RNA干扰技术抑制Polo-like激酶1表达对A549细胞的影响

Polo-like激酶1(Plk1)是参与细胞周期调控的重要分子,已在多种肿瘤中检测到Plk1的高表达,并发现与肿瘤细胞的增殖和预后密切关联.为明确Plk1在肺癌细胞系A549细胞增殖和周期运行中的作用,采用RNA干扰技术,构建能产生siRNA的质粒载体psiRNA-hH1-Plk1并导入A549细胞中.采用RT-PCR检测Plk1mRNA表达的变化,Western印迹检测Plk1、细胞周期蛋白B1、p53蛋白的表达变化,流式细胞术分析细胞周期变化和凋亡;免疫荧光染色检测α微管蛋白的表达.以此观察RNA干扰能否有效抑制Plk1的表达水平,以及抑制后对A549细胞生长的影响.结果表明,psiRNA-hH1-Plk1质粒能特异性地抑制Plk1基因的表达并使其活性下降,细胞周期蛋白B1及p53蛋白的表达水平升高,微管聚集障碍或形成单极的纺锤体,A549细胞增殖减慢,出现G2/M期阻滞并存在细胞凋亡.针对Plk1基因的RNA干扰有望用于肿瘤的基因治疗.     

Polo样激酶1在细胞周期及细胞周期监测点中的功能

Plk1（Polo-like kinase 1）是一类从酵母到人类都高度保守的丝氨酸/苏氨酸蛋白激酶,是真核细胞有丝分裂的重要调控因子.Plk1随有丝分裂进程定位于不同位点,调节分裂期进入、纺锤体形成和胞质分裂等过程.Plk1能够与磷酸化的停靠蛋白结合,从而在不同空间被激活以满足其在细胞周期中的不同功能.Plk1还参与G2和M期DNA损伤监测点的调节,对于DNA损伤恢复后重新进入有丝分裂期是必须的.目前,Plk1的重要功能尤其是在DNA损伤监测点中发挥的重要功能正在被广泛研究.Plk1在多种恶性肿瘤中存在过表达且与肿瘤发生密切相关,对于Plk1功能的深入研究为以Plk1为靶的肿瘤治疗提供理论依据     

氯化锂抑制A549细胞增殖及其对Polo-like激酶1转录活性的影响

利用糖原合成酶激酶3的抑制剂氯化锂作用于A549细胞,观察细胞形态与增殖的改变及其对Polo－like激酶1转录活性的影响.采用细胞计数检测细胞增殖,流式细胞术分析细胞周期变化;Western印迹检测磷酸化GSK3以及细胞周期相关蛋白p53、cyclin B1和Plk1的表达变化;RT-PCR检测Plk1 mRNA的表达;荧光素酶报告基因分析氯化锂对Plk1启动子活性的影响.结果显示,5 mmol/L氯化锂作用48 h后,A549细胞即发生明显的形态学改变,细胞增殖减慢并发生G2/M期阻滞;Plk1 mRNA和蛋白表达均升高,p53蛋白表达增强,而cyclin B1的蛋白表达无明显变化.氯化锂作用24 h后,可见pGL2-Plk1转染组中荧光素酶活性增高（与对照质粒相比,P<0.05）,48 h后更明显.以上结果表明, 氯化锂减慢A549细胞增殖,导致G2/M期阻滞,并能增强Plk1的启动子活性,促进Plk1的表达.     

抑制Plk1表达对食管癌细胞化疗敏感性的影响

研究食管癌细胞中Plk1表达被抑制后对化疗药物敏感性的影响,为进一步研究开发基于Plk1食管癌分子靶向治疗奠定基础。化学合成法合成特异性的短链Plk1siRNA,脂质体转染法将短链siRNA分别导入3代表性的食管癌细胞系;采用RT-PCR和蛋白质印迹法确认siRNA对Plk1表达的抑制效果;MTT分析观察抑制Plk1表达对食管癌细胞化疗药物敏感性的影响。半定量RT-PCR和蛋白质印迹分析结果表明,合成的特异性短链Plk1siRNA在mRNA和蛋白质水平上均能高效、特异性地抑制所使用的3代表性食管癌细胞系中Plk1的表达,抑制程度约90%。MTT分析结果表明,siRNA抑制Plk1表达后,能显著提高3食管癌细胞系对化疗药物阿霉素和顺铂的敏感性。由此证实,siRNA介导的Plk1表达抑制能显著提高食管癌细胞对化疗药物阿霉素和顺铂的敏感性。     

