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Sample records for human checkpoint kinase

  1. Structure and Substrate Recruitment of the Human Spindle Checkpoint Kinase Bub1

    SciTech Connect

    Kang, Jungseog; Yang, Maojun; Li, Bing; Qi, Wei; Zhang, Chao; Shokat, Kevan M.; Tomchick, Diana R.; Machius, Mischa; Yu, Hongtao

    2009-11-10

    In mitosis, the spindle checkpoint detects a single unattached kinetochore, inhibits the anaphase-promoting complex or cyclosome (APC/C), and prevents premature sister chromatid separation. The checkpoint kinase Bub1 contributes to checkpoint sensitivity through phosphorylating the APC/C activator, Cdc20, and inhibiting APC/C catalytically. We report here the crystal structure of the kinase domain of Bub1, revealing the requirement of an N-terminal extension for its kinase activity. Though the activation segment of Bub1 is ordered and has structural features indicative of active kinases, the C-terminal portion of this segment sterically restricts substrate access to the active site. Bub1 uses docking motifs, so-called KEN boxes, outside its kinase domain to recruit Cdc20, one of two known KEN box receptors. The KEN boxes of Bub1 are required for the spindle checkpoint in human cells. Therefore, its unusual active-site conformation and mode of substrate recruitment suggest that Bub1 has an exquisitely tuned specificity for Cdc20.

  2. Targeting checkpoint kinase 1 in cancer therapeutics.

    PubMed

    Tse, Archie N; Carvajal, Richard; Schwartz, Gary K

    2007-04-01

    Progression through the cell cycle is monitored by surveillance mechanisms known as cell cycle checkpoints. Our knowledge of the biochemical nature of checkpoint regulation during an unperturbed cell cycle and following DNA damage has expanded tremendously over the past decade. We now know that dysfunction in cell cycle checkpoints leads to genomic instability and contributes to tumor progression, and most agents used for cancer therapy, such as cytotoxic chemotherapy and ionizing radiation, also activate cell cycle checkpoints. Understanding how checkpoints are regulated is therefore important from the points of view of both tumorigenesis and cancer treatment. In this review, we present an overview of the molecular hierarchy of the checkpoint signaling network and the emerging role of checkpoint targets, especially checkpoint kinase 1, in cancer therapy. Further, we discuss the results of recent clinical trials involving the nonspecific checkpoint kinase 1 inhibitor, UCN-01, and the challenges we face with this new therapeutic approach.

  3. Efficacy of Combined Histone Deacetylase and Checkpoint Kinase Inhibition in a Preclinical Model of Human Burkitt Lymphoma

    PubMed Central

    Kong, YanGuo; Barisone, Gustavo A; Sidhu, Ranjit S; O’Donnell, Robert T; Tuscano, Joseph M

    2015-01-01

    Checkpoint kinase inhibition has been studied as a way of enhancing the effectiveness of DNA-damaging agents. More recently, histone deacetylase inhibitors have shown efficacy in several cancers, including non-Hodgkin lymphoma. To evaluate the effectiveness of this combination for the treatment of lymphoma, we examined the combination of AR42, a histone deacetylase inhibitor, and checkpoint kinase 2 (CHEK2) inhibitor II in vitro and in vivo. The combination resulted in up to 10-fold increase in potency in five Burkitt lymphoma cell lines when compared with either drug alone. Both drugs inhibited tumor progression in xenograft models, but the combination was more effective than either agent alone, resulting in regression of established tumors. No toxicity was observed. These results suggest that the combination of histone deacetylase inhibition and checkpoint kinase inhibition represent an effective and nontoxic treatment option that should be further explored in preclinical and clinical studies. PMID:26322845

  4. Diallyl disulfide selectively causes checkpoint kinase-1 mediated G2/M arrest in human MGC803 gastric cancer cell line.

    PubMed

    Ling, Hui; Lu, Li-Feng; He, Jie; Xiao, Guo-Hua; Jiang, Hao; Su, Qi

    2014-11-01

    Previous studies have shown that diallyl disulfide (DADS), a naturally occurring anticancer agent in garlic, arrested human gastric cancer cells (MGC803) in the G2/M phase of the cell cycle. Due to the importance of cell cycle redistribution in DADS-mediated anticarcinogenic effects, we investigated the role of checkpoint kinases (Chk1 and Chk2) during DADS-induced cell cycle arrest. In the present study, the northern blot analysis showed that mRNA expression of for Chkl and Chk2 was unchanged. Notably, DADS induced the accumulation of phosphorylated Chk1, but not of Chk2, activated phospho-ATR (ATM-RAD3-related gene), and dowregulated CDC25C and cyclin B1 expression. Furthermore, CDC25C was immunoprecipitated by anti-Chk1 but not anti-Chk2. Results of the overexpression and knockdown studies, showed that Chk1 but not Chk2 regulated the DADS-induced G2/M arrest of MGC803 cells. The overexpression of Chk1 resulted in significantly increased DADS-induced G2/M arrest, increased DADS-induced Chk1 phosphorylation and inhibited CDC25C expression. Knockdown of Chk1 reduced DADS‑induced G2/M arrest and blocked the DADS-induced inhibition of CDC25C and cyclin B1 expression. These results suggested that Chk1 is important in DADS‑induced cell cycle G2/M arrest in the human MGC803 gastric cancer cell line. Furthermore, the DADS-induced G2/M checkpoint response is mediated by Chk1 signaling through ATR/Chk1/CDC25C/cyclin B1.

  5. Phosphorylation of Minichromosome Maintenance 3 (MCM3) by Checkpoint Kinase 1 (Chk1) Negatively Regulates DNA Replication and Checkpoint Activation.

    PubMed

    Han, Xiangzi; Mayca Pozo, Franklin; Wisotsky, Jacob N; Wang, Benlian; Jacobberger, James W; Zhang, Youwei

    2015-05-08

    Mechanisms controlling DNA replication and replication checkpoint are critical for the maintenance of genome stability and the prevention or treatment of human cancers. Checkpoint kinase 1 (Chk1) is a key effector protein kinase that regulates the DNA damage response and replication checkpoint. The heterohexameric minichromosome maintenance (MCM) complex is the core component of mammalian DNA helicase and has been implicated in replication checkpoint activation. Here we report that Chk1 phosphorylates the MCM3 subunit of the MCM complex at Ser-205 under normal growth conditions. Mutating the Ser-205 of MCM3 to Ala increased the length of DNA replication track and shortened the S phase duration, indicating that Ser-205 phosphorylation negatively controls normal DNA replication. Upon replicative stress treatment, the inhibitory phosphorylation of MCM3 at Ser-205 was reduced, and this reduction was accompanied with the generation of single strand DNA, the key platform for ataxia telangiectasia mutated and Rad3-related (ATR) activation. As a result, the replication checkpoint is activated. Together, these data provide significant insights into the regulation of both normal DNA replication and replication checkpoint activation through the novel phosphorylation of MCM3 by Chk1.

  6. Targeting lung cancer through inhibition of checkpoint kinases

    PubMed Central

    Syljuåsen, Randi G.; Hasvold, Grete; Hauge, Sissel; Helland, Åslaug

    2015-01-01

    Inhibitors of checkpoint kinases ATR, Chk1, and Wee1 are currently being tested in preclinical and clinical trials. Here, we review the basic principles behind the use of such inhibitors as anticancer agents, and particularly discuss their potential for treatment of lung cancer. As lung cancer is one of the most deadly cancers, new treatment strategies are highly needed. We discuss how checkpoint kinase inhibition in principle can lead to selective killing of lung cancer cells while sparing the surrounding normal tissues. Several features of lung cancer may potentially be exploited for targeting through inhibition of checkpoint kinases, including mutated p53, low ERCC1 levels, amplified Myc, tumor hypoxia and presence of lung cancer stem cells. Synergistic effects have also been reported between inhibitors of ATR/Chk1/Wee1 and conventional lung cancer treatments, such as gemcitabine, cisplatin, or radiation. Altogether, inhibitors of ATR, Chk1, and Wee1 are emerging as new cancer treatment agents, likely to be useful in lung cancer treatment. However, as lung tumors are very diverse, the inhibitors are unlikely to be effective in all patients, and more work is needed to determine how such inhibitors can be utilized in the most optimal ways. PMID:25774168

  7. Inhibition of the checkpoint kinase Chk1 induces DNA damage and cell death in human Leukemia and Lymphoma cells.

    PubMed

    Bryant, Christopher; Scriven, Kirsten; Massey, Andrew J

    2014-06-10

    Chk1 forms a core component of the DNA damage response and small molecule inhibitors are currently being investigated in the clinic as cytotoxic chemotherapy potentiators. Recent evidence suggests that Chk1 inhibitors may demonstrate significant single agent activity in tumors with specific DNA repair defects, a constitutively activated DNA damage response or oncogene induced replicative stress. Growth inhibition induced by the small molecule Chk1 inhibitor V158411 was assessed in a panel of human leukemia and lymphoma cell lines and compared to cancer cell lines derived from solid tumors. The effects on cell cycle and DNA damage response markers were further evaluated. Leukemia and lymphoma cell lines were identified as particularly sensitive to the Chk1 inhibitor V158411 (mean GI50 0.17 μM) compared to colon (2.8 μM) or lung (6.9 μM) cancer cell lines. Chk1 inhibition by V158411 in the leukemia and lymphoma cell lines induced DNA fragmentation and cell death that was both caspase dependent and independent, and prevented cells undergoing mitosis. An analysis of in vitro pharmacodynamic markers identified a dose dependent decrease in Chk1 and cyclin B1 protein levels and Cdc2 Thr15 phosphorylation along with a concomitant increase in H2AX phosphorylation at Ser139 following V158411 treatment. These data support the further evaluation of Chk1 inhibitors in hematopoietic cancers as single agents as well as in combination with standard of care cytotoxic drugs.

