Liver kinase B1 regulates the centrosome via PLK1

Liver kinase B1 (LKB1) is a tumor suppressor mutationally inactivated in Peutz–Jeghers syndrome (PJS) and various sporadic cancers. Although LKB1 encodes a kinase that possesses multiple functions, no individual hypothesis posed to date has convincingly explained how loss of LKB1 contributes to carcinogenesis. In this report we demonstrated that LKB1 maintains genomic stability through the regulation of centrosome duplication. We found that LKB1 colocalized with centrosomal proteins and was situated in the mitotic spindle pole. LKB1 deficiency-induced centrosome amplification was independent of AMP-activated protein kinase (AMPK), a well-defined substrate of LKB1. Cells lacking LKB1 exhibited an increase in phosphorylated and total Polo-like kinase 1 (PLK-1), NIMA-related kinase 2 (NEK2), and ninein-like protein (NLP). Overexpression of active PLK1 (T210D) reversed the inhibition of LKB1 on centrosome amplification. In contrast, depletion of PLK1 with siRNA or suppression of PLK1 kinase activity with BTO-1 (5-Cyano-7-nitro-2-benzothiazolecarboxamide-3-oxide) abrogated LKB1 deficiency-induced centrosome amplification. We further characterized that LKB1 phosphorylated and activated AMPK-related kinase 5 (NUAK1 or ARK5) that in turn increased the phosphorylation of MYPT1, enhanced the binding between MYPT1–PP1 and PLK1, and conferred an effective dephosphorylation of PLK1. More importantly, we noted that LKB1-deficient cells exhibited multiple nuclear abnormalities, such as mitotic delay, binuclear, polylobed, grape, large, and micronuclear. Immediate depletion of LKB1 resulted in the accumulation of multiploidy cells. Expression of LKB1 is reversely correlated with the levels of PLK1 in human cancer tissues. Thus, we have uncovered a novel function of LKB1 in the maintenance of genomic stability through the regulation of centrosome mediated by PLK1.     

PLK2 phosphorylation is critical for CPAP function in procentriole formation during the centrosome cycle

Control of centrosome duplication is tightly linked with the progression of the cell cycle. Recent studies suggest that the fundamental process of centriole duplication is evolutionally conserved. Here, we identified c entrosomal P 4.1‐a ssociated p rotein (CPAP), a human homologue of SAS‐4, as a substrate of PLK2 whose activity oscillates during the cell cycle. PLK2 phosphorylates the S589 and S595 residues of CPAP in vitro and in vivo. This phosphorylation is critical for procentriole formation during the centrosome cycle. PLK4 also phosphorylates S595 of CPAP, but PLK4 phosphorylation is not a critical step in the PLK4 function in procentriole assembly. CPAP is phosphorylated in a cell cycle stage‐specific manner, so that its phosphorylation increases at the G1/S transition phase and decreases during the exit of mitosis. Phosphorylated CPAP is preferentially located at the procentriole. Furthermore, overexpression of a phospho‐resistant CPAP mutant resulted in the failure to form elongated centrioles. On the basis of these results, we propose that phosphorylated CPAP is involved in procentriole assembly, possibly for centriole elongation. This work demonstrates an example of how procentriole formation is linked to the progression of the cell cycle.     

4E-BP1 participates in maintaining spindle integrity and genomic stability via interacting with PLK1

The essential function of eIF4E-binding protein 1 (4E-BP1) in translation initiation has been well established; however, the role of 4E-BP1 in normal cell cycle progression is coming to attention. Here, we revealed the role of 4E-BP1 on mitotic regulation and chromosomal DNA dynamics during mitosis. First, we have observed the co-localization of the phosphorylated 4E-BP1 at T37/46 with Polo-like kinase 1 (PLK1) at the centrosomes during. Depression of 4E-BP1 by small interfering RNA in HepG2 or HeLa cells resulted in an increased outcome of polyploidy and aberrant mitosis, including chromosomal DNA misaligned and multi-polar spindles or multiple centrosomes. We observed that 4E-BP1 interacted with PLK1 directly in vitro and in vivo in mitotic cells, and the C-terminal aa 77–118 of 4E-BP1 mediates its interaction with PLK1. PLK1 can phosphorylate 4E-BP1 in vitro. Furthermore, the depletion of 4E-BP1 sensitized HepG2 and HeLa cells to the microtubule disruption agent paclitaxel. These results demonstrate that 4E-BP1, beyond its role in translation regulation, can function as a regulator of mitosis via interacting with PLK1, and possibly plays a role in genomic stability maintaining.     

