Furry Protein Promotes Aurora A-mediated Polo-like Kinase 1 Activation

Bipolar mitotic spindle organization is fundamental to faithful chromosome segregation. Furry (Fry) is an evolutionarily conserved protein implicated in cell division and morphology. In human cells, Fry localizes to centrosomes and spindle microtubules in early mitosis, and depletion of Fry causes multipolar spindle formation. However, it remains unknown how Fry controls bipolar spindle organization. This study demonstrates that Fry binds to polo-like kinase 1 (Plk1) through the polo-box domain of Plk1 in a manner dependent on the cyclin-dependent kinase 1-mediated Fry phosphorylation at Thr-2516. Fry also binds to Aurora A and promotes Plk1 activity by binding to the polo-box domain of Plk1 and by facilitating Aurora A-mediated Plk1 phosphorylation at Thr-210. Depletion of Fry causes centrosome and centriole splitting in mitotic spindles and reduces the kinase activity of Plk1 in mitotic cells and the accumulation of Thr-210-phosphorylated Plk1 at the spindle poles. Our results suggest that Fry plays a crucial role in the structural integrity of mitotic centrosomes and in the maintenance of spindle bipolarity by promoting Plk1 activity at the spindle poles in early mitosis.     

Degradation of a Non-Cycling Mammalian Polo-like Kinase

Commentary to:

Murine FGF-Inducible Kinase is Rapidly Degraded via the Nuclear Ubiquitin-Proteosome System When Overexpressed in NIH 3T3 Cells

Gregory F. Alberts and Jeffrey A. Winkles     

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     

Acidic Nucleoplasmic DNA-binding Protein (And-1) Controls Chromosome Congression by Regulating the Assembly of Centromere Protein A (CENP-A) at Centromeres

The centromere is an epigenetically designated chromatin domain that is essential for the accurate segregation of chromosomes during mitosis. The incorporation of centromere protein A (CENP-A) into chromatin is fundamental in defining the centromeric loci. Newly synthesized CENP-A is loaded at centromeres in early G₁ phase by the CENP-A-specific histone chaperone Holliday junction recognition protein (HJURP) coupled with other chromatin assembly factors. However, it is unknown whether there are additional HJURP-interacting factor(s) involving in this process. Here we identify acidic nucleoplasmic DNA-binding protein 1 (And-1) as a new factor that is required for the assembly of CENP-A nucleosomes. And-1 interacts with both CENP-A and HJURP in a prenucleosomal complex, and the association of And-1 with CENP-A is increased during the cell cycle transition from mitosis to G₁ phase. And-1 down-regulation significantly compromises chromosome congression and the deposition of HJURP-CENP-A complexes at centromeres. Consistently, overexpression of And-1 enhances the assembly of CENP-A at centromeres. We conclude that And-1 is an important factor that functions together with HJURP to facilitate the cell cycle-specific recruitment of CENP-A to centromeres.     

DNA Topoisomerase III Localizes to Centromeres and Affects Centromeric CENP-A Levels in Fission Yeast

Centromeres are specialized chromatin regions marked by the presence of nucleosomes containing the centromere-specific histone H3 variant CENP-A, which is essential for chromosome segregation. Assembly and disassembly of nucleosomes is intimately linked to DNA topology, and DNA topoisomerases have previously been implicated in the dynamics of canonical H3 nucleosomes. Here we show that Schizosaccharomyces pombe Top3 and its partner Rqh1 are involved in controlling the levels of CENP-A^Cnp1 at centromeres. Both top3 and rqh1 mutants display defects in chromosome segregation. Using chromatin immunoprecipitation and tiling microarrays, we show that Top3, unlike Top1 and Top2, is highly enriched at centromeric central domains, demonstrating that Top3 is the major topoisomerase in this region. Moreover, centromeric Top3 occupancy positively correlates with CENP-A^Cnp1 occupancy. Intriguingly, both top3 and rqh1 mutants display increased relative enrichment of CENP-A^Cnp1 at centromeric central domains. Thus, Top3 and Rqh1 normally limit the levels of CENP-A^Cnp1 in this region. This new role is independent of the established function of Top3 and Rqh1 in homologous recombination downstream of Rad51. Therefore, we hypothesize that the Top3-Rqh1 complex has an important role in controlling centromere DNA topology, which in turn affects the dynamics of CENP-A^Cnp1 nucleosomes.     