  8. Checkpoint kinase 2 is required for efficient immunoglobulin diversification.

    PubMed

    Davari, Kathrin; Frankenberger, Samantha; Schmidt, Angelika; Tomi, Nils-Sebastian; Jungnickel, Berit

    2014-01-01

    Maintenance of genome integrity relies on multiple DNA repair pathways as well as on checkpoint regulation. Activation of the checkpoint kinases Chk1 and Chk2 by DNA damage triggers cell cycle arrest and improved DNA repair, or apoptosis in case of excessive damage. Chk1 and Chk2 have been reported to act in a complementary or redundant fashion, depending on the physiological context. During secondary immunoglobulin (Ig) diversification in B lymphocytes, DNA damage is abundantly introduced by activation-induced cytidine deaminase (AID) and processed to mutations in a locus-specific manner by several error-prone DNA repair pathways. We have previously shown that Chk1 negatively regulates Ig somatic hypermutation by promoting error-free homologous recombination and Ig gene conversion. We now report that Chk2 shows opposite effects to Chk1 in the regulation of these processes. Chk2 inactivation in B cells leads to decreased Ig hypermutation and Ig class switching, and increased Ig gene conversion activity. This is linked to defects in non-homologous end joining and increased Chk1 activation upon interference with Chk2 function. Intriguingly, in the context of physiological introduction of substantial DNA damage into the genome during Ig diversification, the 2 checkpoint kinases thus function in an opposing manner, rather than redundantly or cooperatively.

  9. The protein phosphatase 2A functions in the spindle position checkpoint by regulating the checkpoint kinase Kin4.

    PubMed

    Chan, Leon Y; Amon, Angelika

    2009-07-15

    In budding yeast, a surveillance mechanism known as the spindle position checkpoint (SPOC) ensures accurate genome partitioning. In the event of spindle misposition, the checkpoint delays exit from mitosis by restraining the activity of the mitotic exit network (MEN). To date, the only component of the checkpoint to be identified is the protein kinase Kin4. Furthermore, how the kinase is regulated by spindle position is not known. Here, we identify the protein phosphatase 2A (PP2A) in complex with the regulatory subunit Rts1 as a component of the SPOC. Loss of PP2A-Rts1 function abrogates the SPOC but not other mitotic checkpoints. We further show that the protein phosphatase functions upstream of Kin4, regulating the kinase's phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function.

  10. The protein phosphatase 2A functions in the spindle position checkpoint by regulating the checkpoint kinase Kin4

    PubMed Central

    Chan, Leon Y.; Amon, Angelika

    2009-01-01

    In budding yeast, a surveillance mechanism known as the spindle position checkpoint (SPOC) ensures accurate genome partitioning. In the event of spindle misposition, the checkpoint delays exit from mitosis by restraining the activity of the mitotic exit network (MEN). To date, the only component of the checkpoint to be identified is the protein kinase Kin4. Furthermore, how the kinase is regulated by spindle position is not known. Here, we identify the protein phosphatase 2A (PP2A) in complex with the regulatory subunit Rts1 as a component of the SPOC. Loss of PP2A-Rts1 function abrogates the SPOC but not other mitotic checkpoints. We further show that the protein phosphatase functions upstream of Kin4, regulating the kinase's phosphorylation and localization during an unperturbed cell cycle and during SPOC activation, thus defining the phosphatase as a key regulator of SPOC function. PMID:19605686

  11. The effect of ataxia-telangiectasia mutated kinase-dependent hyperphosphorylation of checkpoint kinase-2 on oligodeoxynucleotide 7909 containing CpG motifs-enhanced sensitivity to X-rays in human lung adenocarcinoma A549 cells

    PubMed Central

    Liu, Xiaoqun; Liu, Xiangdong; Qiao, Tiankui; Chen, Wei; Yuan, Sujuan

    2015-01-01

    Objective The aim of the study reported here was to further investigate the potential effect of ataxia-telangiectasia mutated (ATM) kinase-dependent hyperphosphorylation of checkpoint kinase-2 (Chk2) on radiosensitivity enhanced by oligodeoxynucleotide 7909 containing CpG motifs (CpG ODN7909) in human lung adenocarcinoma A549 cells. Methods In vitro A549 cells were randomly separated into control, CpG, X-ray, CpG+ X-ray, ATM kinase-small interfering RNA (siRNA)+CpG+X-ray (ATM-siRNA), and Chk2-siRNA+CpG+X-ray (Chk2-siRNA) groups. siRNAs were adopted to silence the ATM and Chk2 genes. Expression and phosphorylation of ATM kinase and Chk2 were detected by Western blot assay. Cell colonies were observed under inverted phase-contrast microscopy. Cellular survival curves were fitted using a multi-target single-hitting model. Cell cycle and apoptosis were analyzed by flow cytometry. Results Expression of ATM kinase and Chk2 was similar among the control, CpG, X-ray, and CpG+X-ray groups. Phosphorylated ATM kinase and Chk2 were significantly increased in the CpG+X-ray group compared with in the X-ray group (t=6.00, P<0.01 and t=3.13, P<0.05, respectively), though these were hardly detected in the control and CpG groups. However, expression of ATM kinase and Chk2 was clearly downregulated in the ATM-siRNA and Chk2-siRNA groups, respectively. Similarly, their phosphorylation levels were also significantly decreased in the ATM-siRNA group (t=14.35, P<0.01 and t=8.46, P<0.01, respectively) and a significant decrease in phosphorylated Chk2 was observed in the Chk2-siRNA group (t=7.28, P<0.01) when compared with the CpG+X-ray group. Further, the number of A549 cells at Gap 2/mitotic phase and the apoptosis rate were clearly increased in the CpG+X-ray group compared with in the other groups (all P<0.05). The multi-target single-hitting model showed that the sensitization enhancement ratio calculated by mean death dose was 1.39 in CpG+X-ray group (vs 1.04 and 1.03 in the ATM

  12. Loss of the Greatwall Kinase Weakens the Spindle Assembly Checkpoint

    PubMed Central

    Kitagawa, Mayumi; Caldez, Matias J.; Gunaratne, Jayantha; Lee, Sang Hyun

    2016-01-01

    The Greatwall kinase/Mastl is an essential gene that indirectly inhibits the phosphatase activity toward mitotic Cdk1 substrates. Here we show that although Mastl knockout (MastlNULL) MEFs enter mitosis, they progress through mitosis without completing cytokinesis despite the presence of misaligned chromosomes, which causes chromosome segregation defects. Furthermore, we uncover the requirement of Mastl for robust spindle assembly checkpoint (SAC) maintenance since the duration of mitotic arrest caused by microtubule poisons in MastlNULL MEFs is shortened, which correlates with premature disappearance of the essential SAC protein Mad1 at the kinetochores. Notably, MastlNULL MEFs display reduced phosphorylation of a number of proteins in mitosis, which include the essential SAC kinase MPS1. We further demonstrate that Mastl is required for multi-site phosphorylation of MPS1 as well as robust MPS1 kinase activity in mitosis. In contrast, treatment of MastlNULL cells with the phosphatase inhibitor okadaic acid (OKA) rescues the defects in MPS1 kinase activity, mislocalization of phospho-MPS1 as well as Mad1 at the kinetochore, and premature SAC silencing. Moreover, using in vitro dephosphorylation assays, we demonstrate that Mastl promotes persistent MPS1 phosphorylation by inhibiting PP2A/B55-mediated MPS1 dephosphorylation rather than affecting Cdk1 kinase activity. Our findings establish a key regulatory function of the Greatwall kinase/Mastl->PP2A/B55 pathway in preventing premature SAC silencing. PMID:27631493

  13. Ethanol Metabolism Activates Cell Cycle Checkpoint Kinase, Chk2

    PubMed Central

    Clemens, Dahn L.; Mahan Schneider, Katrina J.; Nuss, Robert F.

    2011-01-01

    Chronic ethanol abuse results in hepatocyte injury and impairs hepatocyte replication. We have previously shown that ethanol metabolism results in cell cycle arrest at the G2/M transition, which is partially mediated by inhibitory phosphorylation of the cyclin-dependent kinase, Cdc2. To further delineate the mechanisms by which ethanol metabolism mediates this G2/M arrest, we investigated the involvement of upstream regulators of Cdc2 activity. Cdc2 is activated by the phosphatase Cdc25C. The activity of Cdc25C can, in turn, be regulated by the checkpoint kinase, Chk2, which is regulated by the kinase ataxia telangiectasia mutated (ATM). To investigate the involvement of these regulators of Cdc2 activity, VA-13 cells, which are Hep G2 cells modified to efficiently express alcohol dehydrogenase, were cultured in the presence or absence of 25 mM ethanol. Immunoblots were performed to determine the effects of ethanol metabolism on the activation of Cdc25C, Chk2, and ATM. Ethanol metabolism increased the active forms of ATM, and Chk2, as well as the phosphorylated form of Cdc25C. Additionally, inhibition of ATM resulted in approximately 50% of the cells being rescued from the G2/M cell cycle arrest, and ameliorated the inhibitory phosphorylation of Cdc2. Our findings demonstrate that ethanol metabolism activates ATM. ATM can activate the checkpoint kinase Chk2, resulting in phosphorylation of Cdc25C, and ultimately in the accumulation of inactive Cdc2. This may, in part, explain the ethanol metabolism-mediated impairment in hepatocyte replication, which may be important in the initiation and progression of alcoholic liver injury. PMID:21924579

  14. Characterization of Spindle Checkpoint Kinase Mps1 Reveals Domain with Functional and Structural Similarities to Tetratricopeptide Repeat Motifs of Bub1 and BubR1 Checkpoint Kinases*

    PubMed Central

    Lee, Semin; Thebault, Philippe; Freschi, Luca; Beaufils, Sylvie; Blundell, Tom L.; Landry, Christian R.; Bolanos-Garcia, Victor M.; Elowe, Sabine

    2012-01-01

    Kinetochore targeting of the mitotic kinases Bub1, BubR1, and Mps1 has been implicated in efficient execution of their functions in the spindle checkpoint, the self-monitoring system of the eukaryotic cell cycle that ensures chromosome segregation occurs with high fidelity. In all three kinases, kinetochore docking is mediated by the N-terminal region of the protein. Deletions within this region result in checkpoint failure and chromosome segregation defects. Here, we use an interdisciplinary approach that includes biophysical, biochemical, cell biological, and bioinformatics methods to study the N-terminal region of human Mps1. We report the identification of a tandem repeat of the tetratricopeptide repeat (TPR) motif in the N-terminal kinetochore binding region of Mps1, with close homology to the tandem TPR motif of Bub1 and BubR1. Phylogenetic analysis indicates that TPR Mps1 was acquired after the split between deutorostomes and protostomes, as it is distinguishable in chordates and echinoderms. Overexpression of TPR Mps1 resulted in decreased efficiency of both chromosome alignment and mitotic arrest, likely through displacement of endogenous Mps1 from the kinetochore and decreased Mps1 catalytic activity. Taken together, our multidisciplinary strategy provides new insights into the evolution, structural organization, and function of Mps1 N-terminal region. PMID:22187426

  15. Tumor suppressor protein C53 antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation.

    PubMed

    Jiang, Hai; Wu, Jianchun; He, Chen; Yang, Wending; Li, Honglin

    2009-04-01

    Cyclin-dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint. More recently, Wang et al. (2007) found that C53/LZAP may function as a tumor suppressor by way of inhibiting NF-kappaB signaling. We report here the identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexpression. Intriguingly, we found that C53 interacts with Chk1 and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell-cycle progression and DNA damage response.