Discovery of 2-(1H-indol-5-ylamino)-6-(2,4-difluorophenylsulfonyl)-8-methylpyrido[2,3-d]pyrimidin-7(8H)-one (7ao) as a potent selective inhibitor of Polo like kinase 2 (PLK2)

Several families of protein kinases have been shown to play a critical role in the regulation of cell cycle progression, particularly progression through mitosis. These kinase families include the Aurora kinases, the Mps1 gene product and the Polo Like family of protein kinases (PLKs). The PLK family consists of five members and of these, the role of PLK1 in human cancer is well documented. PLK2 (SNK), which is highly homologous to PLK1, has been shown to play a critical role in centriole duplication and is also believed to play a regulatory role in the survival pathway by physically stabilizing the TSC1/2 complex in tumor cells under hypoxic conditions. As a part of our research program, we have developed a library of novel ATP mimetic chemotypes that are cytotoxic against a panel of cancer cell lines. We show that one of these chemotypes, the 6-arylsulfonyl pyridopyrimidinones, induces apoptosis of human tumor cell lines in nanomolar concentrations. The most potent of these compounds, 7ao, was found to be a highly specific inhibitor of PLK2 when profiled against a panel of 288 wild type, 55 mutant and 12 lipid kinases. Here, we describe the synthesis, structure activity relationship, in vitro kinase specificity and biological activity of the lead compound, 7ao.     

4E-BP1 participates in maintaining spindle integrity and genomic stability via interacting with PLK1

The essential function of eIF4E-binding protein 1 (4E-BP1) in translation initiation has been well established; however, the role of 4E-BP1 in normal cell cycle progression is coming to attention. Here, we revealed the role of 4E-BP1 on mitotic regulation and chromosomal DNA dynamics during mitosis. First, we have observed the co-localization of the phosphorylated 4E-BP1 at T37/46 with Polo-like kinase 1 (PLK1) at the centrosomes during. Depression of 4E-BP1 by small interfering RNA in HepG2 or HeLa cells resulted in an increased outcome of polyploidy and aberrant mitosis, including chromosomal DNA misaligned and multi-polar spindles or multiple centrosomes. We observed that 4E-BP1 interacted with PLK1 directly in vitro and in vivo in mitotic cells, and the C-terminal aa 77–118 of 4E-BP1 mediates its interaction with PLK1. PLK1 can phosphorylate 4E-BP1 in vitro. Furthermore, the depletion of 4E-BP1 sensitized HepG2 and HeLa cells to the microtubule disruption agent paclitaxel. These results demonstrate that 4E-BP1, beyond its role in translation regulation, can function as a regulator of mitosis via interacting with PLK1, and possibly plays a role in genomic stability maintaining.     

Overlapping roles for PLK1 and Aurora A during meiotic centrosome biogenesis in mouse spermatocytes

The establishment of bipolar spindles during meiotic divisions ensures faithful chromosome segregation to prevent gamete aneuploidy. We analyzed centriole duplication, as well as centrosome maturation and separation during meiosis I and II using mouse spermatocytes. The first round of centriole duplication occurs during early prophase I, and then, centrosomes mature and begin to separate by the end of prophase I to prime formation of bipolar metaphase I spindles. The second round of centriole duplication occurs at late anaphase I, and subsequently, centrosome separation coordinates bipolar segregation of sister chromatids during meiosis II. Using a germ cell‐specific conditional knockout strategy, we show that Polo‐like kinase 1 and Aurora A kinase are required for centrosome maturation and separation prior to metaphase I, leading to the formation of bipolar metaphase I spindles. Furthermore, we show that PLK1 is required to block the second round of centriole duplication and maturation until anaphase I. Our findings emphasize the importance of maintaining strict spatiotemporal control of cell cycle kinases during meiosis to ensure proficient centrosome biogenesis and, thus, accurate chromosome segregation during spermatogenesis.     

Centrobin/NIP2 Is a Microtubule Stabilizer Whose Activity Is Enhanced by PLK1 Phosphorylation during Mitosis

Centrobin/NIP2 is a centrosomal protein that is required for centrosome duplication. It is also critical for microtubule organization in both interphase and mitotic cells. In the present study, we observed that centrobin is phosphorylated in a cell cycle stage-specific manner, reaching its maximum at M phase. PLK1 is a kinase that is responsible for M phase-specific phosphorylation of centrobin. The microtubule forming activity of centrobin was enhanced by PLK1 phosphorylation. Furthermore, mitotic spindles were not assembled properly with the phospho-resistant mutant of centrobin. Based on these results, we propose that centrobin functions as a microtubule stabilizing factor and PLK1 enhances centrobin activity for proper spindle formation during mitosis.     

