Podcasts about plk1

On the bright side, polo-like kinase 1 (PLK1) is considered a master regulator of the ever-important cell cycle. On the dark side, PLK1 expression (at both the mRNA and protein level) has shown to be upregulated in tumor cells, suggesting that PLK1 may also contribute to tumorigenesis. Despite this direct association, researchers studying the role of PLK1 in cancer have encountered a problem: a lack of appropriate animal models for experimentation. “Even though studies have suggested that PLK1 contributes to tumorigenesis, the ability of PLK1 to drive oncogenic transformation on its own in vivo was still questionable due to a lack of sophisticated animal models for experimentation [18, 19].” This problem may have been solved in 2021. In a new editorial paper, researchers Lilia Gheghiani and Zheng Fu from Virginia Commonwealth University discuss a recent study using their team's new genetically engineered mouse (GEM) model to assess the ability of PLK1 to be a sole driver of oncogenic transformation in vivo. Their editorial was published in Oncotarget's Volume 14 on June 27, 2023, and entitled, “The dark side of PLK1: Implications for cancer and genomic instability.” Full blog - https://www.oncotarget.org/2023/06/29/novel-gem-model-unveils-plk1s-role-in-tumorigenesis/ Paper DOI - https://doi.org/10.18632/oncotarget.28456 Correspondence to - Zheng Fu - zheng.fu@vcuhealth.org Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.28456 Subscribe for free publication alerts from Oncotarget - https://www.oncotarget.com/subscribe/ Keywords - cancer, polo-like kinase 1 (PLK1), oncogene, chromosomal instability, cell cycle checkpoints, tumorigenesis About Oncotarget Oncotarget is a primarily oncology-focused, peer-reviewed, open access journal. Papers are published continuously within yearly volumes in their final and complete form, and then quickly released to Pubmed. On September 15, 2022, Oncotarget was accepted again for indexing by MEDLINE. Oncotarget is now indexed by Medline/PubMed and PMC/PubMed. To learn more about Oncotarget, please visit https://www.oncotarget.com and connect with us: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/@OncotargetJournal LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.27.534346v1?rss=1 Authors: Burigotto, M., Vigorito, V., Mattivi, A., Gliech, C., Ghetti, S., Bisio, A., Lolli, G., Holland, A. J., Fava, L. L. Abstract: 53BP1 acts at the crossroads between DNA repair and p53-mediated stress response. With its interactor USP28, it is part of the mitotic surveillance pathway (MSP), a sensor that monitors the duration of cell division, promoting p53-dependent cell cycle arrest when a critical time threshold is surpassed. 53BP1 dynamically associates with kinetochores, being recruited during prophase, and then undergoing a time-dependent loss of affinity. However, the relevance of this behaviour remains unclear. Here, we identify CENP-F as an interaction partner and kinetochore receptor for 53BP1. By engineering human cells with a CENP-F point mutation, we demonstrate that preventing 53BP1 kinetochore localization does not reduce MSP proficiency. Strikingly, however, preventing the loss of 53BP1 from the kinetochore by inhibiting Polo-like kinase 1 (PLK1) restrains MSP activity, a phenomenon that is abrogated in the CENP-F mutant condition. Taken together, we demonstrate that kinetochore-loaded 53BP1 represents an MSP functionally inhibited state and that PLK1-dependent re-localization of 53BP1 represents an important layer of MSP regulation. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.03.17.533134v1?rss=1 Authors: Gelot, C., Kovacs, M. T., Miron, S., Mylne, E., Ghouil, R., Popova, T., Dingli, F., Loew, D., Guirouilh-Barbat, J., Del Nery, E., Zinn-Justin, S., Ceccaldi, R. Abstract: DNA double strand breaks (DSBs) are deleterious lesions that challenge genome integrity. To mitigate this threat, human cells rely on the activity of multiple DNA repair machineries that are tightly regulated throughout the cell cycle. In interphase, DSBs are mainly repaired by non-homologous end joining (NHEJ) and homologous recombination (HR). However, these pathways are completely inhibited in mitosis, leaving the fate of mitotic DSBs unknown. Here we show that DNA polymerase theta (Pol{theta}) repairs mitotic DSBs and thereby maintains genome integrity. In contrast to other DSB repair factors, Pol{theta} function is activated in mitosis upon phosphorylation by the Polo-like kinase 1 (PLK1). Phosphorylated Pol{theta} is recruited to mitotic DSBs, where it mediates joining of broken DNA ends, while halting mitotic progression. The lack of Pol{theta} leads to a shortening of mitotic duration and defective repair of mitotic DSBs, resulting in a loss of genome integrity. In addition, we identify mitotic Pol{theta} repair as the underlying cause of the synthetic lethality between Pol{theta} and HR. Our findings reveal the critical importance of mitotic DSB repair for maintaining genome stability. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.02.25.529946v1?rss=1 Authors: Yang, X., Smith, J. L., Beck, M. T., Wilkinson, J. M., Michaud, A., Vasta, J. D., Robers, M. B., Willson, T. M. Abstract: PLK1 is a protein kinase that regulates mitosis and is both an important oncology drug target and a potential anti target of drugs for the DNA damage response pathway or anti-infective host kinases. To expand the range of live cell NanoBRET target engagement assays to include PLK1 we developed an energy transfer probe based on the anilino-tetrahydropteridine chemotype found in several selective PLK inhibitors. Probe 11 was used to configure NanoBRET target engagement assays for PLK1, PLK2, and PLK3 and measure the potency of several known PLK inhibitors. In cell target engagement for PLK1 was in good agreement with the reported cellular potency for inhibition of cell proliferation. Probe 11 enabled investigation of the promiscuity of adavosertib, which had been described as a dual PLK1/WEE1 inhibitor in biochemical assays. Live cell target engagement analysis of adavosertib by NanoBRET demonstrated PLK activity at micromolar concentrations but only selective engagement of WEE1 at clinically relevant doses. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.01.05.522745v1?rss=1 Authors: Jimenez, A. J., Bousquet, H., Bardin, S., Perez, F., Goud, B., Miserey, S. Abstract: RAB GTPases are key regulators of membrane trafficking in eukaryotic cells. In addition to their role in interphase, several RAB proteins, including Golgi-associated RAB6, have mitotic functions. The aim of this study was to investigate how the interphasic and mitotic functions of RAB6 could be regulated. Since phosphorylation is a key regulatory process in mitosis, we looked for specific mitotic phosphorylation of RAB6 using a phospho-proteomic approach. We found that RAB6 is phosphorylated at position S52 by the mitotic kinase Polo-like kinase 1 (Plk1) in mitosis. Phosphorylated RAB6 localizes at the spindle poles from prophase to anaphase. In metaphase, we observed RAB6A-positive structures containing Mad1 and Mad2 moving along the mitotic spindle via the dynein-dynactin complex. We provide evidence that phosphorylation impairs RAB6A binding to some of its known partners, including p150Glued and Bicaudal-D2. In addition, the overexpression of RAB6A phospho-mutants lead to mitosis and cytokinesis defects. Our results suggest that a cycle of RAB6 phosphorylation/dephosphorylation is required for cell division. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.04.514992v1?rss=1 Authors: Leem, J., Kim, J.-S., Oh, J. S. Abstract: Because DNA double-strand breaks (DSBs) greatly threaten genomic integrity, effective DNA damage sensing and repair are essential for cellular survival in all organisms. However, DSB repair mainly occurs during the interphase and is repressed during mitosis. Here, we show that, unlike mitotic cells, oocytes can repair DSBs during meiosis through microtubule-dependent chromosomal recruitment of the CIP2A-MDC1-TOPBP1 complex from spindle poles. After DSB induction, we observed spindle shrinkage and stabilization, as well as BRCA1 and 53BP1 recruitment to chromosomes and subsequent DSB repair during meiosis I. Moreover, p-MDC1 and p-TOPBP1 were recruited from spindle poles to chromosomes in a CIP2A-dependent manner. This pole-to-chromosome relocation of the CIP2A-MDC1-TOPBP1 complex was impaired not only by depolymerizing microtubules but also by depleting CENP-A or HEC1, indicating that the kinetochore/centromere serves as a structural hub for microtubule-dependent transport of the CIP2A-MDC1-TOPBP1 complex. Mechanistically, DSB-induced CIP2A-MDC1-TOPBP1 relocation is regulated by PLK1 but not by ATM activity. Our data provide new insights into the critical crosstalk between chromosomes and spindle microtubules in response to DNA damage to maintain genomic stability during oocyte meiosis. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.10.24.513562v1?rss=1 Authors: Guerber, L., Pangou, E., Vuidel, A., Liao, Y., Kleiss, C., Grandgirard, E., Sumara, I. Abstract: PLK1 is a key regulator of mitosis whose protein levels and activity fluctuate during cell cycle. PLK1 dynamically localizes to distinct mitotic structures to regulate proper chromosome segregation. However, the molecular mechanisms linking localized PLK1 activity to its protein stability remain elusive. Here, we identify the Ubiquitin-Binding Protein 2-Like (UBAP2L) protein that regulates both dynamic removal of PLK1 from kinetochores and PLK1 protein stability during mitosis. We demonstrate that UBAP2L localizes to kinetochores in a PLK1-dependent manner and that UBAP2L depletion leads to the abnormal retention of PLK1 at kinetochores and segregation defects. We show that C-terminal domain of UBAP2L mediates its function on PLK1 and that UBAP2L specifically regulates PLK1 and no other mitotic factors. We demonstrate that inhibited kinetochore removal of PLK1 in UBAP2L-depleted cells, increases PLK1 stability after mitosis completion and results in aberrant PLK1 kinase activity in interphase and cellular death. Overall, our data suggest that UBAP2L is required to fine-tune PLK1 signaling in human cells. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

The mammalian target of rapamycin (mTOR) operates within two distinct protein complexes—mTOR complex 1 (mTORC1) and complex 2 (mTORC2). These protein complexes are not yet fully understood, as they were only recently identified in humans in 1994. What researchers do know is that mTORC1 is involved in the regulation of many cellular processes and is a key mediator of cell growth and proliferation. mTORC1 is activated by growth factor receptor signals through the PI3K–AKT and RAS–ERK mitogen-activated protein kinase (MAPK) pathways. The PI3K/AKT/mTOR pathway may be an efficacious target in the treatment of patients with non-small cell lung cancer (NSCLC). This theory is based on the identification of particular gene mutations in NSCLC that are associated with the PI3K/AKT/mTOR pathway. However, previous studies have not yet succeeded in defining an effective monotherapy or combination of therapies that targets this pathway while improving NSCLC patient outcome. Researchers from Institut Curie, PSL University, Xentech, BioPôle Alfort, Hôpital Foch, and Centre Léon Bérard designed a study using a new methodology to test treatment combinations based on specific targets identified as biomarkers of resistance to PI3K-targeting treatments, and not based on the NSCLC mutations themselves. Their trending research paper was published by Oncotarget in 2021 and entitled, “High in vitro and in vivo synergistic activity between mTORC1 and PLK1 inhibition in adenocarcinoma NSCLC.” Full blog - https://www.oncotarget.org/2021/09/30/trending-with-impact-unconventional-method-effectively-targets-nsclc/ Press release - https://www.oncotarget.com/news/pr/mtorc1-and-plk1-inhibition-in-adenocarcinoma-nsclc/ Sign up for free Altmetric alerts about this article - https://oncotarget.altmetric.com/details/email_updates?id=10.18632%2Foncotarget.27930 DOI - https://doi.org/10.18632/oncotarget.27930 Full text - https://www.oncotarget.com/article/27930/text/ Correspondence to - Didier Decaudin - Didier.decaudin@curie.fr Keywords - NSCLC, Pi3K signalling pathway, mTORC1, RAD001 (everolimus), PLK1 About Oncotarget Oncotarget is a bi-weekly, peer-reviewed, open access biomedical journal covering research on all aspects of oncology. To learn more about Oncotarget, please visit https://www.oncotarget.com or connect with: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget YouTube - https://www.youtube.com/c/OncotargetYouTube/ LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Oncotarget is published by Impact Journals, LLC please visit https://www.ImpactJournals.com or connect with @ImpactJrnls Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