  16. Disruption of the checkpoint kinase 1/cell division cycle 25A pathway abrogates ionizing radiation-induced S and G2 checkpoints

    PubMed Central

    Zhao, Hui; Watkins, Janis L.; Piwnica-Worms, Helen

    2002-01-01

    Checkpoint kinase (Chk)1 is an evolutionarily conserved protein kinase that was first identified in fission yeast as an essential component of the DNA damage checkpoint. In mice, Chk1 provides an essential function in the absence of environmentally imposed genotoxic stress. Here we show that human cells lacking Chk1 exhibit defects in both the ionizing radiation (IR)-induced S and G2 checkpoints. In addition, loss of Chk1 resulted in the accumulation of a hypophosphorylated form of the Cdc25A protein phosphatase, and Chk1-deficient cells failed to degrade Cdc25A after IR. The IR-induced S and G2 checkpoints were partially restored in Chk1-deficient cells when Cdc25A accumulation was interfered with. Finally, Cdc25A was phosphorylated by Chk1 in vitro on similar sites phosphorylated in vivo, including serine-123. These findings indicate that Chk1 directly phosphorylates Cdc25A during an unperturbed cell cycle, and that phosphorylation of Cdc25A by Chk1 is required for cells to delay cell cycle progression in response to double-strand DNA breaks. PMID:12399544

  17. Elevated Levels of the Polo Kinase Cdc5 Override the Mec1/ATR Checkpoint in Budding Yeast by Acting at Different Steps of the Signaling Pathway

    PubMed Central

    Donnianni, Roberto Antonio; Ferrari, Matteo; Lazzaro, Federico; Clerici, Michela; Tamilselvan Nachimuthu, Benjamin; Plevani, Paolo; Muzi-Falconi, Marco; Pellicioli, Achille

    2010-01-01

    Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends. PMID:20098491

  18. Greatwall and Polo-like Kinase 1 Coordinate to Promote Checkpoint Recovery*

    PubMed Central

    Peng, Aimin; Wang, Ling; Fisher, Laura A.

    2011-01-01

    Checkpoint recovery upon completion of DNA repair allows the cell to return to normal cell cycle progression and is thus a crucial process that determines cell fate after DNA damage. We previously studied this process in Xenopus egg extracts and established Greatwall (Gwl) as an important regulator. Here we show that preactivated Gwl kinase can promote checkpoint recovery independently of cyclin-dependent kinase 1 (Cdk1) or Plx1 (Xenopus polo-like kinase 1), whereas depletion of Gwl from extracts exhibits no synergy with that of Plx1 in delaying checkpoint recovery, suggesting a distinct but related relationship between Gwl and Plx1. In further revealing their functional relationship, we found mutual dependence for activation of Gwl and Plx1 during checkpoint recovery, as well as their direct association. We characterized the protein association in detail and recapitulated it in vitro with purified proteins, which suggests direct interaction. Interestingly, Gwl interaction with Plx1 and its phosphorylation by Plx1 both increase at the stage of checkpoint recovery. More importantly, Plx1-mediated phosphorylation renders Gwl more efficient in promoting checkpoint recovery, suggesting a functional involvement of such regulation in the recovery process. Finally, we report an indirect regulatory mechanism involving Aurora A that may account for Gwl-dependent regulation of Plx1 during checkpoint recovery. Our results thus reveal novel mechanisms underlying the involvement of Gwl in checkpoint recovery, in particular, its functional relationship with Plx1, a well characterized regulator of checkpoint recovery. Coordinated interplays between Plx1 and Gwl are required for reactivation of these kinases from the G2/M DNA damage checkpoint and efficient checkpoint recovery. PMID:21708943

  19. Elm1 kinase activates the spindle position checkpoint kinase Kin4.

    PubMed

    Caydasi, Ayse Koca; Kurtulmus, Bahtiyar; Orrico, Maria I L; Hofmann, Astrid; Ibrahim, Bashar; Pereira, Gislene

    2010-09-20

    Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck-associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4.

  20. Elm1 kinase activates the spindle position checkpoint kinase Kin4

    PubMed Central

    Caydasi, Ayse Koca; Kurtulmus, Bahtiyar; Orrico, Maria I.L.; Hofmann, Astrid; Ibrahim, Bashar

    2010-01-01

    Budding yeast asymmetric cell division relies upon the precise coordination of spindle orientation and cell cycle progression. The spindle position checkpoint (SPOC) is a surveillance mechanism that prevents cells with misoriented spindles from exiting mitosis. The cortical kinase Kin4 acts near the top of this network. How Kin4 kinase activity is regulated and maintained in respect to spindle positional cues remains to be established. Here, we show that the bud neck–associated kinase Elm1 participates in Kin4 activation and SPOC signaling by phosphorylating a conserved residue within the activation loop of Kin4. Blocking Elm1 function abolishes Kin4 kinase activity in vivo and eliminates the SPOC response to spindle misalignment. These findings establish a novel function for Elm1 in the coordination of spindle positioning with cell cycle progression via its control of Kin4. PMID:20855503

  1. Mitotic Checkpoint Kinase Mps1 Has a Role in Normal Physiology which Impacts Clinical Utility

    PubMed Central

    Martinez, Ricardo; Blasina, Alessandra; Hallin, Jill F.; Hu, Wenyue; Rymer, Isha; Fan, Jeffery; Hoffman, Robert L.; Murphy, Sean; Marx, Matthew; Yanochko, Gina; Trajkovic, Dusko; Dinh, Dac; Timofeevski, Sergei; Zhu, Zhou; Sun, Peiquing; Lappin, Patrick B.; Murray, Brion W.

    2015-01-01

    Cell cycle checkpoint intervention is an effective therapeutic strategy for cancer when applied to patients predisposed to respond and the treatment is well-tolerated. A critical cell cycle process that could be targeted is the mitotic checkpoint (spindle assembly checkpoint) which governs the metaphase-to-anaphase transition and insures proper chromosomal segregation. The mitotic checkpoint kinase Mps1 was selected to explore whether enhancement in genomic instability is a viable therapeutic strategy. The basal-a subset of triple-negative breast cancer was chosen as a model system because it has a higher incidence of chromosomal instability and Mps1 expression is up-regulated. Depletion of Mps1 reduces tumor cell viability relative to normal cells. Highly selective, extremely potent Mps1 kinase inhibitors were created to investigate the roles of Mps1 catalytic activity in tumor cells and normal physiology (PF-7006, PF-3837; Ki<0.5 nM; cellular IC50 2–6 nM). Treatment of tumor cells in vitro with PF-7006 modulates expected Mps1-dependent biology as demonstrated by molecular and phenotypic measures (reduced pHH3-Ser10 levels, shorter duration of mitosis, micro-nucleation, and apoptosis). Tumor-bearing mice treated with PF-7006 exhibit tumor growth inhibition concomitant with pharmacodynamic modulation of a downstream biomarker (pHH3-Ser10). Unfortunately, efficacy only occurs at drug exposures that cause dose-limiting body weight loss, gastrointestinal toxicities, and neutropenia. Mps1 inhibitor toxicities may be mitigated by inducing G1 cell cycle arrest in Rb1-competent cells with the cyclin-dependent kinase-4/6 inhibitor palbociclib. Using an isogenic cellular model system, PF-7006 is shown to be selectively cytotoxic to Rb1-deficient cells relative to Rb1-competent cells (also a measure of kinase selectivity). Human bone marrow cells pretreated with palbociclib have decreased PF-7006-dependent apoptosis relative to cells without palbociclib pretreatment

  2. Human cytomegalovirus inhibits a DNA damage response by mislocalizing checkpoint proteins

    NASA Astrophysics Data System (ADS)

    Gaspar, Miguel; Shenk, Thomas

    2006-02-01

    The DNA damage checkpoint pathway responds to DNA damage and induces a cell cycle arrest to allow time for DNA repair. Several viruses are known to activate or modulate this cellular response. Here we show that the ataxia-telangiectasia mutated checkpoint pathway, which responds to double-strand breaks in DNA, is activated in response to human cytomegalovirus DNA replication. However, this activation does not propagate through the pathway; it is blocked at the level of the effector kinase, checkpoint kinase 2 (Chk2). Late after infection, several checkpoint proteins, including ataxia-telangiectasia mutated and Chk2, are mislocalized to a cytoplasmic virus assembly zone, where they are colocalized with virion structural proteins. This colocalization was confirmed by immunoprecipitation of virion proteins with an antibody that recognizes Chk2. Virus replication was resistant to ionizing radiation, which causes double-strand breaks in DNA. We propose that human CMV DNA replication activates the checkpoint response to DNA double-strand breaks, and the virus responds by altering the localization of checkpoint proteins to the cytoplasm and thereby inhibiting the signaling pathway. ionizing radiation | ataxia-telangiectasia mutated pathway

  3. The spindle position checkpoint is coordinated by the Elm1 kinase

    PubMed Central

    Moore, Jeffrey K.; Chudalayandi, Prakash; Heil-Chapdelaine, Richard A.