抗siRNA的GFP-Plk1同义突变表达载体及其稳定细胞系的构建

目的:构建GFP-Plk1同义突变表达载体及其稳定转染细胞系。方法:设计Polo样激酶1(Plk1)si RNA序列及相对应的同义突变引物,并利用二次PCR方法扩增Plk1基因,定向克隆到p Rex-EGFP-IRES-Hygro载体中,构建p Rex-EGFP-r Plk1-IRES-Hygro表达载体;利用逆转录病毒感染的方法,构建He La/GFP-r Plk1稳定细胞系;利用免疫印迹及激光共聚焦显微镜,验证Plk1 si RNA的干扰效果及稳定细胞系的构建。结果:双酶切鉴定和测序结果表明构建的p Rex-EGFP-r Plk1-IRES-Hygro正确;免疫印迹实验证明Plk1 si RNA序列可以有效抑制He La/GFP-r Plk1细胞中内源性Plk1蛋白的表达,但不能干扰掉外源GFP-r Plk1蛋白;在荧光共聚焦显微镜下,观察到有丝分裂的前中期和末期,GFP-r Plk1分别定位于着丝粒和中间体上。结论:构建了Plk1同义突变表达载体p Rex-EGFP-r Plk1-IRES-Hygro和He La/GFP-r Plk1稳定细胞系,为下一步研究Plk1在有丝分裂期的调控机制提供了模型。     

乳腺癌中丝/ 苏氨酸蛋白激酶Plk1 mRNA 的表达及其预后价值

目的:检测乳腺癌细胞和组织中丝/苏氨酸蛋白激酶Plk1基因mRNA的表达情况并分析其预后价值。方法:应用半定量RT-PCR方法分析3株人乳腺癌细胞和1株正常乳腺上皮细胞中Plk1基因mRNA的表达水平。同时分析84例乳腺癌及对应的癌旁正常乳腺上皮组织中Plk1 mRNA的表达水平。统计学分析Plk1 mRNA表达水平与乳腺癌患者年龄、肿瘤大小、组织分化程度、淋巴结转移状况、TNM分期和雌激素受体(ER)等临床病理参数之间的关系,以及与预后之间的关系。结果:Plk1基因mRNA在乳腺癌细胞中的相对表达水平显著高于其在正常乳腺上皮细胞中的相对表达水平(P值均小于<0.05)。另外,Plk1 mRNA在乳腺癌组织中平均表达水平(0.88±0.18)显著高于其在癌旁正常乳腺上皮组织中平均表达水平(0.22±0.10;P<0.01)。统计学分析结果表明:Plk1 mRNA表达水平和乳腺癌患者的淋巴结转移状况及TNM分期密切相关(P=0.009或0.007)。Kaplan-Meier生存曲线分析结果表明:高Plk1 mRNA表达水平的乳腺癌患者的5年无疾病进展率及总体生存率均显著低于低Plk1 mRNA表达水平的乳腺癌患者(P=0.0026及0.0136)。COX模型的多因素预后分析结果表明:Plk1基因mRNA表达水平是乳腺癌患者的一个独立的预后因素(HR=4.764,95%CI:1.341~6.123,P=0.0025)。结论:Plk1在乳腺癌组织呈现高表达水平,其mRNA表达水平有望成为临床乳腺癌患者一个重要的预后判断分子指标。     

