Functional and Spatial Regulation of Mitotic Centromere- Associated Kinesin by Cyclin-Dependent Kinase 1

Sanhaji, Mourad; Friel, Claire T.; Kreis, Nina-Naomi; Krämer, Andrea; Martin, Carolyn; Howard, Jonathon; Strebhardt, Klaus; Yuan, Juping

doi:10.1128/mcb.00098-10

Cited by 54 publications

(68 citation statements)

References 45 publications

(80 reference statements)

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“…17,19,20 The following antibodies were used for Western blot analysis: mouse monoclonal antibodies against caspase-3, caspase-9, and Cdc25C (Santa Cruz Biotechnology, Heidelberg, Germany); rabbit polyclonal antibodies against poly(ADP)ribose polymerase (Cell Signaling, Beverly, MA); mouse monoclonal antibodies against ␤-actin (Sigma-Aldrich, Taufkirchen, Germany); and secondary anti-mouse or anti-rabbit antibodies (Santa Cruz Biotechnology). The following primary antibodies were used for staining: rat polyclonal anti-␣-tubulin (Biozol, Eching, Germany), rabbit polyclonal antibodies against pericentrin (Cell Signaling), mouse monoclonal anti-phospho-histone H3 (p-HH3, or Ser10; Millipore, Schwalbach, Germany), mouse monoclonal anti-Plk1 (Santa Cruz Biotechnology), rabbit polyclonal-anti-Plk1 (Millipore), mouse monoclonal antibodies against BubR1 (BD Biosciences, Heidelberg), human anti-centromere antibody (ImmunoVision, Springdale, AR), and mouse anti-␥-tubulin (Abcam, Cambridge, PA).…”

Section: Western Blot Analysis and Indirect Immunofluorescence Stainingmentioning

confidence: 99%

Polo-Box Domain Inhibitor Poloxin Activates the Spindle Assembly Checkpoint and Inhibits Tumor Growth in Vivo

Yuan

Sanhaji

Krämer

et al. 2011

The American Journal of Pathology

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Polo-like kinase 1 (Plk1) is widely established as one of the most promising targets in oncology. Although the protein kinase domain of Plk1 is highly conserved, the polo-box domain (PBD) of Plk1 provides a much more compelling site to specifically inhibit the localization and target binding of Plk1. We recently identified, via fluorescence polarization assay, the natural product derivative, Poloxin, as the first smallmolecule inhibitor specifically targeting the function of the Plk1 PBD. In this study, we characterized its mitotic phenotype and its function in vitro and in vivo. Poloxin induces centrosome fragmentation and abnormal spindle and chromosome misalignment, which activate the spindle assembly checkpoint and prolong mitosis. Notably, centrosomal fragmentation induced by Poloxin is partially attributable to dysfunctional Kizuna, a key substrate of Plk1 at centrosomes. Moreover, Poloxin strongly inhibits proliferation of a panel of cancer cells by inducing mitotic arrest, followed by a surge of apoptosis. More important, we report, for the first time to our knowledge, that the PBD inhibitor, Poloxin, significantly suppresses tumor growth of cancer cell lines in xenograft mouse models by lowering the proliferation rate and triggering apoptosis in treated tumor tissues. The data highlight that targeting the PBD by Poloxin is a powerful approach for selectively inhibiting Plk1 function in vitro and in vivo.

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Section: Western Blot Analysis and Indirect Immunofluorescence Stainingmentioning

confidence: 99%

Polo-Box Domain Inhibitor Poloxin Activates the Spindle Assembly Checkpoint and Inhibits Tumor Growth in Vivo

Yuan

Sanhaji

Krämer

et al. 2011

The American Journal of Pathology

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“…Depletion of MCAK leads to mal-oriented and misaligned chromosomes in achieving faithful metaphase alignment in addition to aberrant anaphase in mammalian cells (7,11,16,26). On the other hand, hyperactive MCAK causes the formation of multipolar spindle (27,28) and spindle assembly defects in mitosis (7,20,29). Interestingly, overexpression of MCAK has been observed in solid tumors such as gastric and breast cancers (30,31), suggesting that aberrant regulation of MCAK may be involved in tumorigenesis.…”

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confidence: 99%

PLK1 Phosphorylates Mitotic Centromere-associated Kinesin and Promotes Its Depolymerase Activity

Zhang

Shao

Huang

et al. 2011

Journal of Biological Chemistry

100

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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.

