AURKB与MAPK参与调控小鼠受精卵早期发育

Aurora 激酶是肿瘤研究领域的热点, 近年来有研究表明该激酶家族在卵母细胞减数分裂中也起着重要的调节作用, 但对于其在哺乳动物早期胚胎发育中的研究鲜有报道. 本研究通过实时荧光定量PCR、免疫印迹、免疫荧光检测了Aurora 激酶 B(Aurora kinase B, AURKB)在小鼠受精卵中的表达和定位, 运用RNA 干扰技术观察了AURKB 功能缺失后对小鼠受精卵发育早期的影响, 并检测丝裂原激活蛋白激酶 (mitogen-activated protein kinase, MAPK)通路抑制后小鼠受精卵卵裂及AURKB 表达、 活性变化. 结果表明, 在小鼠受精卵第一次卵裂进程中, G2/M 期为AURKB 的稳定表达时相, 其蛋白在G1/S 期少量分布于细胞浆, G2 期聚集于染色质周围, 进入有丝分裂后分布于全细胞. AURKB 的功能缺失可导致受精卵发生异常分裂. MAPK 通路的抑制亦可破坏受精卵的正常卵裂, 并下调AURKB的蛋白表达及活性. 结果提示, Aurora 激酶B 是小鼠受精卵早期发育所必需的, 并与MAPK 通路的激活相关.     

小鼠受精卵早期发育过程中PKA在M/G1期进程中的作用

为研究小鼠体内l-细胞期受精卵蛋白激酶A(PKA)对M/G1期进程的影响,应用热稳定性抑制剂PKI显微注射入l-细胞期受精卵内,观察M期促进因子(MPF)及PKA活性变化以及MPF调节亚基Cyclin B含量情况。发现PKI显微注射后PKA活性低,而MPF活性在hCG后27．5h即达高峰,较对照组提前30分钟。PKI达一定浓度则MPF活性不下降,出现M/G1阻滞;与此同时Western blotting法显示PKI注射后Cyclin B含量在M末期相当于M中期水平。结果表明,PKI显微注射抑制PKA活性后MPF活性呈高峰值,高浓度PKI显微注射可引起M/Gl阻滞,其机制与PKI干扰了Cyclin B降解有关。     

雷帕霉素靶在小鼠受精卵早期发育中的作用研究

哺乳动物雷帕霉素靶(mTOR)是细胞生长的中心调控因子,应用RT-PCR、免疫印迹、放射性同位素体外测定酶活性等方法,研究mTOR在小鼠受精卵第一次有丝分裂过程中在卵中的表达、活性变化以及对卵裂的影响.研究发现mTOR在小鼠卵母细胞和受精卵中都有表达,在mRNA水平,mTOR从G2期开始降解,在蛋白水平,则各期没有明显变化;mTOR的激酶活性在受精后明显升高,并且在整个1-细胞期保持较高活性;mTOR的特异性抑制剂雷帕霉素能抑制卵裂,并且能抑制成熟促进因子MPF的调节亚基cyclin B的表达,从而抑制了MPF的活性.结果表明mTOR可能通过促进MPF的激活而促进小鼠受精卵的分裂.     

丝裂原活化蛋白激酶激活蛋白激酶(MK)研究进展

丝裂原活化蛋白激酶(MAPK)信号通路介导多种重要的细胞生理反应.对下游蛋白激酶的磷酸化是MAPK家族成员发挥生理作用的重要方式.在MAPK的下游存在3个结构上相关的MAPK激活蛋白激酶(MAPKAPKorMK),即MK2,MK3和MK5.在被MAPK激活后,MK可将信号传递至细胞内不同靶标,从而在转录和翻译水平调节基因表达,调控细胞骨架和细胞周期,介导细胞迁移和胚胎发育.最近,在基因敲除研究的基础上,不同MK亚族成员之间的功能区分已经逐渐明晰,使我们对于MK的认识有了长足的进步.     

小鼠受精卵早期发育过程中PKB/Akt对p21蛋白表达及定位的影响

初步探讨在小鼠受精卵早期发育过程中PKB/Akt对p21蛋白表达及定位的影响。通过显微操作技术注射野生型、持续激活型及激酶失活型的PKB的mRNA,用免疫荧光方法检测p21蛋白的细胞定位、Western blot方法检测p21蛋白的表达。结果显示在注射不同形式的PKB mRNA后p21蛋白的表达无明显差别,但是细胞定位发生改变,PKB被激活后,p21蛋白滞留在胞浆中。因而初步认为在小鼠受精卵中,PKB/Akt通过影响p21的细胞定位而影响细胞周期的进程。     

蛋白激酶A对小鼠受精卵早期发育过程中M期促进因子活性的影响

为研究小鼠体内 1 细胞期受精卵M期蛋白激酶A(PKA)对M期促进因子 (MPF)活性的影响 ,应用PKA激动剂cAMP及热稳定性抑制剂PKI显微注射入 1 细胞期受精卵内 ,观察MPF及PKA活性变化 .未经注射的对照组MPF活性在分裂期增高 ,分裂间期下降 ;而PKA活性在进入分裂期下降 ,分裂间期升高 .cAMP组PKA活性维持高峰值 ,直至注射HCG后 2 8h ,MPF活性高峰延迟 30min出现 ;PKI显微注射组PKA活性低 ,而MPF活性在注射HCG后 2 7 5h即达高峰 ,且维持高峰时间达1 5h .结果表明 ,PKA活性在细胞周期中也呈波动性 ,间期活性高 ,分裂期活性低 ;PKA高活性抑制MPF活性 ,而抑制PKA活性则MPF活性高峰提前出现 .     