乳腺癌中丝／苏氨酸蛋白激酶Plk1mRNA的表达及其预后价值

目的：检测乳腺癌细胞和组织中丝／苏氨酸蛋白激酶Plk1基因mRNA的表达情况并分析其预后价值。方法：应用半定量RT-PCR方法分析3株人乳腺癌细胞和1株正常乳腺上皮细胞中Plk1基因mRNA的表达水平。同时分析84例乳腺癌及对应的癌旁正常乳腺上皮组织中Plk1mRNA的表达水平。统计学分析Plk1mRNA表达水平与乳腺癌患者年龄、肿瘤大小、组织分化程度、淋巴结转移状况、TNM分期和雌激素受体（ER）等临床病理参数之间的关系,以及与预后之间的关系。结果：Plk1基因mRNA在乳腺癌细胞中的相对表达水平显著高于其在正常乳腺上皮细胞中的相对表达水平（P值均小于〈0．05）。另外,Plk1mRNA在乳腺癌组织中平均表达水平（0．88±0．18）显著高于其在癌旁正常乳腺上皮组织中平均表达水平（0．22±0．10;P〈0．01）。统计学分析结果袁明：Plk1mRNA表达水平和乳腺癌患者的淋巴结转移状况及TNM分期密切相关（P=0．009或0．007）。Kaplan—Meier生存曲线分析结果表明：高Plk1mRNA表达水平的乳腺癌患者的5年无疾病进展率及总体生存率均显著低于低Plk1mRNA表达水平的乳腺癌患者（P=0．0026及0．0136）。COX模型的多因素预后分析结果表明：Plk1基因mRNA表达水平是乳腺癌患者的一个独立的预后因素（HR=4．764,95％CI：1．341-6．123,P=0．0025）。结论：Plk1在乳腺癌组织呈现高表达水平,其mRNA表达水平有望成为临床乳腺癌患者一个重要的预后判断分子指标。     

人Polo样激酶1基因的克隆表达及其影响c-Abl表达水平功能的初探

目的:克隆并在293细胞中表达人Polo样激酶1(Plk1)基因,探索Plk1对非受体型酪氨酸激酶c-Abl表达水平的影响。方法:利用PCR法扩增Plk1基因,分别定向克隆至pcDNA3-Flag及pCMV-Myc真核表达载体,将上述质粒分别转染293细胞进行瞬时表达,Western印迹检测Plk1蛋白的表达;将上述质粒分别与c-Abl表达质粒共转293细胞,检测带有不同标签的Flag-Plk1或Myc-Plk1对细胞中c-Abl激酶表达的影响。结果:构建了Flag-Plk1和Myc-Plk1真核表达质粒,其在293细胞中均可表达,蛋白的相对分子质量为68×103;与其共转的c-Abl激酶表达水平显著下调。结论:在293细胞中表达了Flag-Plk1和Myc-Plk1蛋白,且初步发现Plk1可以抑制c-Abl的表达。     

保罗样激酶1保罗盒结构域: 肿瘤治疗的新靶标

保罗样激酶1（Polo-like kinase 1, Plk1）与恶性肿瘤的发生与发展密切相关,被认为是肿瘤分子靶向治疗中最具潜力的重要靶标之一。目前,针对Plk1激酶结构域（Kinase domain, KD）设计Plk1抑制剂已成为研究热点,其中部分小分子抑制剂已进入Ⅰ/Ⅱ期临床研究并展现出良好的抗癌前景。尽管Plk1 KD结构域抑制剂具有一定的靶标选择性,但鉴于作为ATP结合口袋的KD结构域在众多激酶结构中的高度保守性和易导致交叉耐药等问题,这使开发高选择性的Plk1 KD结构域抑制剂面临极大的挑战。保罗盒结构域（Polo-Box domain, PBD）作为Plk1特有的底物结合域,在调控Plk1的亚细胞定位中具有重要功能,被认为是未来高选择性Plk1抑制剂开发的理想靶标。文中对Plk1 PBD的分子结构、生物学功能和相关抑制剂的研究进展进行了综述和展望,以期为靶向Plk1 PBD结构域抑制剂的分子设计提供有益的借鉴和参考。     

人源Plk1及其结构域蛋白的融合表达及纯化

目的:表达纯化GST-Plk1及GST-Plk1 KD、GST-Plk1 PBD融合蛋白,以用于Plk1与其结合蛋白相互作用的研究。方法:PCR扩增Plk1全长及其KD、PBD结构域基因,定向克隆至p GEX-4T-1原核表达载体中,在大肠杆菌中分别表达其融合蛋白,并利用GSH交联的琼脂糖珠纯化。结果:人源Plk1及其结构域基因被克隆至p GEX-4T-1载体中;通过亲和纯化获得带有GST标签的Plk1及其结构域的融合蛋白。结论:构建了GST-Plk1、GST-Plk1 KD、GST-Plk1 PBD表达质粒并表达了相应的融合蛋白,为研究Plk1体外相互作用蛋白提供了基础。     

