丝裂原活化蛋白激酶对卵母细胞减数分裂成熟和排卵的调控作用及机制

丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)信号通路是一条从酵母到人中都高度保守的信号转导途径,广泛地存在于各种真核细胞中。近30多年来的研究表明,在几乎所有物种的雌性生殖细胞发育和减数分裂成熟过程中,该信号通路都发挥着至关重要的作用。特别是在包括人、小鼠和家畜的哺乳动物中,MAPK信号通路在卵母细胞恢复减数第一次分裂过程中被激活,调控纺锤体组装和细胞周期进程,并在颗粒细胞中介导促性腺激素的生理作用,促进卵丘扩展、排卵和黄体形成。虽然MAPK信号通路在雌性生殖过程中发挥着如此广泛的生理功能,并且这些功能在进化过程中高度保守,但是对于其作用机制,特别是其直接作用靶分子,在很长一段时间没有被充分研究清楚。近些年,基于一些新的基因编辑小鼠模型和理论研究成果,以及各种组学技术的广泛应用,人们进一步揭示了MAPK在减数分裂恢复过程中直接磷酸化激活RNA结合蛋白——胞质聚腺苷酸化原件结合蛋白1 (cytoplasmic polyadenylation element-binding protein-1,CPEB1),促进母源m RNA的poly(A)尾延伸,介导蛋白翻译激活。这些研究结果不但构成了目前本领域哺乳动物卵母细胞成熟和排卵机制的基本理论,也对本领域其他相关研究提供了可借鉴的研究思路。结合本研究组和其他科学家近年来的系统研究工作,我们对MAPK与卵母细胞成熟和排卵的研究进行了历史回顾,介绍了当前研究进展,提出了新近出现但尚未解决的科学问题,包括MAPK在颗粒细胞中对m RNA翻译和降解的调控,以及对翻译起始复合体、m RNA加尾酶的直接磷酸化激活等。     

β肾上腺素受体的丝裂原活化蛋白激酶信号途径

β肾上腺素受体（β－AR）除了通过经典的信号途径介导细胞生物功能外,还可以激活丝裂原活化蛋白激酶（MAPK）信号途径,活化后的MAPK参与调节多种细胞生物学活动。然而,将β-AR与MAPK信号联系起来的分子机制还需要进一步的研究。     

p21活化蛋白激酶2在卵母细胞成熟中的作用

研究p21活化蛋白激酶2（p21-activated kinase 2,PAK2）在爪蟾卵母细胞成熟中的作用。利用特异性抑制PAK2活性的PAK2-N端（PAK2-N terminal,PAK2-NT）片段显微注射爪蟾卵母细胞。荧光显微镜下比较PAK2-NT mRNA注射组和未注射对照组卵母细胞胚泡破裂发生。共聚焦显微镜下,时间延迟摄影法观察两组卵母细胞胞质分裂过程中肌动蛋白和纺锤体的变化。与未注射PAK2-N端mRNA的对照组卵母细胞相比,注射组卵母细胞胚泡破裂发生无异常,但未见胞质分裂发生和极体形成。结果提示PAK2可能参与爪蟾卵母细胞胞质分裂过程。     

成熟促进因子及其对卵母细胞成熟调控机制研究的新进展

卵母细胞成熟调控机制一直是发育生物学和生殖生物学领域的热点问题。以现代分子生物学理论为基础,科学家们对卵母细胞成熟分裂的分子生物学调控机理进行了大量研究。发现了细胞周期中许多关键的调控因子：cdc基因、周期蛋白依赖性激酶(CDKs)及细胞周期蛋白(cyclin)。本文对卵母细胞成熟调控的核心调控物质——成熟促进因子(maturation—promoting factor,MPF)的分子结构、周期变化及其在卵母细胞成熟过程中与丝裂原激活蛋白激酶(mitogen—activated protein kinase,MAPK)相互作用关系的最新进展进行了综述。     