小鼠受精卵微注射Cdc25b mRNA可提高卵裂率并促进受精卵发育

为了观察Cdc25B蛋白及PKA/Cdc25B 信号途径在小鼠受精卵发育中的作用,将突变型和野生型Cdc25b转录成 mRNA,显微注射到小鼠受精卵中,放入含有或不含有dbcAMP的M16中,相差显微镜下观察受精卵卵裂情况;用蛋白激酶活性测定方法检测MPF的活性;利用Western 印迹检测Cdc2-Tyr15的磷酸化状态.结果显示,未加dbcAMP的Cdc25b- S321A mRNA注射组与Cdc25b-WT组相比,能够提前使受精卵发生G ₂/M期转变,导致卵裂,并明显提高卵裂率;MPF的活性测定和Cdc2-Tyr15磷酸化状态的检测结果也显示,Cdc25b-S321A组先于Cdc25b-WT组提前激活MPF.此外, Cdc25b-S321A mRNA注射组可以有效恢复由PKA引起的受精卵G ₂期阻滞,显著增加卵裂率;MPF的活性测定和Cdc2-Tyr15磷酸化状态的检测结果也显示,在PKA持续激活的情况下,对比于Cdc25b-WT组,Cdc25b-S321A组提前激活MPF.因此,在小鼠受精卵发育过程中PKA主要通过磷酸化Cdc25B的321位丝氨酸,从而调控MPF的激活与失活来控制有丝分裂进程.     

小鼠受精卵早期发育过程中PKB/Akt对p21蛋白表达及定位的影响

初步探讨在小鼠受精卵早期发育过程中PKB/Akt对p21 蛋白表达及定位的影响。通过显微操作技术注射野生型、持续激活型及激酶失活型的PKB的mRNA,用免疫荧光方法检测p21蛋白的细胞定位、Western blot 方法检测p21蛋白的表达。结果显示在注射不同形式的PKB mRNA 后p21蛋白的表达无明显差别,但是细胞定位发生改变,PKB被激活后,p21蛋白滞留在胞浆中。因而初步认为在小鼠受精卵中,PKB/Akt通过影响p21 的细胞定位而影响细胞周期的进程。     

有丝分裂激酶Aurora B的研究进展

真核生物细胞通过有丝分裂将遗传物质均等地分配到两个子细胞中,从而维持基因组的稳定性。有丝分裂的每一环节都需要精准而细致的调控,这依赖于一系列调节机制,尤其需要多个相关激酶的共同协调。Aurora B是一个关键的有丝分裂调控激酶,伴随有丝分裂的进行,其先后在染色体臂、内着丝粒、中央纺锤体、中体上动态分布。与其高度时空动态性相一致的是,Aurora B在有丝分裂的多个环节,如姐妹染色体粘连、动粒微管连接、纺锤体检验点和胞质分裂过程中都发挥着一系列重要功能。本文将概述近年来Aurora B激酶功能与调控方面的研究进展。     

Somatostatin down-regulates the expression and release of endozepines from cultured rat astrocytes via distinct receptor subtypes

Endozepines, a family of regulatory peptides related to diazepam-binding inhibitor (DBI), are synthesized and released by astroglial cells. Because rat astrocytes express various subtypes of somatostatin receptors (sst), we have investigated the effect of somatostatin on DBI mRNA level and endozepine secretion in rat astrocytes in secondary culture. Somatostatin reduced in a concentration-dependent manner the level of DBI mRNA in cultured astrocytes. This inhibitory effect was mimicked by the selective sst4 receptor agonist L803-087 but not by the selective sst1, sst2 and sst3 receptor agonists L779-591, L779-976 and L797-778, respectively. Somatostatin was unable to further reduce DBI mRNA level in the presence of the MEK inhibitor U0126. Somatostatin and the sst1, sst2 and sst4 receptor agonists induced a concentration-dependent inhibition of endozepine release. Somatostatin and the sst1, sst2 and sst4 receptor agonists also inhibited cAMP formation dose-dependently. In addition, somatostatin reduced forskolin-induced endozepine release. H89 mimicked the inhibitory effect of somatostatin on endozepine secretion. In contrast the PLC inhibitor U73122, the PKC activator PMA and the PKC inhibitor calphostin C had no effect on somatostatin-induced inhibition of endozepine release. The present data demonstrate that somatostatin reduces DBI mRNA level mainly through activation of sst4 receptors negatively coupled to the MAPK pathway, and inhibits endozepine release through activation of sst1, sst2 and sst4 receptors negatively coupled to the adenylyl cyclase/PKA pathway.     