Listen to short summaries of the latest oncology-focused research published in this week's issue of Oncotarget. https://www.oncotarget.com/archive/v12/i8/ Oncotarget Volume 12, Issue 8 features: COVER PAPER: “Prognostic and therapeutic value of the Hippo pathway, RABL6A, and p53-MDM2 axes in sarcomas” https://doi.org/10.18632/oncotarget.27928 NEWS: “Immunotherapy and fatigue: what we know and what we don’t know” https://doi.org/10.18632/oncotarget.27946 (PDF Download) EDITORIAL: “Drug exposure: still relevant after all these years” https://doi.org/10.18632/oncotarget.27899 (PDF Download) EDITORIAL: “Up to your NEK2 in CIN” https://doi.org/10.18632/oncotarget.27918 (PDF Download) RESEARCH PAPER: “Analytic validation and clinical utilization of the comprehensive genomic profiling test, GEM ExTra®” https://doi.org/10.18632/oncotarget.27945 RESEARCH PAPER: “Insulin-like growth factor 1/Child-Turcotte-Pugh composite score as a predictor of treatment outcomes in patients with advanced hepatocellular carcinoma treated with sorafenib” https://doi.org/10.18632/oncotarget.27924 RESEARCH PAPER: “Controlling for cellular heterogeneity using single-cell deconvolution of gene expression reveals novel markers of colorectal tumors exhibiting microsatellite instability” https://doi.org/10.18632/oncotarget.27935 RESEARCH PAPER: “Urine protein biomarkers of bladder cancer arising from 16-plex antibody-based screens” https://doi.org/10.18632/oncotarget.27941 RESEARCH PAPER: “The acylfulvene alkylating agent, LP-184, retains nanomolar potency in non-small cell lung cancer carrying otherwise therapy-refractory mutations” https://doi.org/10.18632/oncotarget.27943 RESEARCH PAPER: “Loss of CPAP causes sustained EGFR signaling and epithelial-mesenchymal transition in oral cancer” https://doi.org/10.18632/oncotarget.27932 RESEARCH PAPER: “Carcinoma cells that have undergone an epithelial-mesenchymal transition differentiate into endothelial cells and contribute to tumor growth” https://doi.org/10.18632/oncotarget.27940 RESEARCH PAPER: “Predicting clinical outcomes using cancer progression associated signatures” https://doi.org/10.18632/oncotarget.27934 RESEARCH PAPER: “High in vitro and in vivo synergistic activity between mTORC1 and PLK1 inhibition in adenocarcinoma NSCLC” https://doi.org/10.18632/oncotarget.27930 About Oncotarget: Oncotarget is a bi-weekly, peer-reviewed, open access biomedical journal covering research on all aspects of oncology. To learn more about Oncotarget, please visit https://www.oncotarget.com/ or follow us: SoundCloud - https://soundcloud.com/oncotarget Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget LinkedIn - https://www.linkedin.com/company/oncotarget Instagram - https://www.instagram.com/oncotargetjrnl/ YouTube - https://www.youtube.com/oncotargetyoutube Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget Oncotarget is published by Impact Journals , LLC. Please visit https://www.impactjournals.com/ or connect with us @ImpactJrnls Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957

Volume 11, Issue 25 of @Oncotarget reported that to examine the role of RSK in AML, the authors analyzed apoptosis and the cell cycle profile following treatment with BI-D1870, a potent inhibitor of RSK. BI-D1870 treatment increased the G2/M population and induced apoptosis in Acute Myeloid Leukemia cell lines and patient Acute Myeloid Leukemia cells. Therefore, the authors investigated whether BI-D1870 potentiates the anti-leukemic activity of vincristine by targeting mitotic exit. Combination treatment of BI-D1870 and vincristine synergistically increased mitotic arrest and apoptosis in acute leukemia cells. These data show that BI-D1870 induces apoptosis of AML cells alone and in combination with vincristine through blocking mitotic exit, providing a novel approach to overcoming vincristine resistance in AML cells. Dr. Kathleen M. Sakamoto from Stanford University School of Medicine said, "Acute myeloid leukemia (AML) is a genetically and phenotypically heterogeneous hematologic malignancy characterized by the accumulation of immature myeloid blasts with resultant peripheral blood cytopenia." Treatment of cells with microtubule targeting agents, including paclitaxel and the vinca alkaloid vincristine, blocks the proper formation of the mitotic spindle through inhibition of microtubule dynamics, resulting in the prolonged mitotic arrest of cancer cells. MTAs-treated mitotic arrested cells may undergo apoptosis in mitosis, however, the rapid degradation of Cyclin B due to an insufficient SAC