丝裂原活化蛋白激酶(MAPK)信号通路的研究进展

丝裂原活化蛋白激酶（MAPK）信号通路是广泛存在于各种细胞中的一条信号转导途径,由一组级联活化的丝／苏氨酸蛋白激酶组成,对于细胞周期的运行和基因表达具有重要调控作用。MAPK包括多个成员,活化后向核内迁移,磷酸化包括转录因子在内的核蛋白和膜受体,实现对基因转录和其他事件的调节。MAPK激酶（MAPKK）是MAPK的上游激活分子,催化MAPK的Tyr和Thr残基双特异性磷酸化。Mos是脊椎动物生殖细胞中特有的MAPKK,通过MAPKK／MAPK途径活化成熟促进因子,启动卵母细胞成熟发育并维持中期阻滞。MAPK的下游分子包括MAPK活化的蛋白激酶（MAPKAPK）、核转录因子、热休克蛋白和细胞质磷脂酶A2等,执行由MAPK所介导的细胞生命活动调节功能。     

病原菌调节宿主细胞丝裂原活化蛋白激酶免疫防御信号的机理

丝裂原活化蛋白激酶(MAPK)家族广泛存在于高等生物中,介导多种生物学进程,在固有免疫防御中发挥重要作用,是真核细胞抵御病原菌侵染的第一道防线.越来越多的研究发现,病原菌可以利用多种方式激活或者抑制MAPK信号通路来增强其自身侵染力.简单介绍了MAPK信号通路的背景并详细总结了近几年关于病原菌如何作用于MAPK信号通路的研究工作,希望以此能够拓展对病原菌与宿主细胞作用方式的认识,深化对MAPK重要作用的了解.     

p38丝裂原活化蛋白激酶的功能与调控机制

丝裂原活化蛋白激酶家族可以在一系列细胞外刺激下调控细胞的行为。作为该家族的四个亚家族之一,p38亚族在许多生理过程中扮演着重要角色。p38信号通路可以在紫外照射、热击、高渗透压、炎症因子、生长因子等细胞外刺激时被激活,调控细胞分化、细胞周期、炎症反应等多种生理过程。文章重点讨论了p38亚族各个成员的特性、该信号通路的组成部分、调控机制以及生物学功能。另外,还分析了p38与其他信号通路的联系以及对一些生理过程的影响。     

丝裂原活化蛋白激酶(MAPK)生物学功能的结构基础

丝裂原活化蛋白激酶 (MAPK)是生物体内重要的信号转导系统之一 ,能对广泛的细胞外刺激发生反应 .蛋白激酶的空间构象是其功能的重要决定因素 .对MAPK蛋白结构的研究表明 ,MAPK的结构与功能之间具有密切的关系 .尽管MAPK各亚族的结构非常相似 ,但也存在着一些差异 ,这些差异是不同亚族对不同的细胞外刺激产生特异性反应的结构基础 .某些关键性结构 ,例如Loop12 ,在MAPK对上游激酶的作用、下游底物的选择以及亚细胞定位中都具有重要作用 .进一步深入研究MAPK的空间结构 ,探讨MAPK的生物学功能与其空间构象之间的关系 ,对于开发新的MAPK通路抑制剂用于治疗某些严重疾病有着重要的临床意义     

丝裂原活化蛋白激酶和磷酸酯酶-1在心肌病和细胞凋亡中的作用

ERK、JNK和p38等丝裂原活化蛋白激酶通过生长因子、激动剂或应激反应等介导生长、分化、凋亡以及细胞间相互作用等多种过程。ERK、JNK和p38是参与心衰病理过程的主要信号元件,MKP-1是丝裂原活化蛋白激酶等的去磷酸化因子,是一种应激蛋白,在应激反应中可以抑制ERK、JNK和p38的活性,并通过凋节ERK、JNK和p38的活性,参与对心衰病理过程的调节。本文以转基因研究结果为主要线索,对丝裂原活化蛋白激酶和磷酸酯酶．1在心衰病理过程中的作用进行了综述。     