Examination of the involvement of protein kinase A in D2 dopamine receptor antagonist-induced immediate early gene expression

Immediate early genes (IEGs) are induced by different signaling pathways. It has been proposed that D2 dopamine receptor blockade induces IEG expression through activation of protein kinase A (PKA), although few studies have examined this issue in vivo. We infused the PKA inhibitor H-89 into the striatum of male rats, followed 30 min later by systemic administration of eticlopride. Eticlopride-induced c-fos and zif268 mRNA expression in striatum was not blocked by H-89. In addition, eticlopride did not produce measurable levels of PKA activity in striatum, whereas the cAMP activator Sp-8-Br-cAMPs increased levels of activated PKA. Neither the adenosine A2a receptor agonist CGS 21680 nor the phosphodiesterase-4 inhibitor rolipram, each of which should increase PKA activation, potentiated eticlopride-induced IEG expression. To test whether other signaling pathways are involved in eticlopride-mediated gene induction, we also infused inhibitors of the mitogen-activated and calcium/calmodulin-dependent protein kinases into animals and then treated them with eticlopride. The data suggest that eticlopride-induced IEG expression is not solely dependent on these kinases either. These data suggest that PKA activation may not be necessary for induction of IEGs by D2 dopamine receptor antagonists and that other intracellular signaling pathways may be involved.     

促分裂原活化蛋白激酶磷酸酶

促分裂原活化蛋白激酶磷酸酶（mitogen-activated protein kinase phosphatases,MKPs）是一类丝／苏氨酸和酪氨酸双特异性的磷酸酶。它在细胞分化、增殖和基因表达过程中起着重要的作用。MKPs可以选择性地结合促分裂原活化蛋白激酶（mitogen-activated protein kinase,MAPK）,对MAPK进行去磷酸化,从而调节MAPK信号通路的活性。另一方面,MAPK也可以激活MKPs,它们的相互作用确保了细胞内信号的精确传递,并参与细胞功能的调节。     

Endogenous δ-Opioid and ORL1 Receptors Couple to Phosphorylation and Activation of p38 MAPK in NG108-15 Cells and This Is Regulated by Protein Kinase A and Protein Kinase C

The p38 mitogen-activated protein kinase (MAPK) cascade transduces multiple extracellular signals from cell surface to nucleus and is employed in cellular responses to cellular stresses and apoptotic regulation. The involvement of the p38 MAPK cascade in opioid- and opioid receptor-like receptor-1 (ORL1) receptor-mediated signal transduction was examined in NG108-15 neuroblastoma x glioma hybrid cells. Stimulation of endogenous delta-opioid receptor (DOR) or ORL1 resulted in activation of p38 MAPK. It also induced the activation of extracellular signal-regulated kinases (ERKs), another member of the MAPK family, with slower kinetics. Activation of p38 MAPK was abolished by selective antagonists of DOR or ORL1, pretreatment with pertussis toxin, or SB203580, a specific inhibitor of p38 MAPK. Inhibition of p38 MAPK had no significant effect on opioid-induced ERK activation, indicating that p38 MAPK activity was not required for ERK activation, though its stimulation preceded ERK activation. Inhibition of protein kinase A (PKA) strongly diminished p38 activation mediated by DOR or ORL1 but had no significant effect on ERK activation, and protein kinase C (PKC) inhibitors potentiated stimulation of p38 while inhibiting activation of ERKs. Taken together, our results provide the first evidence for coupling of DOR and ORL1 to the p38 MAPK cascade and clearly demonstrate that receptor-mediated activation of p38 MAPK both involves PKA and is negatively regulated by PKC.     

Delayed but sustained induction of mitogen-activated protein kinase activity is associated with beta-adrenergic receptor-mediated morphological differentiation of astrocytes

Astroglial beta-adrenergic receptors (beta-ARs) are functionally linked to regulate cellular morphology. In primary cultures, the beta-AR agonist isoproterenol (ISP) can transform flat polygonal astrocytes into process-bearing, mature stellate cells by 48 h, an effect that can be blocked by the beta-AR antagonist, propranolol. ISP induced immediate activation of protein kinase A (PKA) which persisted up to 2 h, with no visible change in cell morphology. However, activation of PKA was sufficient to drive the process of transformation to completion, suggesting the involvement of downstream regulators of PKA. In addition to PKA inhibitors, the mitogen-activated protein kinase (MAPK) kinase inhibitor PD098059 also blocked ISP-induced morphological transformation. ISP treatment resulted in a biphasic response of cellular phosphorylated MAPK (phosphorylated extracellular signal-regulated kinase; p-ERK) level: an initial decline in p-ERK level followed by a sustained induction at 12-24 h, both of which were blocked by PKA inhibitor. The induction in pERK level coincided with initiation of morphological differentiation of the astrocytes and nuclear translocation of p-ERK. A long-lasting activation of p-ERK activity by ISP, at a later stage, appears to be critical for the transformation of astrocytes.