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“…Moreover, specific small molecule inhibitor RO-3306 targeting Cdk1 strongly weakens this phospho-signal in cells, whereas the inhibitor ZM447439 against Aurora A/B shows no effect (Figure 5e), indicating Ser315 is mainly the target for Cdk1 in normal mitosis, when cells are not enforced to overexpress Aurora A as reported (Katayama et al, 2004). Moreover, it has been very often observed that phosphorylation by Cdk1 affects the localization and the function of its substrates (Sanhaji et al, 2010). This phosphorylation of p53 by Cdk1 indeed destabilizes the protein p53 (Figures 6a and b) by promoting its release from the nucleus to cytoplasm (Figures 6c and d), where it is degraded.…”

Section: Discussionmentioning

confidence: 84%

“…All mutant constructs were confirmed by DNA sequencing. Transient transfection of plasmid was carried out as described (Kreis et al, 2009;Sanhaji et al, 2010). For quantitative-PCR, total RNAs from HeLa, HeLa GFP and HeLa 776-6 cells were extracted and purified using RNeasy Kit and RNase-free DNase Set (Qiagen, Hilden, Germany).…”

Section: Construction Of Dna Plasmids Transient Transfections and Qumentioning

confidence: 99%

Restoration of the tumor suppressor p53 by downregulating cyclin B1 in human papillomavirus 16/18-infected cancer cells

et al. 2010

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Abrogation of functional p53 is responsible for malignant cell transformation and the maintenance of malignant state of human papillomavirus-infected cancer cells. Thus, restoration of p53 has been regarded as an important strategy for molecular intervention combating papillomavirus-associated malignancies. We show here that depleting cyclin B1 stabilizes and reactivates p53 in papillomavirusinfected cervical cancer cell lines HeLa and CaSki. HeLa cells depleted of cyclin B1 exhibit mitotic defects in spindle formation and chromosome alignment. Downregulation of cyclin B1 increases p14 alternative reading frame of p16, the positive regulator of p53, and decreases phosphorylation of Ser315 in p53. Whereas RO-3306, a selective inhibitor of cyclin-dependent kinase 1 (Cdk1), suppresses this phosphorylation at Ser315 of p53, ZM447439, targeting Aurora A/B kinases, shows no effect. Further analyses in HeLa cells and HCT116 p53(À/À) cells suggest that the Ser315 phosphorylation by Cdk1 regulates negatively the protein stability and the function of p53. Moreover, increased p53 in HeLa cells is functional by showing its increased downstream effectors p21, mouse double minute 2 and Bax. Restoration of p53 and silencing cyclin B1 render cervical carcinoma cells more susceptible to DNA damage agent camptothecin. Taken together, targeting cyclin B1 might be an attractive strategy for preventing and treating papillomavirus-associated cancer by reactivating p53 and by reducing the Cdk1 activity.

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Functional and Spatial Regulation of Mitotic Centromere- Associated Kinesin by Cyclin-Dependent Kinase 1

Cited by 54 publications

References 45 publications

Polo-Box Domain Inhibitor Poloxin Activates the Spindle Assembly Checkpoint and Inhibits Tumor Growth in Vivo

Polo-Box Domain Inhibitor Poloxin Activates the Spindle Assembly Checkpoint and Inhibits Tumor Growth in Vivo

PLK1 Phosphorylates Mitotic Centromere-associated Kinesin and Promotes Its Depolymerase Activity

Restoration of the tumor suppressor p53 by downregulating cyclin B1 in human papillomavirus 16/18-infected cancer cells

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