PLK1 Interacts and Phosphorylates Axin That Is Essential for Proper Centrosome Formation

Abnormal amplification of centrosomes could lead to improper chromosome segregation and aneuploidy and is implicated in cancer development. Here, we demonstrate that Axin, a scaffolding protein in Wnt signaling, is phosphorylated by PLK1 during mitosis. Phosphorylation of Axin Ser-157 by PLK1 abolished Axin association with γ-tubulin, while substitution of Ser-157 with alanine exhibited sustained interaction with γ-tubulin. In addition, overexpression of Axin-S157A significantly increased the number of cells with multi-centrosomes. These results suggest that the phosphorylation status of Axin, mediated by PLK1, dynamically regulates its association with γ-tubulin and centrosome formation and segregation.     

Centrioles generate a local pulse of Polo/PLK1 activity to initiate mitotic centrosome assembly

Mitotic centrosomes are formed when centrioles start to recruit large amounts of pericentriolar material (PCM) around themselves in preparation for mitosis. This centrosome “maturation” requires the centrioles and also Polo/PLK1 protein kinase. The PCM comprises several hundred proteins and, in Drosophila, Polo cooperates with the conserved centrosome proteins Spd‐2/CEP192 and Cnn/CDK5RAP2 to assemble a PCM scaffold around the mother centriole that then recruits other PCM client proteins. We show here that in Drosophila syncytial blastoderm embryos, centrosomal Polo levels rise and fall during the assembly process—peaking, and then starting to decline, even as levels of the PCM scaffold continue to rise and plateau. Experiments and mathematical modelling indicate that a centriolar pulse of Polo activity, potentially generated by the interaction between Polo and its centriole receptor Ana1 (CEP295 in humans), could explain these unexpected scaffold assembly dynamics. We propose that centrioles generate a local pulse of Polo activity prior to mitotic entry to initiate centrosome maturation, explaining why centrioles and Polo/PLK1 are normally essential for this process.     

特异小干扰RNA敲除PLK1基因的表达

为研究特异小干扰RNA（siRNA）作用于大肠癌细胞株SW480中PLK1 (Polo-like kinase 1)基因表达的mRNA对该细胞分裂生长的影响,设计了对应于PLK1基因表达mRNA不同位点的10种特异siRNA,经化学合成后,用脂质体转染SW480细胞,实时定量PCR检测PLK1基因的表达,观察不同的siRNA作用强度,并计数细胞了解相应细胞的生长情况,western-blot观察PLK1表达蛋白的变化和流式细胞计数分析细胞周期改变。发现10种siRNA均可敲除PLK1基因表达的20 %以上,其中P1、P4和P9 3组敲除mRNA达80 ％以上,这3种siRNA及其混合物对PLK1基因mRNA的作用具有相应浓度效应,在25 nmol/L时达到最佳作用效果,而且相同浓度的混合物作用效果更好（超过95%）,PLK1表达蛋白质明显降低,细胞周期在G2期受到阻碍。72 h后的各种siRNA浓度下细胞生长变化与PLK1基因的mRNA水平变化相一致。结果表明化学合成的特异siRNA对SW480细胞中PLK1基因表达具有消除作用,混合物作用更强,在细胞水平上抑制了SW480细胞的分裂生长。     

PLK1 phosphorylates mitotic centromere-associated kinesin and promotes its depolymerase activity

During cell division, interaction between kinetochores and dynamic spindle microtubules governs chromosome movements. The microtubule depolymerase mitotic centromere-associated kinesin (MCAK) is a key regulator of mitotic spindle assembly and dynamics. However, the regulatory mechanisms underlying its depolymerase activity during the cell cycle remain elusive. Here, we showed that PLK1 is a novel regulator of MCAK in mammalian cells. MCAK interacts with PLK1 in vitro and in vivo. The neck and motor domain of MCAK associates with the kinase domain of PLK1. MCAK is a novel substrate of PLK1, and the phosphorylation stimulates its microtubule depolymerization activity of MCAK in vivo. Overexpression of a polo-like kinase 1 phosphomimetic mutant MCAK causes a dramatic increase in misaligned chromosomes and in multipolar spindles in mitotic cells, whereas overexpression of a nonphosphorylatable MCAK mutant results in aberrant anaphase with sister chromatid bridges, suggesting that precise regulation of the MCAK activity by PLK1 phosphorylation is critical for proper microtubule dynamics and essential for the faithful chromosome segregation. We reasoned that dynamic regulation of MCAK phosphorylation by PLK1 is required to orchestrate faithful cell division, whereas the high levels of PLK1 and MCAK activities seen in cancer cells may account for a mechanism underlying the pathogenesis of genomic instability.     