Combination of chemical genetics and phosphoproteomics for kinase signaling analysis enables confident identification of cellular downstream targets

Delineation of phosphorylation-based signaling networks requires reliable data about the underlying cellular kinase-substrate interactions. We report a chemical genetics and quantitative phosphoproteomics approach that encompasses cellular kinase activation in combination with comparative replicate mass spectrometry analyses of cells expressing either inhibitor-sensitive or resistant kinase variant. We applied this workflow to Plk1 (Polo-like kinase 1) in mitotic cells and induced cellular Plk1 activity by wash-out of the bulky kinase inhibitor 3-MB-PP1, which targets a mutant kinase version with an enlarged catalytic pocket while not interfering with wild-type Plk1. We quantified more than 20,000 distinct phosphorylation sites by SILAC, approximately half of which were measured in at least two independent experiments in cells expressing mutant and wild-type Plk1. Based on replicate phosphorylation site quantifications in both mutant and wild-type Plk1 cells, our chemical genetic proteomics concept enabled stringent comparative statistics by significance analysis of microarrays, which unveiled more than 350 cellular downstream targets of Plk1 validated by full concordance of both statistical and experimental data. Our data point to hitherto poorly characterized aspects in Plk1-controlled mitotic progression and provide a largely extended resource for functional studies. We anticipate the described strategies to be of general utility for systematic and confident identification of cellular protein kinase substrates.     

Enabling and disabling polo-like kinase 1 inhibition through chemical genetics

Polo-like kinase 1 (Plk1) is a core regulator of cell division and an emerging target for cancer therapy. Pharmacologic inhibitors of Plk1 exist but affect other kinases, complicating their in vivo validation. To address this, we examined effects of two structurally unrelated Plk1 inhibitors (BI-2536 and TAL) against isogenic human cell lines that solely express wildtype (wt) or analogue-sensitive (as) Plk1 alleles. Unexpectedly, Plk1(as) cells displayed profound biochemical and functional resistance to both inhibitors. Cells that co-express Plk1(wt) and Plk1(as) exhibit loss-of-function phenotypes only when both kinase alleles are inhibited. Resistance to BI-2536 is linked to an intragenic suppressor mutation (C67V) that restores an otherwise invariant valine to the kinase active site. Structural modeling demonstrates that this mutation not only enables Plk1(as) to function in vivo but also occludes BI-2536 from the ATP-binding pocket. Our results reveal the molecular basis of Plk inhibitor selectivity and a potential mechanism for tumor cell resistance.     

Heterologous expression of mammalian Plk1 in Drosophila reveals divergence from Polo during late mitosis

Drosophila Polo kinase is the founder member of a conserved kinase family required for multiple stages of mitosis. We assessed the ability of mouse Polo-like kinase 1 (Plk1) to perform the multiple mitotic functions of Polo kinase, by expressing a Plk1-GFP fusion in Drosophila. Consistent with the previously reported localization of Polo kinase, Plk1-GFP was strongly localized to centrosomes and recruited to the centromeric regions of condensing chromosomes during early mitosis. However, in contrast to a functional Polo-GFP fusion, Plk1-GFP failed to localize to the central spindle midzone in both syncytial embryo mitosis and the conventional mitoses of cellularized embryos and S2 cells. Moreover, unlike endogenous Polo kinase and Polo-GFP, Plk1-GFP failed to associate with the contractile ring. Expression of Plk1-GFP enhanced the lethality of hypomorphic polo mutants and disrupted the organization of the actinomyosin cytoskeleton in a dominant-negative manner. Taken together, our results suggest that endogenous Polo kinase has specific roles in regulating actinomyosin rearrangements during Drosophila mitoses that its mammalian counterpart, Plk1, cannot fulfill. Consistent with this hypothesis, we observed defects in the cortical recruitment of myosin and myosin regulatory light chain in Polo deficient cells.     