    2010-01-01

    How dividing cells monitor the effective transmission of genomes during mitosis is poorly understood. Budding yeast use a signaling pathway known as the spindle position checkpoint (SPC) to ensure the arrival of one end of the mitotic spindle in the nascent daughter cell. An important question is how SPC activity is coordinated with mother–daughter polarity. We sought to identify factors at the bud neck, the junction between mother and bud, which contribute to checkpoint signaling. In this paper, we show that the protein kinase Elm1 is an obligate regulator of the SPC, and this function requires localization of Elm1 to the bud neck. Furthermore, we show that Elm1 promotes the activity of the checkpoint kinase Kin4. These findings reveal a novel function for Elm1 in the SPC and suggest how checkpoint activity may be linked to cellular organization. PMID:21041444

  4. The fission yeast meiotic checkpoint kinase Mek1 regulates nuclear localization of Cdc25 by phosphorylation.

    PubMed

    Pérez-Hidalgo, Livia; Moreno, Sergio; San-Segundo, Pedro A

    2008-12-01

    In eukaryotic cells, fidelity in transmission of genetic information during cell division is ensured by the action of cell cycle checkpoints. Checkpoints are surveillance mechanisms that arrest or delay cell cycle progression when critical cellular processes are defective or when the genome is damaged. During meiosis, the so-called meiotic recombination checkpoint blocks entry into meiosis I until recombination has been completed, thus avoiding aberrant chromosome segregation and the formation of aneuploid gametes. One of the key components of the meiotic recombination checkpoint is the meiosis-specific Mek1 kinase, which belongs to the family of Rad53/Cds1/Chk2 checkpoint kinases containing forkhead-associated domains. In fission yeast, several lines of evidence suggest that Mek1 targets the critical cell cycle regulator Cdc25 to delay meiotic cell cycle progression. Here, we investigate in more detail the molecular mechanism of action of the fission yeast Mek1 protein. We demonstrate that Mek1 acts independently of Cds1 to phosphorylate Cdc25, and this phosphorylation is required to trigger cell cycle arrest. Using ectopic overexpression of mek1(+) as a tool to induce in vivo activation of Mek1, we find that Mek1 promotes cytoplasmic accumulation of Cdc25 and results in prolonged phosphorylation of Cdc2 at tyrosine 15. We propose that at least one of the mechanisms contributing to the cell cycle delay when the meiotic recombination checkpoint is activated in fission yeast is the nuclear exclusion of the Cdc25 phosphatase by Mek1-dependent phosphorylation.

  5. p73 induction after DNA damage is regulated by checkpoint kinases Chk1 and Chk2

    PubMed Central

    Urist, Marshall; Tanaka, Tomoaki; Poyurovsky, Masha V.; Prives, Carol

    2004-01-01

    The checkpoint kinases Chk1 and Chk2 are central to the induction of cell cycle arrest, DNA repair, and apoptosis as elements in the DNA-damage checkpoint. We report here that in several human tumor cell lines, Chk1 and Chk2 control the induction of the p53 related transcription factor p73 in response to DNA damage. Multiple experimental systems were used to show that interference with or augmentation of Chk1 or Chk2 signaling strongly impacts p73 accumulation. Furthermore, Chk1 and Chk2 control p73 mRNA accumulation after DNA damage. We demonstrate as well that E2F1 directs p73 expression in the presence and absence of DNA damage. Chk1 and Chk2, in turn, are vital to E2F1 stabilization and activity after genotoxic stress. Thus, Chk1, Chk2, E2F1, and p73 function in a pathway mediating p53-independent cell death produced by cytotoxic drugs. Since p53 is often obviated through mutation as a cellular port for anticancer intervention, this pathway controlling p53 autonomous pro-apoptotic signaling is of potential therapeutic importance. PMID:15601819

  6. A tumor suppressor C53 protein antagonizes checkpoint kinases to promote cyclin-dependent kinase 1 activation

    PubMed Central

    Jiang, Hai; Wu, Jianchun; He, Chen; Yang, Wending; Li, Honglin

    2009-01-01

    Cyclin dependent kinase 1 (Cdk1)/cyclin B1 complex is the driving force for mitotic entry, and its activation is tightly regulated by the G2/M checkpoint. We originally reported that a novel protein C53 (also known as Cdk5rap3 and LZAP) potentiates DNA damage-induced cell death by modulating the G2/M checkpoint (1). More recently, Wang et al (2007) found that C53/LZAP may function as a tumor suppressor via inhibiting NF-κB signaling (2). We report here identification of C53 protein as a novel regulator of Cdk1 activation. We found that knockdown of C53 protein causes delayed Cdk1 activation and mitotic entry. During DNA damage response, activation of checkpoint kinase 1 and 2 (Chk1 and Chk2) is partially inhibited by C53 overexrepsssion. Intriguingly, we found that C53 interacts with checkpoint kinase 1 (Chk1) and antagonizes its function. Moreover, a portion of C53 protein is localized at the centrosome, and centrosome-targeting C53 potently promotes local Cdk1 activation. Taken together, our results strongly suggest that C53 is a novel negative regulator of checkpoint response. By counteracting Chk1, C53 promotes Cdk1 activation and mitotic entry in both unperturbed cell cycle progression and DNA damage response. PMID:19223857

  7. Centrosome-Dependent Bypass of the DNA Damage Checkpoint by the Polo Kinase Cdc5.

    PubMed

    Ratsima, Hery; Serrano, Diego; Pascariu, Mirela; D'Amours, Damien

    2016-02-16

    Cell-cycle checkpoints are essential feedback mechanisms that promote genome integrity. However, in the face of unrepairable DNA lesions, bypass mechanisms can suppress checkpoint activity and allow cells to resume proliferation. The molecular mechanisms underlying this biological response are currently not understood. Taking advantage of unique separation-of-function mutants, we show that the Polo-like kinase (PLK) Cdc5 uses a phosphopriming-based interaction mechanism to suppress G2/M checkpoint arrest by targeting Polo kinase activity to centrosomes. We also show that key subunits of the evolutionarily conserved RSC complex are critical downstream effectors of Cdc5 activity in checkpoint suppression. Importantly, the lethality and checkpoint defects associated with loss of Cdc5 Polo box activity can be fully rescued by artificially anchoring Cdc5 kinase domain to yeast centrosomes. Collectively, our results highlight a previously unappreciated role for centrosomes as key signaling centers for the suppression of cell-cycle arrest induced by persistent or unrepairable DNA damage.

  8. Src Family Kinases Promote Silencing of ATR-Chk1 Signaling in Termination of DNA Damage Checkpoint*

    PubMed Central

    Fukumoto, Yasunori; Morii, Mariko; Miura, Takahito; Kubota, Sho; Ishibashi, Kenichi; Honda, Takuya; Okamoto, Aya; Yamaguchi, Noritaka; Iwama, Atsushi; Nakayama, Yuji; Yamaguchi, Naoto

    2014-01-01

    The DNA damage checkpoint arrests cell cycle progression to allow time for repair. Once DNA repair is completed, checkpoint signaling is terminated. Currently little is known about the mechanism by which checkpoint signaling is terminated, and the disappearance of DNA lesions is considered to induce the end of checkpoint signaling; however, here we show that the termination of checkpoint signaling is an active process promoted by Src family tyrosine kinases. Inhibition of Src activity delays recovery from the G2 phase DNA damage checkpoint following DNA repair. Src activity is required for the termination of checkpoint signaling, and inhibition of Src activity induces persistent activation of ataxia telangiectasia mutated (ATM)- and Rad3-related (ATR) and Chk1 kinases. Src-dependent nuclear protein tyrosine phosphorylation and v-Src expression suppress the ATR-mediated Chk1 and Rad17 phosphorylation induced by DNA double strand breaks or DNA replication stress. Thus, Src family kinases promote checkpoint recovery through termination of ATR- and Chk1-dependent G2 DNA damage checkpoint. These results suggest a model according to which Src family kinases send a termination signal between the completion of DNA repair and the initiation of checkpoint termination. PMID:24634213

  9. Re-purposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints

    PubMed Central

    Beeharry, Neil; Banina, Eugenia; Hittle, James; Skobeleva, Natalia; Khazak, Vladimir; Deacon, Sean; Andrake, Mark; Egleston, Brian L; Peterson, Jeffrey R; Astsaturov, Igor; Yen, Timothy J

    2014-01-01

    Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as “authentic” bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents’ gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient’s tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers. PMID:24955955

  10. Re-purposing clinical kinase inhibitors to enhance chemosensitivity by overriding checkpoints.

    PubMed

    Beeharry, Neil; Banina, Eugenia; Hittle, James; Skobeleva, Natalia; Khazak, Vladimir; Deacon, Sean; Andrake, Mark; Egleston, Brian L; Peterson, Jeffrey R; Astsaturov, Igor; Yen, Timothy J

    2014-01-01

    Inhibitors of the DNA damage checkpoint kinase, Chk1, are highly effective as chemo- and radio-sensitizers in preclinical studies but are not well-tolerated by patients. We exploited the promiscuous nature of kinase inhibitors to screen 9 clinically relevant kinase inhibitors for their ability to sensitize pancreatic cancer cells to a sub-lethal concentration of gemcitabine. Bosutinib, dovitinib, and BEZ-235 were identified as sensitizers that abrogated the DNA damage checkpoint. We further characterized bosutinib, an FDA-approved Src/Abl inhibitor approved for chronic myelogenous leukemia. Unbeknownst to us, we used an isomer (Bos-I) that was unknowingly synthesized and sold to the research community as "authentic" bosutinib. In vitro and cell-based assays showed that both the authentic bosutinib and Bos-I inhibited DNA damage checkpoint kinases Chk1 and Wee1, with Bos-I showing greater potency. Imaging data showed that Bos-I forced cells to override gemcitabine-induced DNA damage checkpoint arrest and destabilized stalled replication forks. These inhibitors enhanced sensitivity to the DNA damaging agents' gemcitabine, cisplatin, and doxorubicin in pancreatic cancer cell lines. The in vivo efficacy of Bos-I was validated using cells derived directly from a pancreatic cancer patient's tumor. Notably, the xenograft studies showed that the combination of gemcitabine and Bos-I was significantly more effective in suppressing tumor growth than either agent alone. Finally, we show that the gatekeeper residue in Wee1 dictates its sensitivity to the 2 compounds. Our strategy to screen clinically relevant kinase inhibitors for off-target effects on cell cycle checkpoints is a promising approach to re-purpose drugs as chemosensitizers.

