PLK激酶家族新成员PLK5的克
发布时间：2018-07-18 17:05
【摘要】： Polo-like kinase(PLK)家族是一类高度保守的丝氨酸／苏氨酸蛋白激酶，在细胞周期调控中发挥至关重要的作用。目前已在哺乳类细胞中鉴定出4个PLK，分别是：Polo-like kinase 1(PLK1)，Polo-like kinase 2／SNK(serum-inducible kinase)，Polo-like kinase 3／FNK／PRK(fibroblast-growth-factor-inducible kina或proliferation related kinase)及Polo-like kinase 4／SAK(SNK akin kinase)。PLK1及其同源蛋白在细胞周期的不同时相中，都有十分重要的作用：如在G2晚期／M初期参与激活Cyclin B／CDK1复合体，使细胞进入M期，协助中心体的功能成熟以及双极纺锤体的形成，活化细胞分裂后期促进复合体(anaphase-promoting complex，APC)，促进染色体正常分离，分配；决定细胞能否按期退出M期及调节而进入胞质分裂等事件。PLK2在G1期中表达最高，可能参与调控细胞进入S期。PLK3主要在S期和G2期表达，可能参与DNA损伤检查点信号通路和细胞进入S期。而PLK4则主要与有丝分裂的退出有关。同时，PLK家族与肿瘤的转移及预后密切相关，，是一个很有前景的抗癌药物靶点，其小分子化合物抑制剂已经进入临床Ⅰ期实验。人们在研究以上四个PLK家族成员的同时，也一直在关注该激酶家族中是否还存在其它新成员，及其在细胞周期调控中的作用。然而自1997年以来，再也没有新的PLK基因被克隆的报道，在最新的人类蛋白激酶图谱中也没有新的PLK记录。 我们综合应用生物信息学和RT-PCR等方法，在小鼠中克隆了一种未知基因，其N端具有一个与PLK家族催化结构域高度同源的激酶结构域(Kinase domain)，C端则有PLK家族特征性Polo-box结构域(polo-box domain，PBD)，序列同源性分析显示该基因所编码的蛋白与PLK1的相似性为53％，同源性为33％，这比PLK4与PLK1的相似性(32％)和同源性(16％)还高。因而，我们初步将其归为PLK激酶家族的新成员，并命名为Mouse Polo-like kinase 5(mPLK5，mammalian PLK5)。我们通过生物信息学分析，RT-PCR、原位杂交、细胞免疫组织化学实验以及细胞转基因实验等分子细胞生物学方法和现代成像技术，对mPLK5的一些基本生化特征，组织表达、分布，亚细胞定位以及功能进行了初步的研究。我们期望通过对这个新基因的深入研究，进一步明确PLK家族在细胞周期调控中所发挥的作用，并为细胞周期调控异常的疾病(如肿瘤)提供新的治疗靶点。 结果：1、生物信息学初步分析提示：小鼠PLK5 cDNA为2089 bp，定位于第10号染色体上。它含有两个保守的结构域：其N端具有一个与PLK家族催化结构域高度同源的激酶结构域——丝氨酸／苏氨酸激酶结构域，C端则有PLK家族特征性Polo-box结构域。序列同源性分析显示该基因所编码的蛋白与PLK1的相似性为53％，同源性为33％。2、RT-PCR结果显示：mPLK5的分布具有组织特异性，主要在小鼠的大脑、小脑、脑干、眼睛、肝脏、肾脏和睾丸等组织表达，而小鼠的心脏、大肠、脾脏、肌肉等组织中未见表达。3、原位杂交结果表明：mPLK5在小鼠脑部各区域广泛分布，其中大脑皮层、海马等区域表达丰度较高。4、mPLK5在小鼠发育过程中脑部分布的区域略有改变。在胎鼠时期，mPLK5主要在脑部的脑干、小脑皮层和延髓表达，而在大脑皮层和海马等区域未见表达；新生鼠、幼鼠、成年鼠和老年鼠在大脑皮层、海马、小脑皮层、脑干和延髓均有稳定表达。5、细胞免疫组织化学研究提示，外源性表达的mPLK5(pGFP-mPLK5)主要定位于细胞核，mPLK5的Polo-box结构域的关键保守氨基酸位的点突变(pGFP-mPLK5 W417F)不改变mPLK5的亚细胞定位。7、初步细胞转基因实验发现与转染pGFP-C1空载体相比，过表达pGFP-mPLK5的细胞增殖受到明显的抑制。 结论：我们首次成功克隆了哺乳类PLK家族新成员mPLK5，其与PLK家族中的其他成员在组织分布和可能的作用等方面具有显著的差别，如mPLK5分布具有组织特异性，主要在神经系统、肾脏和睾丸等组织表达；初步细胞转基因实验提示mPLK5与其它PLKs促进细胞增殖作用不同，可能具有抑制细胞增殖 的作用。
[Abstract]:Polo - like kinase ( PLK ) family is a kind of highly conserved serine / threonine protein kinase , which plays a crucial role in cell cycle regulation . Four PLK have been identified in mammalian cells : Polo - like kinase 1 ( PLK1 ) , Polo - like kinase 2 / SNK ( serum - inducible kinase ) , Polo - like kinase 3 / FNK / keratectomy ( fibroblast - like kinase ) and Polo - like kinase 4 / SAK ( SNK akin kinase ) . PLK1 and its homologous proteins play an important role in the cell cycle , such as the activation of the Cyclin B / CDK1 complex at the early stage of G2 / M , the cell entering M phase , the function maturation of the central body and the formation of the bipolar spindle , activating the complex ( APC ) at the later stage of the cell division , promoting the normal separation and distribution of the chromosomes ;
PLK3 has the highest expression in G1 phase and may be involved in the regulation of cell entry S phase . PLK3 is mainly involved in S phase and G2 phase . PLK4 may be involved in DNA damage checkpoint signaling pathway and cell entry S phase . PLK4 is a promising anticancer drug target , and its role in cell cycle regulation has been studied . However , no new PLK gene has been cloned since 1997 , and no new PLK record has been reported in the latest human protein kinase map .