Genetic depletion of Polo‐like kinase 1 leads to embryonic lethality due to mitotic aberrancies

Polo‐like kinase 1 (PLK1) is a serine/threonine kinase that plays multiple and essential roles during the cell division cycle. Its inhibition in cultured cells leads to severe mitotic aberrancies and cell death. Whereas previous reports suggested that Plk1 depletion in mice leads to a non‐mitotic arrest in early embryos, we show here that the bi‐allelic Plk1 depletion in mice certainly results in embryonic lethality due to extensive mitotic aberrations at the morula stage, including multi‐ and mono‐polar spindles, impaired chromosome segregation and cytokinesis failure. In addition, the conditional depletion of Plk1 during mid‐gestation leads also to severe mitotic aberrancies. Our data also confirms that Plk1 is completely dispensable for mitotic entry in vivo. On the other hand, Plk1 haploinsufficient mice are viable, and Plk1‐heterozygous fibroblasts do not harbor any cell cycle alterations. Plk1 is overexpressed in many human tumors, suggesting a therapeutic benefit of inhibiting Plk1, and specific small‐molecule inhibitors for this kinase are now being evaluated in clinical trials. Therefore, the different Plk1 mouse models here presented are a valuable tool to reexamine the relevance of the mitotic kinase Plk1 during mammalian development and animal physiology.     

The Functional Significance of Posttranslational Modifications on Polo-Like Kinase 1 Revealed by Chemical Genetic Complementation

Mitosis is coordinated by carefully controlled phosphorylation and ubiquitin-mediated proteolysis. Polo-like kinase 1 (Plk1) plays a central role in regulating mitosis and cytokinesis by phosphorylating target proteins. Yet, Plk1 is itself a target for posttranslational modification by phosphorylation and ubiquitination. We developed a chemical-genetic complementation assay to evaluate the functional significance of 34 posttranslational modifications (PTMs) on human Plk1. To do this, we used human cells that solely express a modified analog-sensitive Plk1 (Plk1^AS) and complemented with wildtype Plk1. The wildtype Plk1 provides cells with a functional Plk1 allele in the presence of 3-MB-PP1, a bulky ATP-analog inhibitor that specifically inhibits Plk1^AS. Using this approach, we evaluated the ability of 34 singly non-modifiable Plk1 mutants to complement Plk1^AS in the presence of 3-MB-PP1. Mutation of the T-loop activating residue T210 and adjacent T214 are lethal, but surprisingly individual mutation of the remaining 32 posttranslational modification sites did not disrupt the essential functions of Plk1. To evaluate redundancy, we simultaneously mutated all phosphorylation sites in the kinase domain except for T210 and T214 or all sites in the C-terminal polo-box domain (PBD). We discovered that redundant phosphorylation events within the kinase domain are required for accurate chromosome segregation in anaphase but those in the PBD are dispensable. We conclude that PTMs within the T-loop of Plk1 are essential and nonredundant, additional modifications in the kinase domain provide redundant control of Plk1 function, and those in the PBD are dispensable for essential mitotic functions of Plk1. This comprehensive evaluation of Plk1 modifications demonstrates that although phosphorylation and ubiquitination are important for mitotic progression, many individual PTMs detected in human tissue may have redundant, subtle, or dispensable roles in gene function.     

Distinct regulators for Plk1 activation in starfish meiotic and early embryonic cycles

The Polo-like kinase, Plk, has multiple roles in regulating mitosis. In particular, Plk1 has been postulated to function as a trigger kinase that phosphorylates and activates Cdc25C prior to the activation of cyclin B-Cdc2 and thereby initiates its activation. However, the upstream regulation of Plk1 activation remains unclear. Here we have studied the interplay between Plk1 and Cdc2 through meiotic and early embryonic cycles in starfish. Distinct kinases, cyclin B-Cdc2, MAPK along with cyclin B- and/or cyclin A-Cdc2 and cyclin A-Cdc2, were unique upstream regulators for Plk1 activation at meiosis I, meiosis II and embryonic M-phase, respectively, indicating that Plk1 is not the trigger kinase at meiotic reinitiation. When Plk1 was required for cyclin B-Cdc2 activation, the action of Plk1 was mediated primarily through suppression of Myt1 rather than through activation of Cdc25. We propose that Plk1 can be activated by either cyclin A- or cyclin B-Cdc2, and its primary target is Myt1.     