卵母细胞成熟调控的分子机制及进展

卵母细胞的成熟是人类配子发育成熟,进而形成胚胎的必然阶段。目前已知有多种因素调控卵母细胞的成熟。成熟促进因子(MPF)是卵母细胞成熟调控的最重要分子,它通过CDK1亚基的磷酸化及cyclin B累积合成调节卵母细胞的成熟。MAPK/Mos及cAMP均可通过影响MPF的活性从而间接调控卵母细胞成熟。这三者之间又存在相互影响相互作用,形成一个复杂的调控网络。阐明卵母细胞成熟的分子机制有利于为治疗女性不孕症及卵母细胞体外培养成熟提供可靠的理论依据。     

c-erbB2 and c-myb induce mouse oocyte maturation involving activation of maturation promoting factor

Proto-oncogenes are involved in cell growth, proliferation, and differentiation. In the present study, we investigated the roles and mediating pathways of proto-oncogenes c-erbB(2) and c-myb in mouse oocyte maturation by RT-PCR, real-time quantitative PCR, western blot, and recombinant proto-oncogene protein microinjection. Results showed that both c-erbB(2) and c-myb antisense oligodeoxynucleotides (c-erbB(2) ASODN and c-myb ASODN) inhibited germinal vesicle breakdown and the first polar body extrusion in a dose-dependent manner. However, microinjection of recombinant c-erbB(2) or c-myb protein into germinal vesicle stage oocytes stimulated oocyte meiotic maturation. In addition, the expression of c-erbB(2) and c-myb mRNA was detected in oocytes; and c-erbB(2) ASODN and c-myb ASODN inhibited c-erbB(2) mRNA and c-myb mRNA expression, respectively. Maturation promoting factor (MPF) inhibitor roscovitine did not affect the expression of c-erbB(2) mRNA and c-myb mRNA, but blocked the effects of recombinant c-erbB(2) and c-myb protein-induced oocyte maturation. Further, cyclin B1 protein expression in oocytes was remarkably inhibited by c-erbB(2) ASODN, c-myb ASODN, and roscovitine. Nonsense tat ODN had no effect on the expression of c-erbB(2), c-myb, and cyclin B1. These results suggest that c-erbB(2) and c-myb may induce oocyte maturation through mediating a pathway involving the activation of MPF.     

Multiple roles of the mitogen-activated protein kinase kinase/mitogen-activated protein kinase cascade in Xenopus laevis

Mitogen-activated protein kinase (MAPK) was originally identified as a serine/threonine protein kinase that is rapidly activated in response to various growth factors and tumor promoters in mammalian cultured cells. The kinase cascade including MAPK and its direct activator, MAPK kinase (MAPKK), is now believed to transmit various extracellular signals into their intracellular targets in eukaryotic cells. It has been reported that activation of MAPKK and MAPK occurs during the meiotic maturation of oocytes in several species, including Xenopus laevis . Studies with neutralizing antibodies against MAPKK, MAPK phosphatases and constitutively active MAPKK or MAPK have revealed a crucial role of the MAPKK/MAPK cascade in a number of developmental processes in Xenopus oocytes and embryos.     