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。     

Polo样激酶1功能及其在肿瘤靶向治疗中的应用

Polo样激酶1(polo-like kinase1,PLK1)是一种广泛存在于真核细胞中的丝/苏氨酸激酶,在细胞周期调控中发挥关键的作用。其主要功能包括参与激活cyclin B/CDK1复合体,协助中心体的功能成熟,活化细胞分裂后期促进复合物(anaphase promoting complex,APC),促进染色体正常分离、分配和调控胞质分裂等。现已发现PLK1在多种肿瘤中表达增高并与某些肿瘤的预后密切相关。利用反义寡核苷酸、RNA干扰技术和化学合成PLK1小分子抑制剂等方法阻断PLK1的表达或降低其激酶活性,能够有效抑制肿瘤细胞的增殖并介导肿瘤细胞的凋亡,但对正常细胞没有明显影响,因此PLK1在肿瘤靶向治疗中具有重要的应用前景。     

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。     

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-like Kinase 4 Autodestructs by Generating Its Slimb-Binding Phosphodegron

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Phosphorylation of Myosin II-interacting Guanine Nucleotide Exchange Factor (MyoGEF) at Threonine 544 by Aurora B Kinase Promotes the Binding of Polo-like Kinase 1 to MyoGEF

We previously reported that phosphorylation of myosin II-interacting guanine nucleotide exchange factor (MyoGEF) by polo-like kinase 1 (Plk1) promotes the localization of MyoGEF to the central spindle and increases MyoGEF activity toward RhoA during mitosis. In this study we report that aurora B-mediated phosphorylation of MyoGEF at Thr-544 creates a docking site for Plk1, leading to the localization and activation of MyoGEF at the central spindle. In vitro kinase assays show that aurora B can phosphorylate MyoGEF. T544A mutation drastically decreases aurora B-mediated phosphorylation of MyoGEF in vitro and in transfected HeLa cells. Coimmunoprecipitation and in vitro pulldown assays reveal that phosphorylation of MyoGEF at Thr-544 enhances the binding of Plk1 to MyoGEF. Immunofluorescence analysis shows that aurora B colocalizes with MyoGEF at the central spindle and midbody during cytokinesis. Suppression of aurora B activity by an aurora B inhibitor disrupts the localization of MyoGEF to the central spindle. In addition, T544A mutation interferes with the localization of MyoGEF to the cleavage furrow and decreases MyoGEF activity toward RhoA during mitosis. Taken together, our results suggest that aurora B coordinates with Plk1 to regulate MyoGEF activation and localization, thus contributing to the regulation of cytokinesis.     

Phosphorylation of Synucleins by Members of the Polo-like Kinase Family

Phosphorylation of α-synuclein (α-syn) at Ser-129 is a hallmark of Parkinson disease and related synucleinopathies. However, the identity of the natural kinases and phosphatases responsible for regulating α-syn phosphorylation remain unknown. Here we demonstrate that three closely related members of the human Polo-like kinase (PLK) family (PLK1, PLK2, and PLK3) phosphorylate α-syn and β-syn specifically at Ser-129 and Ser-118, respectively. Unlike other kinases reported to partially phosphorylate α-syn at Ser-129 in vitro, phosphorylation by PLK2 and PLK3 is quantitative (>95% conversion). Only PLK1 and PLK3 phosphorylate β-syn at Ser-118, whereas no phosphorylation of γ-syn was detected by any of the four PLKs (PLK1 to -4). PLK-mediated phosphorylation was greatly reduced in an isolated C-terminal fragment (residues 103–140) of α-syn, suggesting substrate recognition via the N-terminal repeats and/or the non-amyloid component domain of α-syn. PLKs specifically co-localized with phosphorylated Ser-129 (Ser(P)-129) α-syn in various subcellular compartments (cytoplasm, nucleus, and membranes) of mammalian cell lines and primary neurons as well as in α-syn transgenic mice, especially cortical brain areas involved in synaptic plasticity. Furthermore, we report that the levels of PLK2 are significantly increased in brains of Alzheimer disease and Lewy body disease patients. Taken together, these results provide biochemical and in vivo evidence of α-syn and β-syn phosphorylation by specific PLKs. Our results suggest a need for further studies to elucidate the potential role of PLK-syn interactions in the normal biology of these proteins as well as their involvement in the pathogenesis of Parkinson disease and other synucleinopathies.     

Inhibition of Polo-like Kinase 1 (Plk1) Enhances the Antineoplastic Activity of Metformin in Prostate Cancer

The widely used anti-diabetic drug metformin has been shown to exert strong antineoplastic actions in numerous tumor types, including prostate cancer (PCa). In this study, we show that BI2536, a specific Plk1 inhibitor, acted synergistically with metformin in inhibiting PCa cell proliferation. Furthermore, we also provide evidence that Plk1 inhibition makes PCa cells carrying WT p53 much more sensitive to low-dose metformin treatment. Mechanistically, we found that co-treatment with BI2536 and metformin induced p53-dependent apoptosis and further activated the p53/Redd-1 pathway. Moreover, we also show that BI2536 treatment inhibited metformin-induced glycolysis and glutamine anaplerosis, both of which are survival responses of cells against mitochondrial poisons. Finally, we confirmed the cell-based observations using both cultured cell-derived and patient-derived xenograft studies. Collectively, our findings support another promising therapeutic strategy by combining two well tolerated drugs against PCa proliferation and the progression of androgen-dependent PCa to the castration-resistant stage.     

The Polo-Box Domain: A Molecular Integrator of Mitotic Kinase Cascades and Polo-like Kinase Function

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