leads to the mitotic slippage into tetraploid G1 stage in resistant cells. Though vinca alkaloid microtubule-destabilizing compounds have shown clinical efficacy against various hematological malignancies and were included in combination chemotherapy of the VAPA study, they are not currently used in induction chemotherapy for AML due to their high toxicity against lymphoid cells and rapid degradation by myeloperoxidase in AML cells. In this study, they demonstrate that BI-D1870, a potent inhibitor of RSK, induces mitotic arrest, and apoptosis in AML cells without inhibiting CDC2 and CDC25C. Furthermore, BI-D1870 synergizes with vinca alkaloid vincristine in AML cells, suggesting that inhibition of mitotic exit with BI-D1870 could be a promising novel approach for AML therapy in combination with MTAs. The Sakamoto Research Team concluded in their Oncotarget Research Paper that BI-D1870 is a reversible pan-RSK inhibitor, showing > 500-fold higher activity for RSK than other AGC kinases. BI-D1870 also inhibits the activity of PLK1, Aurora-B, MELK, PIM3, MST2, and GSK3β at higher concentrations than for RSK. BI-D1870 and BRD7389 have been reported to inhibit proliferation and significantly increase the G2/M population in melanoma cells. BI-D1870 does not have proper physicochemical properties for clinical application. Future structure-activity relationships study for BI-D1870 is required to improve solubility and pharmacokinetic profiles for in vivo preclinical and clinical studies. Sign up for free Altmetric alerts about this article DOI - https://doi.org/10.18632/oncotarget.27630 Full text - https://www.oncotarget.com/article/27630/text/ Correspondence to - Kathleen M. Sakamoto - kmsakamo@stanford.edu Keywords - acute myeloid leukemia, BI-D1870, RSK, vincristine, spindle assembly checkpoint About Oncotarget Oncotarget is a weekly, peer-reviewed, open access biomedical journal covering research on all aspects of oncology. To learn more about Oncotarget, please visit https://www.oncotarget.com or connect with: Facebook - https://www.facebook.com/Oncotarget/ Twitter - https://twitter.com/oncotarget LinkedIn - https://www.linkedin.com/company/oncotarget Pinterest - https://www.pinterest.com/oncotarget/ Reddit - https://www.reddit.com/user/Oncotarget/ Oncotarget is published by Impact Journals, LLC please visit http://www.ImpactJournals.com or connect with @ImpactJrnls Media Contact MEDIA@IMPACTJOURNALS.COM 18009220957x105

Dr. Mark Erlander, CEO, Cardiff Oncology identifies the unmet needs of those suffering from metastatic colorectal cancer who have the oncogene KRAS mutation that hyper-drives the tumor growth.  Mark talks about the company's investigational drug, onvansertib that is being evaluated in metastatic colorectal cancer (mCRC), acute myeloid leukemia (AML), and metastatic castrate-resistant prostate cancer (mCRPC).  Onvansertib is a first-in-class, third-generation, oral and highly-selective inhibitor of PLK1, an enzyme that is over-expressed in most types of cancers. Onvansertib works synergistically in combination with approved chemotherapies and targeted therapeutics. @CardiffOncology #onvansertib #cancer #oncology #drugdevelopment #colorectalcancer #KRAS Cardiffoncology.com Download the transcript here  

Dr. Mark Erlander, CEO, Cardiff Oncology identifies the unmet needs of those suffering from metastatic colorectal cancer who have the oncogene KRAS mutation that hyper-drives the tumor growth.  Mark talks about the company's investigational drug, onvansertib that is being evaluated in metastatic colorectal cancer (mCRC), acute myeloid leukemia (AML), and metastatic castrate-resistant prostate cancer (mCRPC).  Onvansertib is a first-in-class, third-generation, oral and highly-selective inhibitor of PLK1, an enzyme that is over-expressed in most types of cancers. Onvansertib works synergistically in combination with approved chemotherapies and targeted therapeutics. @CardiffOncology #onvansertib #cancer #oncology #drugdevelopment #colorectalcancer #KRAS Cardiffoncology.com Listen to the podcast here  

Dr. Mark Erlander, CEO of Cardiff Oncology discusses their lead drug candidate Onvansertib and the three active clinical trials in colorectal cancer, acute myeloid leukemia, and prostate cancer. Onvansertib specifically targets and inhibits a downstream enzyme, PLK1, effectively stopping cancer cell division.

