary for the development of mouse fertilized eggs.2.AKT1 loacted in cytomembrane of mouse fertilized eggs.3.AKT1 may regulate G₂/M transition of mouse fertilized eggs.4.PIPP regulates G₂/M transition by decreasing AKT activity,but inactive PIPP-H557A can not regulate AKT activity.更多还原