  11. Transient knock down of checkpoint kinase 1 in hematopoietic progenitors is linked to bone marrow toxicity.

    PubMed

    Hu, Wenyue; Zong, Qing; John-Baptiste, Annette; Jessen, Bart

    2011-07-28

    Checkpoint kinase 1 (Chk1) is required for both intra-S phase and G2/M checkpoints in cell cycle, and plays critical roles in maintaining genomic stability and transducing DNA damage response. Chk1 deficiency has been shown to inhibit T-cell differentiation and resulted in severe anemia in a Chk1 heterozygous mouse model. To date, there has been a good correlation between Chk1 inhibition and in vitro bone marrow toxicity among small molecule inhibitors. To better understand the role of Chk1 in hematopoiesis, we conducted transient Chk1 gene silencing in human bone marrow progenitor cells using siRNA and electroporation. At 48h post electroporation, approximately 70% inhibition of Chk1 was confirmed using real-time RT-PCR and immunoblotting, which resulted in more than 60% reduction in cell count when compared to the non-specific siRNA control on day 6 post-electroporation. This result was confirmed using a colony forming unit assay, where reduced number in both erythroid and granulocyte colonies was observed with Chk1 siRNA treatment. The Chk1 gene inhibition in bone marrow progenitor cells resulted in significant induction of apoptosis, but not cell cycle arrest, as assessed using flow cytometry. In this study an effective method to knock down a gene of interest was established in hard-to-transfect hematopoietic stem cells. Furthermore, our results support a direct role of Chk1 in maintaining normal hematopoiesis in the bone marrow. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

  12. The Protein Kinase Cδ Catalytic Fragment Is Critical for Maintenance of the G2/M DNA Damage Checkpoint*

    PubMed Central

    LaGory, Edward L.; Sitailo, Leonid A.; Denning, Mitchell F.

    2010-01-01

    Protein kinase Cδ (PKCδ) is an essential component of the intrinsic apoptotic program. Following DNA damage, such as exposure to UV radiation, PKCδ is cleaved in a caspase-dependent manner, generating a constitutively active catalytic fragment (PKCδ-cat), which is necessary and sufficient for keratinocyte apoptosis. We found that in addition to inducing apoptosis, expression of PKCδ-cat caused a pronounced G2/M cell cycle arrest in both primary human keratinocytes and immortalized HaCaT cells. Consistent with a G2/M arrest, PKCδ-cat induced phosphorylation of Cdk1 (Tyr15), a critical event in the G2/M checkpoint. Treatment with the ATM/ATR inhibitor caffeine was unable to prevent PKCδ-cat-induced G2/M arrest, suggesting that PKCδ-cat is functioning downstream of ATM/ATR in the G2/M checkpoint. To better understand the role of PKCδ and PKCδ-cat in the cell cycle response to DNA damage, we exposed wild-type and PKCδ null mouse embryonic fibroblasts (MEFs) to UV radiation. Wild-type MEFs underwent a pronounced G2/M arrest, Cdk1 phosphorylation, and induction of apoptosis following UV exposure, whereas PKCδ null MEFs were resistant to these effects. Expression of PKCδ-green fluorescent protein, but not caspase-resistant or kinase-inactive PKCδ, was able to restore G2/M checkpoint integrity in PKCδ null MEFs. The function of PKCδ in the DNA damage-induced G2/M cell cycle checkpoint may be a critical component of its tumor suppressor function. PMID:19917613

  13. Role of Intrinsic and Extrinsic Factors in the Regulation of the Mitotic Checkpoint Kinase Bub1

    PubMed Central

    Breit, Claudia; Bange, Tanja; Petrovic, Arsen; Weir, John R.; Müller, Franziska; Vogt, Doro; Musacchio, Andrea

    2015-01-01

    The spindle assembly checkpoint (SAC) monitors microtubule attachment to kinetochores to ensure accurate sister chromatid segregation during mitosis. The SAC members Bub1 and BubR1 are paralogs that underwent significant functional specializations during evolution. We report an in-depth characterization of the kinase domains of Bub1 and BubR1. BubR1 kinase domain binds nucleotides but is unable to deliver catalytic activity in vitro. Conversely, Bub1 is an active kinase regulated by intra-molecular phosphorylation at the P+1 loop. The crystal structure of the phosphorylated Bub1 kinase domain illustrates a hitherto unknown conformation of the P+1 loop docked into the active site of the Bub1 kinase. Both Bub1 and BubR1 bind Bub3 constitutively. A hydrodynamic characterization of Bub1:Bub3 and BubR1:Bub3 demonstrates both complexes to have 1:1 stoichiometry, with no additional oligomerization. Conversely, Bub1:Bub3 and BubR1:Bub3 combine to form a heterotetramer. Neither BubR1:Bub3 nor Knl1, the kinetochore receptor of Bub1:Bub3, modulate the kinase activity of Bub1 in vitro, suggesting autonomous regulation of the Bub1 kinase domain. We complement our study with an analysis of the Bub1 substrates. Our results contribute to the mechanistic characterization of a crucial cell cycle checkpoint. PMID:26658523

  14. Polo-like kinase-1 regulates kinetochore–microtubule dynamics and spindle checkpoint silencing

    PubMed Central

    Liu, Dan; Davydenko, Olga

    2012-01-01

    Polo-like kinase-1 (Plk1) is a highly conserved kinase with multiple mitotic functions. Plk1 localizes to prometaphase kinetochores and is reduced at metaphase kinetochores, similar to many checkpoint signaling proteins, but Plk1 is not required for spindle checkpoint function. Plk1 is also implicated in stabilizing kinetochore–microtubule attachments, but these attachments are most stable when kinetochore Plk1 levels are low at metaphase. Therefore, it is unclear how Plk1 function at kinetochores can be understood in the context of its dynamic localization. In this paper, we show that Plk1 activity suppresses kinetochore–microtubule dynamics to stabilize initial attachments in prometaphase, and Plk1 removal from kinetochores is necessary to maintain dynamic microtubules in metaphase. Constitutively targeting Plk1 to kinetochores maintained high activity at metaphase, leading to reduced interkinetochore tension and intrakinetochore stretch, a checkpoint-dependent mitotic arrest, and accumulation of microtubule attachment errors. Together, our data show that Plk1 dynamics at kinetochores control two critical mitotic processes: initially establishing correct kinetochore–microtubule attachments and subsequently silencing the spindle checkpoint. PMID:22908307

  15. Lack of Casein Kinase 1 Delta Promotes Genomic Instability - The Accumulation of DNA Damage and Down-Regulation of Checkpoint Kinase 1

    PubMed Central

    Greer, Yoshimi Endo; Gao, Bo; Yang, Yingzi; Nussenzweig, Andre; Rubin, Jeffrey S.

    2017-01-01

    Casein kinase 1 delta (CK1δ) is a conserved serine/threonine protein kinase that regulates diverse cellular processes. Mice lacking CK1δ have a perinatal lethal phenotype and typically weigh 30% less than their wild type littermates. However, the causes of death and small size are unknown. We observed cells with abnormally large nuclei in tissue from Csnk1d null embryos, and multiple centrosomes in mouse embryo fibroblasts (MEFs) deficient in CK1δ (MEFCsnk1d null). Results from γ-H2AX staining and the comet assay demonstrated significant DNA damage in MEFCsnk1d null cells. These cells often contain micronuclei, an indicator of genomic instability. Similarly, abrogation of CK1δ expression in control MEFs stimulated micronuclei formation after doxorubicin treatment, suggesting that CK1δ loss increases vulnerability to genotoxic stress. Cellular levels of total and activated checkpoint kinase 1 (Chk1), which functions in the DNA damage response and mitotic checkpoints, and its downstream effector, Cdc2/CDK1 kinase, were often decreased in MEFCsnk1d null cells as well as in control MEFs transfected with CK1δ siRNA. Hydroxyurea-induced Chk1 activation, as measured by Ser345 phosphorylation, and nuclear localization also were impaired in MEF cells following siRNA knockdown of CK1δ. Similar results were observed in the MCF7 human breast cancer cell line. The decreases in phosphorylated Chk1 were rescued by concomitant expression of siRNA-resistant CK1δ. Experiments with cycloheximide demonstrated that the stability of Chk1 protein was diminished in cells subjected to CK1δ knockdown. Together, these findings suggest that CK1δ contributes to the efficient repair of DNA damage and the proper functioning of mitotic checkpoints by maintaining appropriate levels of Chk1. PMID:28125685

  16. SUMOylation regulates polo-like kinase 1-interacting checkpoint helicase (PICH) during mitosis.

    PubMed

    Sridharan, Vinidhra; Park, Hyewon; Ryu, Hyunju; Azuma, Yoshiaki

    2015-02-06

    Mitotic SUMOylation has an essential role in faithful chromosome segregation in eukaryotes, although its molecular consequences are not yet fully understood. In Xenopus egg extract assays, we showed that poly(ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that its enzymatic activity could be regulated by SUMOylation. To determine the molecular consequence of mitotic SUMOylation, we analyzed SUMOylated PARP1-specific binding proteins. We identified Polo-like kinase 1-interacting checkpoint helicase (PICH) as an interaction partner of SUMOylated PARP1 in Xenopus egg extract. Interestingly, PICH also bound to SUMOylated topoisomerase IIα (TopoIIα), a major centromeric small ubiquitin-like modifier (SUMO) substrate. Purified recombinant human PICH interacted with SUMOylated substrates, indicating that PICH directly interacts with SUMO, and this interaction is conserved among species. Further analysis of mitotic chromosomes revealed that PICH localized to the centromere independent of mitotic SUMOylation. Additionally, we found that PICH is modified by SUMO2/3 on mitotic chromosomes and in vitro. PICH SUMOylation is highly dependent on protein inhibitor of activated STAT, PIASy, consistent with other mitotic chromosomal SUMO substrates. Finally, the SUMOylation of PICH significantly reduced its DNA binding capability, indicating that SUMOylation might regulate its DNA-dependent ATPase activity. Collectively, our findings suggest a novel SUMO-mediated regulation of the function of PICH at mitotic centromeres.