Fibroblast growth factor receptor 3 kinase domain mutation increases cortical progenitor proliferation via mitogen-activated protein kinase activation

We have previously shown that mice carrying the K644E kinase domain mutation in fibroblast growth factor receptor 3 (Fgfr3) (EIIa;Fgfr3(+/K644E)) have enlarged brains with increased proliferation and decreased apoptosis of the cortical progenitors. Despite its unique rostral-low caudal-high gradient expression in the cortex, how Fgfr3 temporally and spatially influences progenitor proliferation is unknown. In vivo BrdU labelling now showed that progenitor proliferation was 10-46% higher in the EIIa;Fgfr3(+/K644E) cortex compared with wild type during embryonic day 11.5 (E11.5)-E13.5. The difference in proliferation between the EIIa;Fgfr3(+/K644E) and wild-type cortices was the greatest in the caudal cortex at E12.5 and E13.5. Inhibition of mitogen-activated or extracellular signal-regulated protein kinase (MEK) in vitro at E11.5 reduced the proliferation rate of the EIIa;Fgfr3(+/K644E) cortical progenitors to similar levels observed in the wild type, indicating that the majority of the increase in cell proliferation caused by the Fgfr3 mutation is mitogen-activated protein kinase (MAPK) pathway-dependent at this stage. In addition, elevated levels of Sprouty were observed in the EIIa;Fgfr3(+/K644E) telencephalon at E14.5, indicating the presence of negative feedback that may have suppressed further MAPK activation. We suggest that temporal activation of MAPK is largely responsible for cell proliferation caused by the Fgfr3 mutation during early stages of cortical development.     

Bovine embryo cloning: Characterization of the recipient cytoplasts by phosphorylation patterns and kinase activities

When in vitro -matured oocytes were enucleated, aged and kept at 10°C before reconstitution, the in vitro development of nuclear transfer embryos to the blastocyst stage did not differ from that obtained with in vitro fertilization. This suggests that these recipient cytoplasts constitute a suitable environment for the development of the nuclear transplant. The aim of the present study was to investigate, at the biochemical level, the result of the preparation of recipient oocytes, including enucleation, ageing and cooling. For this purpose the phosphorylation profiles of four groups of in vitro -matured bovine oocytes (aged oocytes, aged-cooled oocytes, enucleated-aged oocytes and enucleated-aged-cooled oocytes (recipient cytoplasts)) were analyzed. These recipient cytoplasts exhibited a phosphorylation profile similar to that of activated oocytes. Maturation promoting factor (MPF) activity, which was high in young metaphase II oocytes, in aged oocytes, in enucleated-aged oocytes and in aged-cooled oocytes, dropped to the basal level in enucleated-aged-cooled oocytes (recipient cytoplasts), while mitogen-activated protein kinase (MAPK) activity remained elevated. The combination of enucleation, ageing and cooling following oocyte in vitro maturation resulted in an interphase-like stage cytoplasm having a phosphorylation profile and low MPF activity similar to activated oocytes, but exhibiting high MAPK activity.     

The role of brain-derived neurotrophic factor in mouse oocyte maturation in vitro involves activation of protein kinase B

Brain-derived neurotrophic factor (BDNF) can promote developmental competence in mammalian oocytes during in vitro maturation, but the signal transduction pathways are not clear. In this study, we investigated (using western blots) the effects of BDNF on the phosphorylation of protein kinase B (PKB) and mitogen-activated protein kinase (MAPK) in mouse oocytes and cumulus cells cultured in vitro. Treatment with BDNF enhanced phosphorylation of PKB in oocytes at 2 h (P = 0.0006) and 3 h (P < 0.0001) of in vitro maturation, compared with control oocytes. However, the pan-specific tyrosine kinase (Trk) inhibitor K252a together with BDNF completely inhibited phosphorylation of PKB in the oocytes. Furthermore, BDNF increased phosphorylation of MAPK in oocytes at 16 h of in vitro maturation (P = 0.0041), but K252a together with BDNF did not reduce phosphorylation of MAPK in the oocytes. For cumulus cells, BDNF significantly prolonged the phosphorylation of PKB and MAPK and increased the total amounts of PKB and MAPK proteins after 16 h of in vitro maturation. However, BDNF did not affect apoptosis of the cumulus cells during oocyte maturation in vitro. In conclusion, the PKB pathway is likely to be one signaling cascade activated by BDNF in combination with the TrkB receptor, whereas the MAPK pathway is not involved. These findings may have relevance for BDNF-induced promotion of developmental capacity of in vitro-matured oocytes.     