Investigation on PLK2 and PLK3 substrate recognition

Analyses of human phosphoproteome based on primary structure of the aminoacids surrounding the phosphor Ser/Thr suggest that a significant proportion of phosphosites is generated by a restricted number of acidophilic kinases, among which protein kinase CK2 plays a prominent role. Recently, new acidophilic kinases belonging to the Polo like kinase family have been characterized, with special reference to PLK1, PLK2, and PLK3 kinases. While some progress has been made in deciphering the PLK1-dependent phosphoproteome, very little is known about the targets of PLK2 and PLK3 kinases. In this report by using an in vitro approach, consisting of cell lysate phosphorylation, phosphoprotein separation by 2D gel electrophoresis and mass spectrometry, we describe the identification of new potential substrates of PLK2 and PLK3 kinases. We have identified and validated as in vitro PLK2 and PLK3 substrates HSP90, GRP-94, β-tubulin, calumenin, and 14-3-3 epsilon. The phosphosites generated by PLK3 in these proteins have been identified by mass spectrometry analysis to get new insights about PLKs specificity determinants. These latter have been further corroborated by an in silico analysis of the PLKs substrate binding region.     

PLK1 regulates centrosome migration and spindle dynamics in male mouse meiosis

Cell division requires the regulation of karyokinesis and cytokinesis, which includes an essential role of the achromatic spindle. Although the functions of centrosomes are well characterised in somatic cells, their role during vertebrate spermatogenesis remains elusive. We have studied the dynamics of the meiotic centrosomes in male mouse during both meiotic divisions. Results show that meiotic centrosomes duplicate twice: first duplication occurs in the leptotene/zygotene transition, while the second occurs in interkinesis. The maturation of duplicated centrosomes during the early stages of prophase I and II are followed by their separation and migration to opposite poles to form bipolar spindles I and II. The study of the genetic mouse model Plk1(Δ/Δ) indicates a central role of Polo‐like kinase 1 in pericentriolar matrix assembly, in centrosome maturation and migration, and in the formation of the bipolar spindles during spermatogenesis. In addition, in vitro inhibition of Polo‐like kinase 1 and Aurora A in organotypic cultures of seminiferous tubules points out to a prominent role of both kinases in the regulation of the formation of meiotic bipolar spindles.     

P53, cyclin-dependent kinase and abnormal amplification of centrosomes

Centrosomes play a critical role in formation of bipolar mitotic spindles, an essential event for accurate chromosome segregation into daughter cells. Numeral abnormalities of centrosomes (centrosome amplification) occur frequently in cancers, and are considered to be the major cause of chromosome instability, which accelerates acquisition of malignant phenotypes during tumor progression. Loss or mutational inactivation of p53 tumor suppressor protein, one of the most common mutations found in cancers, results in a high frequency of centrosome amplification in part via allowing the activation of the cyclin-dependent kinase (CDK) 2-cyclin E (as well as CDK2-cyclin A) which is a key factor for the initiation of centrosome duplication. In this review, the role of centrosome amplification in tumor progression, and mechanistic view of how centrosomes are amplified in cells through focusing on loss of p53 and aberrant activities of CDK2-cyclins will be discussed.     

中心体蛋白Centlein的敲除促进细胞周期进程

中心体是大部分动物细胞的微管组织中心,它确保了有序的细胞周期进程以及染色体的精确分离,我们之前报道了中心体蛋白Centlein作为一个分子连接,与C-Nap1和Cep68一起形成复合物维持中心体的连接. 然而,关于Centlein的其他功能我们还知之甚少. 在本研究中,建立了Centlein的敲除细胞系,并且运用RNA-seq技术分析了敲除细胞系和正常野生型细胞系之间转录水平的差异. 发现Centlein敲除细胞系中细胞周期相关基因PLK1、CCNB1、CCNA2和CDC20的表达量上调,流式结果又表明Centlein的敲除促进了细胞周期进程. 同时发现Centlein与PLK1之间存在细胞内相互作用,于是我们提出了Centlein通过与PLK1的作用参与细胞周期进程.     