Mechanisms Underlying Plk1 Polo-Box Domain-Mediated Biological Processes and Their Physiological Significance

Mammalian polo-like kinase 1 (Plk1) has been studied intensively as a key regulator of various cell cycle events that are critical for proper M-phase progression. The polo-box domain (PBD) present in Plk1’s C-terminal non-catalytic region has been shown to play a central role in targeting the N-terminal kinase domain of Plk1 to specific subcellular locations. Subsequent studies reveal that PBD binds to a phosphorylated motif generated by one of the two mechanisms - self-priming by Plk1 itself or non-self-priming by a Pro-directed kinase, such as Cdc2. Here, we comparatively review the differences in the biochemical steps of these mechanisms and discuss their physiological significance. Considering the diverse functions of Plk1 during the cell cycle, a better understanding of how the catalytic activity of Plk1 functions in concert with its cis-acting PBD and how this coordinated process is intricately regulated to promote Plk1 functions will be important for providing new insights into different mechanisms underlying various Plk1-mediated biological events that occur at the multiple stages of the cell cycle.     

Proteomic screen defines the Polo-box domain interactome and identifies Rock2 as a Plk1 substrate

Polo-like kinase-1 (Plk1) phosphorylates a number of mitotic substrates, but the diversity of Plk1-dependent processes suggests the existence of additional targets. Plk1 contains a specialized phosphoserine-threonine binding domain, the Polo-box domain (PBD), postulated to target the kinase to its substrates. Using the specialized PBD of Plk1 as an affinity capture agent, we performed a screen to define the mitotic Plk1-PBD interactome by mass spectrometry. We identified 622 proteins that showed phosphorylation-dependent mitosis-specific interactions, including proteins involved in well-established Plk1-regulated processes, and in processes not previously linked to Plk1 such as translational control, RNA processing, and vesicle transport. Many proteins identified in our screen play important roles in cytokinesis, where, in mammalian cells, the detailed mechanistic role of Plk1 remains poorly defined. We go on to characterize the mitosis-specific interaction of the Plk1-PBD with the cytokinesis effector kinase Rho-associated coiled-coil domain-containing protein kinase 2 (Rock2), demonstrate that Rock2 is a Plk1 substrate, and show that Rock2 colocalizes with Plk1 during cytokinesis. Finally, we show that Plk1 and RhoA function together to maximally enhance Rock2 kinase activity in vitro and within cells, and implicate Plk1 as a central regulator of multiple pathways that synergistically converge to regulate actomyosin ring contraction during cleavage furrow ingression.     

Mitotic entry: The interplay between Cdk1, Plk1 and Bora

Polo-like kinase 1 (Plk1) is an important mitotic kinase that is crucial for entry into mitosis after recovery from DNA damage-induced cell cycle arrest. Plk1 activation is promoted by the conserved protein Bora (SPAT-1 in C. elegans), which stimulates the phosphorylation of a conserved residue in the activation loop by the Aurora A kinase. In a recent article published in Cell Reports, we show that the master mitotic kinase Cdk1 contributes to Plk1 activation through SPAT-1/Bora phosphorylation. We identified 3 conserved Sp/Tp residues that are located in the N-terminal, most conserved part, of SPAT-1/Bora. Phosphorylation of these sites by Cdk1 is essential for Plk1 function in mitotic entry in C. elegans embryos and during DNA damage checkpoint recovery in mammalian cells. Here, using an untargeted Förster Resonance Energy Transfer (FRET) biosensor to monitor Plk1 activation, we provide additional experimental evidence supporting the importance of these phosphorylation sites for Plk1 activation and subsequent mitotic entry after DNA damage. We also briefly discuss the mechanism of Plk1 activation and the potential role of Bora phosphorylation by Cdk1 in this process. As Plk1 is overexpressed in cancer cells and this correlates with poor prognosis, understanding how Bora contributes to Plk1 activation is paramount for the development of innovative therapeutical approaches.