Oncogenic K-Ras and basic fibroblast growth factor prevent Fas-mediated apoptosis in fibroblasts through activation of mitogen-activated protein kinase

By an expression cloning method using Fas-transgenic Balb3T3 cells, we tried to obtain inhibitory genes against Fas-mediated apoptosis and identified proto-oncogene c-K-ras. Transient expression of K-Ras mutants revealed that oncogenic mutant K-Ras (RasV12) strongly inhibited, whereas dominant-inhibitory mutant K-Ras (RasN17) enhanced, Fas-mediated apoptosis by inhibiting Fas-triggered activation of caspases without affecting an expression level of Fas. Among the target molecules of Ras, including Raf (mitogen-activated protein kinase kinase kinase [MAPKKK]), phosphatidylinositol 3 (PI-3) kinase, and Ral guanine nucleotide exchange factor (RalGDS), only the constitutively active form of Raf (Raf-CAAX) could inhibit Fas-mediated apoptosis. In addition, the constitutively active form of MAPKK (SDSE-MAPKK) suppressed Fas-mediated apoptosis, and MKP-1, a phosphatase specific for classical MAPK, canceled the protective activity of oncogenic K-Ras (K-RasV12), Raf-CAAX, and SDSE-MAPKK. Furthermore, physiological activation of Ras by basic fibroblast growth factor (bFGF) protected Fas-transgenic Balb3T3 cells from Fas-mediated apoptosis. bFGF protection was also dependent on the activation of the MAPK pathway through Ras. All the results indicate that the activation of MAPK through Ras inhibits Fas-mediated apoptosis in Balb3T3 cells, which may play a role in oncogenesis.     

Blockade of the translocation and activation of mitogen-activated protein kinase kinase 4 (MKK4) signaling attenuates neuronal damage during later ischemia-reperfusion

Mitogen-activated protein kinase kinase 4 (MKK4), as an upstream activator of c-Jun NH(2)-terminal kinase (JNK), plays a critical role in response to cellular stresses and pro-inflammatory cytokines. In this study, we investigated the subcellular localization and activation of MKK4 in response to global cerebral ischemia. Our results indicated that MKK4 had two activation peaks in both the cytosol and the nucleus, and translocated from the cytosol to the nucleus at 30 min and 6 h of reperfusion. We also detected the interaction of JNK-interacting protein 3 (JIP3) and MKK4, which reached a maximum at 6 h of reperfusion. To elucidate the mechanism of translocation and activation, we administered N-acetylcysteine, an antioxidant reagent, and a glutamate receptor 6 C-terminus-containing peptide (Tat-GluR6-9c) to rats. The data showed that N-acetylcysteine limited the translocation and activation at 30 min of reperfusion; however, the peptide perturbed the subcellular localization and activation at 6 h of reperfusion, and subsequently provided a protective role against delayed neuronal cell death. Taken together, these results demonstrate that the translocation and activation of MKK4 during early reperfusion are closely associated with reactive oxygen species, whereas, at late reperfusion, MKK4 activation may be involved in brain ischemic injury.     

Mitogen-activated protein kinase (MAP kinase) activation in Xenopus oocytes: Roles of MPF and protein synthesis