We used bioinformatics and RT - PCR to clone an unknown gene in mice . The N - terminus has a kinase domain highly homologous to the PLK family catalytic domain , and the C - terminal has a PLK family characteristic Polo - box domain ( PBD ) . The homology of the protein encoded by the gene with PLK1 is 53 % , and the homology is 33 % , which is higher than that of PLK4 and PLK1 ( 32 % ) and homology ( 16 % ) . Therefore , we initially classified them as a new member of PLK kinase family and named Mouse Polo - like kinase 5 ( mPL5 ) . We hope to further clarify the role of PLK family in cell cycle regulation by means of bioinformatics analysis , RT - PCR , in situ hybridization , cellular immune histochemistry experiment and cell transgenic experiment and so on . We expect to further clarify the role of PLK family in cell cycle regulation and provide a new treatment target for diseases such as tumors .
Results : 1 . Preliminary analysis of biological information showed that the cDNA of PLK5 was 2089 bp , located on chromosome 10 . It contained two conserved domains : its N - terminal has a kinase domain _ serine / threonine kinase domain which is highly homologous to the catalytic domain of PLK family . The homology is 33 % . 2 . The results of RT - PCR show that the expression of the gene is higher in the brain , cerebellum , brain stem , eye , liver , kidney and testis .
The expression of pGFP - mPLK562 was mainly localized in the nucleus , and the point mutation of the key conserved amino acid position of the Polo - box domain ( pGFP - mPL5W417F ) was significantly inhibited compared with that of pGFP - C1 empty vector transfected with pGFP - C1 empty vector .
Conclusion : The new member of PLK family of mammals has been successfully cloned , which is different from other members of PLK family in the aspects of tissue distribution and possible roles , such as mPL5 distribution with tissue specificity , which is mainly expressed in nervous system , kidney and testis .
The results suggest that mPL5 can inhibit the proliferation of cells and inhibit the proliferation of cells .
and the like .
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346
本文编号：2132572
[Abstract]:Polo - like kinase ( PLK ) family is a kind of highly conserved serine / threonine protein kinase , which plays a crucial role in cell cycle regulation . Four PLK have been identified in mammalian cells : Polo - like kinase 1 ( PLK1 ) , Polo - like kinase 2 / SNK ( serum - inducible kinase ) , Polo - like kinase 3 / FNK / keratectomy ( fibroblast - like kinase ) and Polo - like kinase 4 / SAK ( SNK akin kinase ) . PLK1 and its homologous proteins play an important role in the cell cycle , such as the activation of the Cyclin B / CDK1 complex at the early stage of G2 / M , the cell entering M phase , the function maturation of the central body and the formation of the bipolar spindle , activating the complex ( APC ) at the later stage of the cell division , promoting the normal separation and distribution of the chromosomes ;
PLK3 has the highest expression in G1 phase and may be involved in the regulation of cell entry S phase . PLK3 is mainly involved in S phase and G2 phase . PLK4 may be involved in DNA damage checkpoint signaling pathway and cell entry S phase . PLK4 is a promising anticancer drug target , and its role in cell cycle regulation has been studied . However , no new PLK gene has been cloned since 1997 , and no new PLK record has been reported in the latest human protein kinase map .
We used bioinformatics and RT - PCR to clone an unknown gene in mice . The N - terminus has a kinase domain highly homologous to the PLK family catalytic domain , and the C - terminal has a PLK family characteristic Polo - box domain ( PBD ) . The homology of the protein encoded by the gene with PLK1 is 53 % , and the homology is 33 % , which is higher than that of PLK4 and PLK1 ( 32 % ) and homology ( 16 % ) . Therefore , we initially classified them as a new member of PLK kinase family and named Mouse Polo - like kinase 5 ( mPL5 ) . We hope to further clarify the role of PLK family in cell cycle regulation by means of bioinformatics analysis , RT - PCR , in situ hybridization , cellular immune histochemistry experiment and cell transgenic experiment and so on . We expect to further clarify the role of PLK family in cell cycle regulation and provide a new treatment target for diseases such as tumors .
Results : 1 . Preliminary analysis of biological information showed that the cDNA of PLK5 was 2089 bp , located on chromosome 10 . It contained two conserved domains : its N - terminal has a kinase domain _ serine / threonine kinase domain which is highly homologous to the catalytic domain of PLK family . The homology is 33 % . 2 . The results of RT - PCR show that the expression of the gene is higher in the brain , cerebellum , brain stem , eye , liver , kidney and testis .
The expression of pGFP - mPLK562 was mainly localized in the nucleus , and the point mutation of the key conserved amino acid position of the Polo - box domain ( pGFP - mPL5W417F ) was significantly inhibited compared with that of pGFP - C1 empty vector transfected with pGFP - C1 empty vector .
Conclusion : The new member of PLK family of mammals has been successfully cloned , which is different from other members of PLK family in the aspects of tissue distribution and possible roles , such as mPL5 distribution with tissue specificity , which is mainly expressed in nervous system , kidney and testis .
The results suggest that mPL5 can inhibit the proliferation of cells and inhibit the proliferation of cells .
and the like .
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R346
【参考文献】
相关期刊论文 前1条
1 林莉,王升启,管伟,杨秉呼,胡小电;Plk1基因为靶的反义寡核苷酸对HepG2肝癌细胞的体内外抗肿瘤作用[J];癌症;2001年11期
本文编号：2132572
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