Mon, 6 Dec 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12444/ https://edoc.ub.uni-muenchen.de/12444/1/Huebner_Nadja.pdf Huebner, Nadja

Cells drastically alter their structure at the onset of mitosis. Gavet and Pines reveal that the mitotic kinase CyclinB1-Cdk1 triggers its own nuclear import in prophase to ensure reorganization of the nucleus and cytoplasm is synchronized. This biosights episode presents the paper by Gavet and Pines from the April 19, 2010 issue of The Journal of Cell Biology, and includes an interview with senior author Jonathon Pines. Produced by Justin Paul and Ben Short.   Subscribe to biosights via iTunes or RSS View biosights archive The Rockefeller University Press biosights@rockefeller.edu

Mitosis is the process by which sister chromatids are equally segregated into two daughter cells. Tight control in various events during mitotic progression is essential for maintaining chromosome stability. Mitotic kinases including Cyclin dependent kinase 1 (Cdk1) and Aurora family are required for regulating proper mitotic progression by phosphorylating mitotic substrates thereby, controlling their activities, localization or abundance. On the other hand, these mitotic kinases are modulated by de-novo synthesis, activators, phosphorylation and ubiquitin-dependent proteolysis. A thorough understanding of the function and regulation of mitotic kinases could further our knowledge on mitotic progression. In the first part of the thesis, we investigated the expression, localization and regulation of human Lats1 kinase, which is a close homologue of the yeast Dbf2 kinase family involved in the mitotic exit network (MEN). Despite the fact that Lats1 has been suggested to be a spindle protein that binds and inactivates Cdk1, we found that Lats1 is mainly cytoplasmic throughout the cell cycle by immunofluorescence microscopy. Both yeast two-hybrid and coimmunoprecipitation showed no significant interaction between Lats1 and Cdk1. Although Lats1 was highly phosphorylated during mitosis, no detectable kinase activity was observed. However, we identified Ste20 like kinase MST2 as the upstream regulator of human Lats1. Phosphorylation of Lats1 by Mst2 resulted in the activation of Lats1 kinase activity both in vivo and in vitro. This kinase-substrate relation was proven to be specific, as another distant Mst2 homolog, Mst4, did not possess this ability. Subsequent mass-spectrometry-based phosphosites analysis revealed that Mst2 phosphorylates Lats1 on more than five residues. Alanine mutations on Lats1T1079 and S909 impaired Lats1 kinase activity. Thus, we could not confirm the suggested role of Lat1 in mitosis. Instead, we show that similar to its Drosophila ortholog, Lats1 is involved in the Mst2 signaling pathway and might control developmentally regulated cell proliferation and apoptosis in mammals. In the second part of this thesis, we characterized hBora, a novel Aurora A interactor originally found in Drosophila. We show that hBora is upregulated and phosphorylated during mitosis. siRNA-mediated knockdown of hBora led to spindle formation defects and aneuploidy. hBora overexpression caused monoastral spindle formation and mislocalization not only of Aurora A but also Plk1. Further investigations showed that Cdk1 phosphorylation on hBoraSer252 leads to Plk1 binding and this may promote the SCF-mediated proteolysis of hBora. Indeed, Plk1 depletion led to an increase in hBora levels. Interestingly, the co-depletion of both hBora and Plk1 (to lower hBora levels in Plk1 depleted cells) rescued the localization of Aurora A to the centrosomes and bipolar spindle formation. Thus, we propose that hBora is a functional link between Plk1 and Aurora A and that by modulating the proteolysis of hBora, Plk1 could regulate Aurora A localization and activity. At the end, we also investigated the function of Aurora A and could show that Aurora A is required for centriole cohesion and centrosome separation.

To investigate the requirements of mitotic, kinetochore-associated proteins for human chromosome segregation we analyzed the human Ndc80 complex, a core structural component of the outer kinetochore. The Ndc80 complex contains Hec1 and Nuf2 that interact via their N-termini and a smaller subcomplex of Spc24 and Spc25 is linked to Hec1. The complex is required for faithful chromosome congression and the recruitment of several proteins of the outer kinetochore, including the mitotic regulatory kinase Plk1. Pull down experiments with Plk1 identified a novel kinetochore/centromere associated DNA translocase, which we termed PICH, as an interactor of Plk1. The localization of PICH to kinetochores and centromeres is controlled by Plk1; and moreover, Plk1 phosphorylation on PICH negatively regulates its localization / chromatin association. PICH associates with conspicuous threads that persist into anaphase where Topoisomerase II causes their resolution. Moreover, PICH is a novel component of the spindle assembly checkpoint and PICH-dependent checkpoint signaling is likely to be mediated via kinetochore associated Mad2. This study raises questions as to the fate of centromeric DNA in sister chromatid separation and its timing of decatenation. We speculate that this enzyme associates with catenated, centromeric DNA from prometaphase where it may be required to sense the tension between sister kinetochores.