Mitogen-activated protein kinase (MAP kinase) is a serine/threonine kinase whose enzymatic activity is thought to play a crucial role in mitogenic signal transduction and also in the progesterone-induced meiotic maturation of Xenopus oocytes. We have purified MAP kinase from Xenopus oocytes and have shown that the protein is present in metaphase ll oocytes under two different forms: an inactive 41-kD protein able to autoactivate and to autophosphorylate in vitro, and an active 42-kD kinase resolved into two tyrosine phosphorylated isoforms on 2D gels. During meiotic maturation, MAP kinase becomes tyrosine phosphorylated and activated following the activation of the M-phase promoting factor (MPF), a complex between the p34^cdc2 kinase and cyclin B. In vivo, MAP kinase activity displays a different stability in metaphase l and in metaphase II: protein synthesis is required to maintain MAP kinase activity in metaphase I but not in metaphase II oocytes. Injection of either MPF or cyclin B into prophase oocytes promotes tyrosine phosphorylation of MAP kinase, indicating that its activation is a downstream event of MPF activation. In contrast, injection of okadaic acid, which induces in vivo MPF activation, promotes only a very weak tyrosine phosphorylation of MAP kinase, suggesting that effectors other than MPF are required for the MAP kinase activation. Moreover, in the absence of protein synthesis, cyclin B and MPF are unable to promote in vivo activation of MAP kinase, indicating that this activation requires the synthesis of new protein(s). © 1993 Wiley-Liss, Inc.     

Dobesilate diminishes activation of the mitogen-activated protein kinase ERK1/2 in glioma cells

Fibroblast growth factors (FGFs) and their receptors, regularly expressed at high levels in gliomas, are further upregulated during the transition of the tumor from low- to high-grade malignancy, and are essential for glioma progression. FGFs induce upregulation of the mitogen-activated protein kinase (MAPK) signaling cascade in cultured glioma cells, which suggests that MAPK pathway participates in the FGF-dependent glioma development. Recently, it has been shown that dobesilate, an inhibitor of FGF mitogenic activity, shows antiproliferative and proapoptotic activities in glioma cell cultures. Accordingly, it should be expected this new synthetic FGF inhibitor to affect the activation levels of MAPK. Here we report that immunocytochemical and Western blot data unequivocally show that treatment of cell cultures with dobesilate causes a significant decrease of the intracellular levels of ERK1/2 activation, one of the components of the MAPK signalling cascade. This finding supports an important role for dobesilate in glioma growth, suggesting that dobesilate should be a treatment to be born in mind for glioma management.     

AMP-activated protein kinase is involved in hormone-induced mouse oocyte meiotic maturation in vitro

We have previously shown that AMP-activated protein kinase (AMPK) can induce the resumption of meiosis in mouse oocytes maintained in meiotic arrest in vitro. The present study was carried out to determine whether AMPK activation is involved in hormone-induced maturation. Follicle-stimulating hormone (FSH) and the EGF-like peptide, amphiregulin (AR), are potent inducers of maturation in cumulus cell-enclosed oocytes (CEO). Within 3 h of FSH treatment, phospho-acetyl CoA carboxylase (ACC) levels were increased in germinal vesicle (GV)-stage oocytes when compared to non-stimulated controls and remained elevated throughout 9 h of culture, indicating AMPK activation. A similar response to AR was observed after 6 h of culture. Using anti-PT172 antibody (binds only to activated AMPK), Western analysis demonstrated active AMPK in both FSH- or AR-treated GV-stage oocytes within 6 h. The AMPK inhibitors, compound C and adenine 9-beta-d-arabinofuranoside (araA), blocked FSH- or AR-induced meiotic resumption and ACC phosphorylation, further supporting a causal role for AMPK in hormone-induced meiotic resumption. Immunocytochemistry using anti-PT172-AMPK antibody showed an increased diffuse cytoplasmic staining and more intense punctate staining in the germinal vesicles of oocytes following treatment with the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR) or with FSH or AR, and this staining was eliminated by compound C or a blocking peptide for the anti-PT172 antibody. Staining of oocytes from hCG-stimulated mice with the anti-PT172 antibody also showed pronounced label in the germinal vesicles within 1-2 h. Furthermore, in oocytes from all groups, active AMPK was always observed in association with the condensed chromosomes of maturing oocytes. Taken together, these results support a role for AMPK in FSH and AR-induced maturation in vitro and hCG-induced maturation in vivo.