  17. SUMOylation Regulates Polo-like Kinase 1-interacting Checkpoint Helicase (PICH) during Mitosis*

    PubMed Central

    Sridharan, Vinidhra; Park, Hyewon; Ryu, Hyunju; Azuma, Yoshiaki

    2015-01-01

    Mitotic SUMOylation has an essential role in faithful chromosome segregation in eukaryotes, although its molecular consequences are not yet fully understood. In Xenopus egg extract assays, we showed that poly(ADP-ribose) polymerase 1 (PARP1) is modified by SUMO2/3 at mitotic centromeres and that its enzymatic activity could be regulated by SUMOylation. To determine the molecular consequence of mitotic SUMOylation, we analyzed SUMOylated PARP1-specific binding proteins. We identified Polo-like kinase 1-interacting checkpoint helicase (PICH) as an interaction partner of SUMOylated PARP1 in Xenopus egg extract. Interestingly, PICH also bound to SUMOylated topoisomerase IIα (TopoIIα), a major centromeric small ubiquitin-like modifier (SUMO) substrate. Purified recombinant human PICH interacted with SUMOylated substrates, indicating that PICH directly interacts with SUMO, and this interaction is conserved among species. Further analysis of mitotic chromosomes revealed that PICH localized to the centromere independent of mitotic SUMOylation. Additionally, we found that PICH is modified by SUMO2/3 on mitotic chromosomes and in vitro. PICH SUMOylation is highly dependent on protein inhibitor of activated STAT, PIASy, consistent with other mitotic chromosomal SUMO substrates. Finally, the SUMOylation of PICH significantly reduced its DNA binding capability, indicating that SUMOylation might regulate its DNA-dependent ATPase activity. Collectively, our findings suggest a novel SUMO-mediated regulation of the function of PICH at mitotic centromeres. PMID:25564610

  18. Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways.

    PubMed

    Sanchez, Y; Desany, B A; Jones, W J; Liu, Q; Wang, B; Elledge, S J

    1996-01-19

    Mutants of the Saccharomyces cerevisiae ataxia telangiectasia mutated (ATM) homolog MEC1/SAD3/ESR1 were identified that could live only if the RAD53/SAD1 checkpoint kinase was overproduced. MEC1 and a structurally related gene, TEL1, have overlapping functions in response to DNA damage and replication blocks that in mutants can be provided by overproduction of RAD53. Both MEC1 and TEL1 were found to control phosphorylation of Rad53p in response to DNA damage. These results indicate that RAD53 is a signal transducer in the DNA damage and replication checkpoint pathways and functions downstream of two members of the ATM lipid kinase family. Because several members of this pathway are conserved among eukaryotes, it is likely that a RAD53-related kinase will function downstream of the human ATM gene product and play an important role in the mammalian response to DNA damage.

  19. Replication Checkpoint Kinase Cds1 Regulates Recombinational Repair Protein Rad60

    PubMed Central

    Boddy, Michael N.; Shanahan, Paul; Hayes McDonald, W.; Lopez-Girona, Antonia; Noguchi, Eishi; Yates III, John R.; Russell, Paul

    2003-01-01

    Genome integrity is protected by Cds1 (Chk2), a checkpoint kinase that stabilizes arrested replication forks. How Cds1 accomplishes this task is unknown. We report that Cds1 interacts with Rad60, a protein required for recombinational repair in fission yeast. Cds1 activation triggers Rad60 phosphorylation and nuclear delocalization. A Rad60 mutant that inhibits regulation by Cds1 renders cells specifically sensitive to replication fork arrest. Genetic and biochemical studies indicate that Rad60 functions codependently with Smc5 and Smc6, subunits of an SMC (structural maintenance of chromosomes) complex required for recombinational repair. These studies indicate that regulation of Rad60 is an important part of the replication checkpoint response controlled by Cds1. We propose that control of Rad60 regulates recombination events at stalled forks. PMID:12897162

  20. Tethering DNA damage checkpoint mediator proteins topoisomerase IIbeta-binding protein 1 (TopBP1) and Claspin to DNA activates ataxia-telangiectasia mutated and RAD3-related (ATR) phosphorylation of checkpoint kinase 1 (Chk1).

    PubMed

    Lindsey-Boltz, Laura A; Sancar, Aziz

    2011-06-03

    The ataxia-telangiectasia mutated and RAD3-related (ATR) kinase initiates DNA damage signaling pathways in human cells after DNA damage such as that induced upon exposure to ultraviolet light by phosphorylating many effector proteins including the checkpoint kinase Chk1. The conventional view of ATR activation involves a universal signal consisting of genomic regions of replication protein A-covered single-stranded DNA. However, there are some indications that the ATR-mediated checkpoint can be activated by other mechanisms. Here, using the well defined Escherichia coli lac repressor/operator system, we have found that directly tethering the ATR activator topoisomerase IIβ-binding protein 1 (TopBP1) to DNA is sufficient to induce ATR phosphorylation of Chk1 in an in vitro system as well as in vivo in mammalian cells. In addition, we find synergistic activation of ATR phosphorylation of Chk1 when the mediator protein Claspin is also tethered to the DNA with TopBP1. Together, these findings indicate that crowding of checkpoint mediator proteins on DNA is sufficient to activate the ATR kinase.

  1. Lyn tyrosine kinase promotes silencing of ATM-dependent checkpoint signaling during recovery from DNA double-strand breaks

    SciTech Connect

    Fukumoto, Yasunori Kuki, Kazumasa; Morii, Mariko; Miura, Takahito; Honda, Takuya; Ishibashi, Kenichi; Hasegawa, Hitomi; Kubota, Sho; Ide, Yudai; Yamaguchi, Noritaka; Nakayama, Yuji; Yamaguchi, Naoto

    2014-09-26

    Highlights: • Inhibition of Src family kinases decreased γ-H2AX signal. • Inhibition of Src family increased ATM-dependent phosphorylation of Chk2 and Kap1. • shRNA-mediated knockdown of Lyn increased phosphorylation of Kap1 by ATM. • Ectopic expression of Src family kinase suppressed ATM-mediated Kap1 phosphorylation. • Src is involved in upstream signaling for inactivation of ATM signaling. - Abstract: DNA damage activates the DNA damage checkpoint and the DNA repair machinery. After initial activation of DNA damage responses, cells recover to their original states through completion of DNA repair and termination of checkpoint signaling. Currently, little is known about the process by which cells recover from the DNA damage checkpoint, a process called checkpoint recovery. Here, we show that Src family kinases promote inactivation of ataxia telangiectasia mutated (ATM)-dependent checkpoint signaling during recovery from DNA double-strand breaks. Inhibition of Src activity increased ATM-dependent phosphorylation of Chk2 and Kap1. Src inhibition increased ATM signaling both in G2 phase and during asynchronous growth. shRNA knockdown of Lyn increased ATM signaling. Src-dependent nuclear tyrosine phosphorylation suppressed ATM-mediated Kap1 phosphorylation. These results suggest that Src family kinases are involved in upstream signaling that leads to inactivation of the ATM-dependent DNA damage checkpoint.

  2. Structure and Activation Mechanism of the CHK2 DNA Damage Checkpoint Kinase

    SciTech Connect

    Cai, Z.; Chehab, N; Pavletich, N

    2009-01-01

    The CHK2 protein kinase is an important transducer of DNA damage checkpoint signals, and its mutation contributes to hereditary and sporadic cancer. CHK2 activation is triggered by the phosphorylation of Thr68 by the DNA damage-activated ATM kinase. This leads to transient CHK2 dimerization, in part through intermolecular phosphoThr68-FHA domain interactions. Dimerization promotes kinase activation through activation-loop autophosphorylation, but the mechanism of this process has not been clear. The dimeric crystal structure of CHK2, described here, in conjunction with biochemical and mutational data reveals that productive CHK2 dimerization additionally involves intermolecular FHA-kinase domain and FHA-FHA interactions. Ile157, mutated in the Li-Fraumeni cancer-predisposition syndrome, plays a central role in the FHA-kinase domain interface, explaining the lack of dimerization and autophosphorylation of this mutant. In the dimer, the kinase active sites face each other in close proximity, indicating that dimerization may also serve to optimally position the kinase active sites for efficient activation loop transphosphorylation.

  3. The Meiotic Recombination Checkpoint Suppresses NHK-1 Kinase to Prevent Reorganisation of the Oocyte Nucleus in Drosophila

    PubMed Central

    Lancaster, Oscar M.; Breuer, Manuel; Cullen, C. Fiona; Ito, Takashi; Ohkura, Hiroyuki

    2010-01-01

    The meiotic recombination checkpoint is a signalling pathway that blocks meiotic progression when the repair of DNA breaks formed during recombination is delayed. In comparison to the signalling pathway itself, however, the molecular targets of the checkpoint that control meiotic progression are not well understood in metazoans. In Drosophila, activation of the meiotic checkpoint is known to prevent formation of the karyosome, a meiosis-specific organisation of chromosomes, but the molecular pathway by which this occurs remains to be identified. Here we show that the conserved kinase NHK-1 (Drosophila Vrk-1) is a crucial meiotic regulator controlled by the meiotic checkpoint. An nhk-1 mutation, whilst resulting in karyosome defects, does so independent of meiotic checkpoint activation. Rather, we find unrepaired DNA breaks formed during recombination suppress NHK-1 activity (inferred from the phosphorylation level of one of its substrates) through the meiotic checkpoint. Additionally DNA breaks induced by X-rays in cultured cells also suppress NHK-1 kinase activity. Unrepaired DNA breaks in oocytes also delay other NHK-1 dependent nuclear events, such as synaptonemal complex disassembly and condensin loading onto chromosomes. Therefore we propose that NHK-1 is a crucial regulator of meiosis and that the meiotic checkpoint suppresses NHK-1 activity to prevent oocyte nuclear reorganisation until DNA breaks are repaired. PMID:21060809

  4. Crystal Structure of Checkpoint Kinase 2 in Complex with Nsc 109555, a Potent and Selective Inhibitor

    SciTech Connect

    Lountos, George T.; Tropea, Joseph E.; Zhang, Di; Jobson, Andrew G.; Pommier, Yves; Shoemaker, Robert H.; Waugh, David S.