Um eine Parthenogenese zu verhindern, arretieren reife Oozyten von Wirbeltieren in der Metaphase der Meiose II. Diese biochemische Aktivität wurde 1971 als Zytostatischer Faktor (engl. Cytostatic Factor; CSF) beschrieben. Einzelne wichtige Komponenten wurden im Laufe der Zeit identifiziert, aber deren Zusammenspiel noch nicht aufgeklärt. Eine wichtige Rolle spielt dabei der Anaphase Fördernder Komplex (engl.Anaphase promoting complex/Cyclosome;APC/C), eine Ubiquitin-Ligase welche Zellzyklus regulierende Proteine dem Abbau zuführt und somit den Beginn der Anaphase ermöglicht. Der APC/C ist in reifen Oozyten inaktiv und wird nach der Befruchtung aktiviert, so dass der Arrest aufgehoben wird. Des Weiteren sind für den Eintritt in die Anaphase II die Aktivitäten zweier Kinasen nötig. Erstens erfolgt während der Befruchtung ein Anstieg der Konzentration des intrazellulären Calciums, dies führt zur Aktivierung der Calmodulin-abhängigen-kinase-II (CaMKII). Allerdings waren die Substrate dieser Kinase bis jetzt unbekannt. Zweitens ist die Polo-like-kinase-1 (Plk1) essentiell für die Aufhebung des Metaphase II - Arrests. In Xenopus Eiextrakt konnte gezeigt werden, dass die Aktivität der Xenopus Plk1 (Plx1) essentiell für den Eintritt in die Anaphase ist. Kürzlich wurde ein Inhibitor des APC/C in einem Yeast-Two-Hybrid-Screen mit inaktiver Plx1 als bait gefunden – Xenopus-Emi1-verwandtes-Protein-1 (engl. Xenopus-Emi1-related protein-1; XErp1). Die Depletion dieses Proteins in Xenopus-Ei-Extrakt führt zu einem verfrühten Eintritt in die Anaphase. Im Rahmen meiner Doktorarbeit konnte gezeigt werden, dass CaMKII und Plx1 kooperieren, um XErp1 nach der Befruchtung zu inaktivieren, indem sie XErp1 für den Abbau markieren. Auch das humane Protein wurde kloniert und es wurde damit begonnen Versuche in Säugetierzelllinien durchzuführen. Erste Hinweise lassen darauf schließen, dass das humane Protein in gleicher Weise reguliert wird wie XErp1.

Cytokinesis is the process that divides the cytoplasm of a parent cell into two. In animal cells, cytokinesis requires the formation of the central spindle and the contractile ring structures. The onset of cytokinesis is marked during anaphase with the specification of the division site, followed by cleavage furrow formation and ingression, midbody formation and abscission. The astral microtubules that originate from the centrosomes and the anti-parallel microtubules of the central spindle are proposed to determine the site of cleavage furrow formation (Bringmann and Hyman, 2005). The acto-myosin based contractile ring assembles at the division site and constricts the cytoplasm which is supported by the fusion of membrane vesicles to the ingressing plasma membrane. All these processes together result in the formation of two daughter cells. The small GTPase RhoA is one of the most upstream regulators of contractile ring assembly at the cortex. Rho proteins are activated by GEF’s (guanine nucleotide exchange factors) and one GEF that is required for cytokinesis is Ect2 (epithelial cell transforming protein2) (Tatsumoto et al., 1999). The Drosophila pebble (pbl) gene product is the founding member of the Ect2 protein family and has been shown to be required for cytokinesis (Lehner, 1992). In mammals, Ect2 was originally identified as a transforming protein in an expression cloning assay (Miki et al., 1993) and subsequently shown to be essential for cytokinesis. In this study, we have explored the temporal and spatial mechanisms that regulate Ect2 function. In agreement with previous studies, we show that Ect2 is a cell cycle regulated protein and is phosphorylated during mitosis. We identify a number of potentially interesting endogenous phosphorylation sites in Ect2, including potential Plk1 and Cdk1 sites. Although we have not been able to determine the function of these phosphorylation sites, their strong conservation among different species implies that they accomplish evolutionarily conserved roles.The identification of these phosphorylation sites sets the stage for future functional analyses. In complementary studies, we have shown that the central spindle and cell cortex localizations of Ect2 are facilitated by the BRCT and PH domains, respectively. The targeting of Ect2 to the central spindle is mediated by the MKlp1/MgcRacGAP and MKlp2/Aurora-B complexes. Of the two complexes, we show that Ect2 interacts and colocalizes only with the MKlp1/MgcRacGAP complex in telophase and propose that this interaction is mediated by a phosphorylation dependent docking mechanism that targets Ect2 to the central spindle. Interestingly, the displacement of Ect2 from the central spindle did not prevent cytokinesis, suggesting that localized GEF activity is not absolutely essential for cleavage furrow ingression and cytokinesis. In the second part of this thesis, we have explored