PLK4 overexpression and its effect on centrosome regulation and chromosome stability in human gastric cancer

Polo-like kinase 4 (PLK4) is a centrosomal protein that is involved in the regulation of centrosome duplication. This study aimed to determine whether the genetic abnormality of PLK4 is involved in human gastric cancer. First, we examined the status of PLK4 mRNA expression in 7 gastric cancer cell lines and 48 primary gastric cancers using an RT-PCR analysis. The upregulation of PLK4 mRNA expression was detected in 57.1 % (4/7) of the gastric cancer cell lines, and a novel PLK4 variant with exon 4, but without exon 5, was identified. In the primary gastric cancers, the upregulation of PLK4 mRNA expression in the cancerous cells was detected in 50.0 % (24/48) of the cases, and this upregulation was statistically significant (P value = 0.0139). Next, we established AGS gastric cancer cells capable of inducibly expressing PLK4 using the piggyBac transposon vector system and showed that PLK4 overexpression induced centrosome amplification and chromosome instability using immunofluorescence and FISH analyses, respectively. Furthermore, PLK4 overexpression suppressed primary cilia formation. Our current findings suggested that PLK4 is upregulated in a subset of primary gastric cancers and that PLK4 overexpression induces centrosome amplification and chromosome instability and causes the suppression of primary cilia formation.     

Aurora-A overexpression reveals tetraploidization as a major route to centrosome amplification in p53-/- cells

Aberrations in centrosome numbers have long been implicated in aneuploidy and tumorigenesis, but their origins are unknown. Here we have examined how overexpression of Aurora-A kinase causes centrosome amplification in cultured cells. We show that excess Aurora-A does not deregulate centrosome duplication but gives rise to extra centrosomes through defects in cell division and consequent tetraploidization. Over expression of other mitotic kinases (Polo-like kinase 1 and Aurora-B) also causes multinucleation and concomitant increases in centrosome numbers. Absence of a p53 checkpoint exacerbates this phenotype, providing a plausible explanation for the centrosome amplification typical of p53-/- cells. We propose that errors during cell division, combined with the inability to detect the resulting hyperploidy, constitute a major cause for numerical centrosome aberrations in tumors.     

CUL3 and protein kinases: Insights from PLK1/KLHL22 interaction

Posttranslational mechanisms drive fidelity of cellular processes. Phosphorylation and ubiquitination of substrates represent very common, covalent, posttranslational modifications and are often co-regulated. Phosphorylation may play a critical role both by directly regulating E3-ubiquitin ligases and/or by ensuring specificity of the ubiquitination substrate. Importantly, many kinases are not only critical regulatory components of these pathways but also represent themselves the direct ubiquitination substrates. Recent data suggest the role of CUL3-based ligases in both proteolytic and non-proteolytic regulation of protein kinases. Our own recent study identified the mitotic kinase PLK1 as a direct target of the CUL3 E3-ligase complex containing BTB-KELCH adaptor protein KLHL22.¹ In this study, we aim at gaining mechanistic insights into CUL3-mediated regulation of the substrates, in particular protein kinases, by analyzing mechanisms of interaction between KLHL22 and PLK1. We find that kinase activity of PLK1 is redundant for its targeting for CUL3-ubiquitination. Moreover, CUL3/KLHL22 may contact 2 distinct motifs within PLK1 protein, consistent with the bivalent mode of substrate targeting found in other CUL3-based complexes. We discuss these findings in the context of the existing knowledge on other protein kinases and substrates targeted by CUL3-based E3-ligases.     

Interaction between ROCK II and nucleophosmin/B23 in the regulation of centrosome duplication

Nucleophosmin (NPM)/B23 has been implicated in the regulation of centrosome duplication. NPM/B23 localizes between two centrioles in the unduplicated centrosome. Upon phosphorylation on Thr¹⁹⁹ by cyclin-dependent kinase 2 (CDK2)/cyclin E, the majority of centrosomal NPM/B23 dissociates from centrosomes, but some NPM/B23 phosphorylated on Thr¹⁹⁹ remains at centrosomes. It has been shown that Thr¹⁹⁹ phosphorylation of NPM/B23 is critical for the physical separation of the paired centrioles, an initial event of the centrosome duplication process. Here, we identified ROCK II kinase, an effector of Rho small GTPase, as a protein that localizes to centrosomes and physically interacts with NPM/B23. Expression of the constitutively active form of ROCK II promotes centrosome duplication, while down-regulation of ROCK II expression results in the suppression of centrosome duplication, especially delaying the initiation of centrosome duplication during the cell cycle. Moreover, ROCK II regulates centrosome duplication in its kinase and centrosome localization activity-dependent manner. We further found that ROCK II kinase activity is significantly enhanced by binding to NPM/B23 and that NPM/B23 acquires a higher binding affinity to ROCK II upon phosphorylation on Thr¹⁹⁹. Moreover, physical interaction between ROCK II and NPM/B23 in vivo occurs in association with CDK2/cyclin E activation and the emergence of Thr¹⁹⁹-phosphorylated NPM/B23. All these findings point to ROCK II as the effector of the CDK2/cyclin E-NPM/B23 pathway in the regulation of centrosome duplication.