    2009-03-05

    Checkpoint kinase 2 (Chk2), a ser/thr kinase involved in the ATM-Chk2 checkpoint pathway, is activated by genomic instability and DNA damage and results in either arrest of the cell cycle to allow DNA repair to occur or apoptosis if the DNA damage is severe. Drugs that specifically target Chk2 could be beneficial when administered in combination with current DNA-damaging agents used in cancer therapy. Recently, a novel inhibitor of Chk2, NSC 109555, was identified that exhibited high potency (IC{sub 50} = 240 nM) and selectivity. This compound represents a new chemotype and lead for the development of novel Chk2 inhibitors that could be used as therapeutic agents for the treatment of cancer. To facilitate the discovery of new analogs of NSC 109555 with even greater potency and selectivity, we have solved the crystal structure of this inhibitor in complex with the catalytic domain of Chk2. The structure confirms that the compound is an ATP-competitive inhibitor, as the electron density clearly reveals that it occupies the ATP-binding pocket. However, the mode of inhibition differs from that of the previously studied structure of Chk2 in complex with debromohymenialdisine, a compound that inhibits both Chk1 and Chk2. A unique hydrophobic pocket in Chk2, located very close to the bound inhibitor, presents an opportunity for the rational design of compounds with higher binding affinity and greater selectivity.

  5. The metabolic checkpoint kinase mTOR is essential for interleukin-15 signaling during NK cell development and activation

    PubMed Central

    Marçais, Antoine; Degouve, Sophie; Viel, Sébastien; Fenis, Aurore; Rabilloud, Jessica; Mayol, Katia; Tavares, Armelle; Bienvenu, Jacques; Gangloff, Yann-Gaël; Gilson, Eric; Vivier, Eric; Walzer, Thierry

    2014-01-01

    Interleukin-15 (IL-15) controls both the homeostasis and the peripheral activation of Natural Killer (NK) cells. The molecular basis for this duality of action remains unknown. Here we report that the metabolic checkpoint kinase mTOR is activated and boosts bioenergetic metabolism upon NK cell exposure to high concentrations of IL-15 whereas low doses of IL-15 only triggers the phosphorylation of the transcription factor STAT5. mTOR stimulates NK cell growth and nutrient uptake and positively feeds back onto the IL-15 receptor. This process is essential to sustain NK cell proliferation during development and acquisition of cytolytic potential upon inflammation or virus infection. The mTORC1 inhibitor rapamycin inhibits NK cell cytotoxicity both in mouse and human, which likely contribute to the immunosuppressant activities of this drug in different clinical settings. PMID:24973821

  6. Casein kinase II is required for the spindle assembly checkpoint by regulating Mad2p in fission yeast

    SciTech Connect

    Shimada, Midori; Yamamoto, Ayumu; Murakami-Tonami, Yuko; Nakanishi, Makoto; Yoshida, Takashi; Aiba, Hirofumi; Murakami, Hiroshi

    2009-10-23

    The spindle checkpoint is a surveillance mechanism that ensures the fidelity of chromosome segregation in mitosis. Here we show that fission yeast casein kinase II (CK2) is required for this checkpoint function. In the CK2 mutants mitosis occurs in the presence of a spindle defect, and the spindle checkpoint protein Mad2p fails to localize to unattached kinetochores. The CK2 mutants are sensitive to the microtubule depolymerising drug thiabendazole, which is counteracted by ectopic expression of mad2{sup +}. The level of Mad2p is low in the CK2 mutants. These results suggest that CK2 has a role in the spindle checkpoint by regulating Mad2p.

  7. DW-MRI as a Predictive Biomarker of Radiosensitization of GBM through Targeted Inhibition of Checkpoint Kinases.

    PubMed

    Williams, Terence M; Galbán, Stefanie; Li, Fei; Heist, Kevin A; Galbán, Craig J; Lawrence, Theodore S; Holland, Eric C; Thomae, Tami L; Chenevert, Thomas L; Rehemtulla, Alnawaz; Ross, Brian D

    2013-04-01

    The inherent treatment resistance of glioblastoma (GBM) can involve multiple mechanisms including checkpoint kinase (Chk1/2)-mediated increased DNA repair capability, which can attenuate the effects of genotoxic chemotherapies and radiation. The goal of this study was to evaluate diffusion-weighted magnetic resonance imaging (DW-MRI) as a biomarker for Chk1/2 inhibitors in combination with radiation for enhancement of treatment efficacy in GBM. We evaluated a specific small molecule inhibitor of Chk1/2, AZD7762, in combination with radiation using in vitro human cell lines and in vivo using a genetically engineered GBM mouse model. DW-MRI and T1-contrast MRI were used to follow treatment effects on intracranial tumor cellularity and growth rates, respectively. AZD7762 inhibited clonal proliferation in a panel of GBM cell lines and increased radiosensitivity in p53-mutated GBM cell lines to a greater extent compared to p53 wild-type cells. In vivo efficacy of AZD7762 demonstrated a dose-dependent inhibitory effect on GBM tumor growth rate and a reduction in tumor cellularity based on DW-MRI scans along with enhancement of radiation efficacy. DW-MRI was found to be a useful imaging biomarker for the detection of radiosensitization through inhibition of checkpoint kinases. Chk1/2 inhibition resulted in antiproliferative activity, prevention of DNA damage-induced repair, and radiosensitization in preclinical GBM tumor models, both in vitro and in vivo. The effects were found to be maximal in p53-mutated GBM cells. These results provide the rationale for integration of DW-MRI in clinical translation of Chk1/2 inhibition with radiation for the treatment of GBM.

  8. The Aurora B Kinase in Chromosome Bi-Orientation and Spindle Checkpoint Signaling

    PubMed Central

    Krenn, Veronica; Musacchio, Andrea

    2015-01-01

    Aurora B, a member of the Aurora family of serine/threonine protein kinases, is a key player in chromosome segregation. As part of a macromolecular complex known as the chromosome passenger complex, Aurora B concentrates early during mitosis in the proximity of centromeres and kinetochores, the sites of attachment of chromosomes to spindle microtubules. There, it contributes to a number of processes that impart fidelity to cell division, including kinetochore stabilization, kinetochore–microtubule attachment, and the regulation of a surveillance mechanism named the spindle assembly checkpoint. In the regulation of these processes, Aurora B is the fulcrum of a remarkably complex network of interactions that feed back on its localization and activation state. In this review, we discuss the multiple roles of Aurora B during mitosis, focusing in particular on its role at centromeres and kinetochores. Many details of the network of interactions at these locations remain poorly understood, and we focus here on several crucial outstanding questions. PMID:26528436

  9. Rad53 kinase activation-independent replication checkpoint function of the N-terminal forkhead-associated (FHA1) domain.

    PubMed

    Pike, Brietta L; Tenis, Nora; Heierhorst, Jörg

    2004-09-17

    Saccharomyces cerevisiae Rad53 has crucial functions in many aspects of the cellular response to DNA damage and replication blocks. To coordinate these diverse roles, Rad53 has two forkhead-associated (FHA) phosphothreonine-binding domains in addition to a kinase domain. Here, we show that the conserved N-terminal FHA1 domain is essential for the function of Rad53 to prevent the firing of late replication origins in response to replication blocks. However, the FHA1 domain is not required for Rad53 activation during S phase, and as a consequence of defective downstream signaling, Rad53 containing an inactive FHA1 domain is hyperphosphorylated in response to replication blocks. The FHA1 mutation dramatically hypersensitizes strains with defects in the cell cycle-wide checkpoint pathways (rad9Delta and rad17Delta) to DNA damage, but it is largely epistatic with defects in the replication checkpoint (mrc1Delta). Altogether, our data indicate that the FHA1 domain links activated Rad53 to downstream effectors in the replication checkpoint. The results reveal an important mechanistic difference to the homologous Schizosaccharomyces pombe FHA domain that is required for Mrc1-dependent activation of the corresponding Cds1 kinase. Surprisingly, despite the severely impaired replication checkpoint and also G(2)/M checkpoint functions, the FHA1 mutation by itself leads to only moderate viability defects in response to DNA damage, highlighting the importance of functionally redundant pathways.

  10. Design checkpoint kinase 2 inhibitors by pharmacophore modeling and virtual screening techniques.

    PubMed

    Wang, Yen-Ling; Lin, Chun-Yuan; Shih, Kuei-Chung; Huang, Jui-Wen; Tang, Chuan-Yi

    2013-12-01

    Damage to DNA is caused by ionizing radiation, genotoxic chemicals or collapsed replication forks. When DNA is damaged or cells fail to respond, a mutation that is associated with breast or ovarian cancer may occur. Mammalian cells control and stabilize the genome using a cell cycle checkpoint to prevent damage to DNA or to repair damaged DNA. Checkpoint kinase 2 (Chk2) is one of the important kinases, which strongly affects DNA-damage and plays an important role in the response to the breakage of DNA double-strands and related lesions. Therefore, this study concerns Chk2. Its purpose is to find potential inhibitors using the pharmacophore hypotheses (PhModels) and virtual screening techniques. PhModels can identify inhibitors with high biological activities and virtual screening techniques are used to screen the database of the National Cancer Institute (NCI) to retrieve compounds that exhibit all of the pharmacophoric features of potential inhibitors with high interaction energy. Ten PhModels were generated using the HypoGen best algorithm. The established PhModel, Hypo01, was evaluated by performing a cost function analysis of its correlation coefficient (r), root mean square deviation (RMSD), cost difference, and configuration cost, with the values 0.955, 1.28, 192.51, and 16.07, respectively. The result of Fischer's cross-validation test for the Hypo01 model yielded a 95% confidence level, and the correlation coefficient of the testing set (rtest) had a best value of 0.81. The potential inhibitors were then chosen from the NCI database by Hypo01 model screening and molecular docking using the cdocker docking program. Finally, the selected compounds exhibited the identified pharmacophoric features and had a high interaction energy between the ligand and the receptor. Eighty-three potential inhibitors for Chk2 are retrieved for further study.