the role of Ect2 during cytokinesis and show that, in Ect2 depleted cells, levels of RhoA and Citron kinase are diminished at the cleavage site, concomitant with the impairment of cleavage furrow formation and ingression. Additionally, overexpression of appropriate amino-terminal Ect2 fragments in cells also hinders cytokinesis. In these cells, RhoA and Citron kinase localize to the cortex and cleavage furrow ingression occurs, but, the subsequent abscission fails. Taken together, these results suggest that proper function of Ect2 is not only important for cleavage furrow ingression, but also for cell abscission. Finally, we investigate the overexpression phenotypes of different Ect2 truncation mutants. We show that abscission failure correlates with the persistence of amino-terminal Ect2 fragments at striking ring-like structures surrounding the midbody, indicating that completion of cell division requires the displacement of Ect2 from the contractile ring and its re-import into the reforming cell nucleus. Collectively, our data indicate that multiple mechanisms cooperate to regulate Ect2 in a spatial-temporal manner.

The centrosome is the major microtubule organizing centre (MTOC) in animal cells. Most microtubules (MTs) emanate from the centrosome, where gamma-tubulin ring complexes (gammaTuRCs) act as templates for MT nucleation. During interphase, the centrosome organizes a MT array that imparts shape and polarity to the cell and is essential for intracellular transport and positioning of organelles such as the Golgi apparatus. During mitosis, centrosomes ensure bipolarity and correct orientation of the spindle by forming the spindle poles. In order to switch from the interphasic to the mitotic state, the centrosome undergoes a structural reorganization, termed maturation, which is mainly characterized by an increase in MT nucleation activity. A full appreciation of how centrosomes contribute to cellular functions requires the isolation and characterization of unknown centrosome-associated molecules. Here we describe the identification and characterization of a novel centrosomal component, the human protein Nlp (ninein-like protein) related to the previously characterized MT-anchoring protein ninein. In the first part of the present thesis we describe the identification of Nlp as a novel centrosomal substrate of Polo-like kinase 1 (Plk1), an important regulator of mitosis whose activity is required for centrosome maturation. Nlp interacts with two distinct gammaTuRC components, gamma-tubulin and hGCP4, and stimulates MT nucleation. Plk1 phosphorylates Nlp and disrupts its centrosomal association. Overexpression of an Nlp mutant lacking Plk1 phosphorylation sites induces defects in mitotic spindle formation. We propose that Nlp acts as a gammaTuRC binding protein (GTBP), contributing to the MT nucleation activity of the centrosome during interphase. At the onset of mitosis, the displacement of Nlp from the centrosome triggered by Plk1 phosphorylation could represent an important step in the maturation process which allows the centrosome to switch from the interphasic to the mitotic state. Thus, we conclude that Nlp, as well as the related protein ninein, plays an important role in MT organization. However the function of these two proteins possibly diverged during evolution: whilst Nlp gained a more prominent role in MT nucleation, ninein became principally involved in MT anchoring. In the second part of this thesis we report the initial characterization of the molecular mechanisms underlying the ability of Nlp and ninein to induce the fragmentation of the Golgi apparatus when overexpressed in human cells. We show that the ability of these two centrosomal proteins to affect the organization of the Golgi clearly depends on their capacity to associate with the cytoplasmic dynein-dynactin complex, a molecular motor complex primarly involved in the maintainance of Golgi architecture. We propose that the excess of Nlp and ninein could induce the disruption of the Golgi apparatus by sequestering the dynein-dynactin complexes. Future investigations should be aimed at understanding whether the dissociation of the Golgi apparatus from the centrosome induced by the excess of Nlp and ninein could interfere with cell migration and cell polarization processes, which require a highly coordinated action of these two organelles. Cell migration and cell polarization represent critical events for immune responses as well as for embryonic development, invasive growth and metastasis. Thus, our findings raise the interesting possibility that an upregulation in the expression levels of structural centrosomal proteins could represent the molecular basis for developmental disorders and malfunctioning of the immune system and, on the other hand, modulate the acquisition of invasive properties by neoplastic cells.