  11. VISTA is an immune checkpoint molecule for human T cells

    PubMed Central

    Lines, J. Louise; Sempere, Lorenzo F.; Wang, Li; Pantazi, Eirini; Mak, Justin; O’Connell, Samuel; Ceeraz, Sabrina; Suriawinata, Arief A.; Yan, Shaofeng; Ernstoff, Marc S.; Noelle, Randolph

    2014-01-01

    VISTA is a potent negative regulator of T cell function that is expressed on hematopoietic cells and leukocytes. VISTA levels are heightened within the tumor microenvironment where its blockade can enhance anti-tumor immune responses in mice. In humans, blockade of the related PD-1 pathway has shown great potential in clinical immunotherapy trials. Here we report the structure of human VISTA and examine its function in lymphocyte negative regulation in cancer. VISTA is expressed predominantly within the hematopoietic compartment with highest expression within the myeloid lineage. VISTA-Ig suppressed proliferation of T cells but not B cells, blunted production of T cell cytokines and activation markers. Our results establish VISTA as a negative checkpoint regulator that suppresses T cell activation, induces Foxp3 expression and is highly expressed within the tumor microenvironment. By analogy to PD-1 and PD-L1 blockade, VISTA blockade may offer an immunotherapeutic strategy for human cancer. PMID:24691993

  12. Role of the ATM-Checkpoint Kinase 2 Pathway in CDT-Mediated Apoptosis of Gingival Epithelial Cells

    PubMed Central

    Alaoui-El-Azher, Mounia; Mans, Jeffrey J.; Baker, Henry V.; Chen, Casey; Progulske-Fox, Ann; Lamont, Richard J.; Handfield, Martin

    2010-01-01

    The cytolethal distending toxin (CDT) of the oral pathogen Aggregatibacter actinomycetemcomitans induces cell cycle arrest and apoptosis in various cell types. Western analysis, pharmacological inhibition and siRNA silencing were performed in human immortalized gingival keratinocytes (HIGK) to dissect the functional role of the ataxia telangiectasia mutated (ATM) pathway in the signal transduction steps triggered by the CDT. Infection of HIGK was associated with a time-dependent induction of cytoplasmic histone-associated DNA fragmentation. However, in the absence of CDT, infected HIGK underwent reversible DNA strand breaks but not apoptosis, while caspase 3 activity, p21 levels, and HIGK viability were unaffected. Caspase 9 activity was attenuated in the CDT mutant-infected HIGK compared to wild-type infected cells. Pharmacological inhibition and siRNA-silencing of the ATM downstream effector, the protein kinase checkpoint kinase 2 (Chk2), significantly impacted CDT-mediated apoptosis. Together, these findings provide insight on the specificity of the ATM-Chk2 pathway in response to the CDT of A. actinomycetemcomitans in oral epithelial cells, which ultimately leads to apoptosis. We further propose the existence of an unidentified factor that is distinct from the CDT, and involved with a reversible DNA fragmentation that does not trigger terminal apoptosis in oral epithelial cells. This model potentially explains conflicting reports on the biological activity of the A. actinomycetemcomitans CDT. PMID:20668524

  13. Role of the ATM-checkpoint kinase 2 pathway in CDT-mediated apoptosis of gingival epithelial cells.

    PubMed

    Alaoui-El-Azher, Mounia; Mans, Jeffrey J; Baker, Henry V; Chen, Casey; Progulske-Fox, Ann; Lamont, Richard J; Handfield, Martin

    2010-07-23

    The cytolethal distending toxin (CDT) of the oral pathogen Aggregatibacter actinomycetemcomitans induces cell cycle arrest and apoptosis in various cell types. Western analysis, pharmacological inhibition and siRNA silencing were performed in human immortalized gingival keratinocytes (HIGK) to dissect the functional role of the ataxia telangiectasia mutated (ATM) pathway in the signal transduction steps triggered by the CDT. Infection of HIGK was associated with a time-dependent induction of cytoplasmic histone-associated DNA fragmentation. However, in the absence of CDT, infected HIGK underwent reversible DNA strand breaks but not apoptosis, while caspase 3 activity, p21 levels, and HIGK viability were unaffected. Caspase 9 activity was attenuated in the CDT mutant-infected HIGK compared to wild-type infected cells. Pharmacological inhibition and siRNA-silencing of the ATM downstream effector, the protein kinase checkpoint kinase 2 (Chk2), significantly impacted CDT-mediated apoptosis. Together, these findings provide insight on the specificity of the ATM-Chk2 pathway in response to the CDT of A. actinomycetemcomitans in oral epithelial cells, which ultimately leads to apoptosis. We further propose the existence of an unidentified factor that is distinct from the CDT, and involved with a reversible DNA fragmentation that does not trigger terminal apoptosis in oral epithelial cells. This model potentially explains conflicting reports on the biological activity of the A. actinomycetemcomitans CDT.

  14. Chronic signaling via the metabolic checkpoint kinase mTORC1 induces macrophage granuloma formation and marks sarcoidosis progression.

    PubMed

    Linke, Monika; Pham, Ha Thi Thanh; Katholnig, Karl; Schnöller, Thomas; Miller, Anne; Demel, Florian; Schütz, Birgit; Rosner, Margit; Kovacic, Boris; Sukhbaatar, Nyamdelger; Niederreiter, Birgit; Blüml, Stephan; Kuess, Peter; Sexl, Veronika; Müller, Mathias; Mikula, Mario; Weckwerth, Wolfram; Haschemi, Arvand; Susani, Martin; Hengstschläger, Markus; Gambello, Michael J; Weichhart, Thomas

    2017-03-01

    The aggregation of hypertrophic macrophages constitutes the basis of all granulomatous diseases, such as tuberculosis or sarcoidosis, and is decisive for disease pathogenesis. However, macrophage-intrinsic pathways driving granuloma initiation and maintenance remain elusive. We found that activation of the metabolic checkpoint kinase mTORC1 in macrophages by deletion of the gene encoding tuberous sclerosis 2 (Tsc2) was sufficient to induce hypertrophy and proliferation, resulting in excessive granuloma formation in vivo. TSC2-deficient macrophages formed mTORC1-dependent granulomatous structures in vitro and showed constitutive proliferation that was mediated by the neo-expression of cyclin-dependent kinase 4 (CDK4). Moreover, mTORC1 promoted metabolic reprogramming via CDK4 toward increased glycolysis while simultaneously inhibiting NF-κB signaling and apoptosis. Inhibition of mTORC1 induced apoptosis and completely resolved granulomas in myeloid TSC2-deficient mice. In human sarcoidosis patients, mTORC1 activation, macrophage proliferation and glycolysis were identified as hallmarks that correlated with clinical disease progression. Collectively, TSC2 maintains macrophage quiescence and prevents mTORC1-dependent granulomatous disease with clinical implications for sarcoidosis.

  15. Morphogenesis checkpoint kinase Swe1 is the executor of lipolysis-dependent cell-cycle progression

    PubMed Central

    Chauhan, Neha; Visram, Myriam; Cristobal-Sarramian, Alvaro; Sarkleti, Florian

    2015-01-01

    Cell growth and division requires the precise duplication of cellular DNA content but also of membranes and organelles. Knowledge about the cell-cycle–dependent regulation of membrane and storage lipid homeostasis is only rudimentary. Previous work from our laboratory has shown that the breakdown of triacylglycerols (TGs) is regulated in a cell-cycle–dependent manner, by activation of the Tgl4 lipase by the major cyclin-dependent kinase Cdc28. The lipases Tgl3 and Tgl4 are required for efficient cell-cycle progression during the G1/S (Gap1/replication phase) transition, at the onset of bud formation, and their absence leads to a cell-cycle delay. We now show that defective lipolysis activates the Swe1 morphogenesis checkpoint kinase that halts cell-cycle progression by phosphorylation of Cdc28 at tyrosine residue 19. Saturated long-chain fatty acids and phytosphingosine supplementation rescue the cell-cycle delay in the Tgl3/Tgl4 lipase-deficient strain, suggesting that Swe1 activity responds to imbalanced sphingolipid metabolism, in the absence of TG degradation. We propose a model by which TG-derived sphingolipids are required to activate the protein phosphatase 2A (PP2ACdc55) to attenuate Swe1 phosphorylation and its inhibitory effect on Cdc28 at the G1/S transition of the cell cycle. PMID:25713391

  16. DNA damage checkpoint kinase ATM regulates germination and maintains genome stability in seeds.

    PubMed

    Waterworth, Wanda M; Footitt, Steven; Bray, Clifford M; Finch-Savage, William E; West, Christopher E

    2016-08-23

    Genome integrity is crucial for cellular survival and the faithful transmission of genetic information. The eukaryotic cellular response to DNA damage is orchestrated by the DNA damage checkpoint kinases ATAXIA TELANGIECTASIA MUTATED (ATM) and ATM AND RAD3-RELATED (ATR). Here we identify important physiological roles for these sensor kinases in control of seed germination. We demonstrate that double-strand breaks (DSBs) are rate-limiting for germination. We identify that desiccation tolerant seeds exhibit a striking transcriptional DSB damage response during germination, indicative of high levels of genotoxic stress, which is induced following maturation drying and quiescence. Mutant atr and atm seeds are highly resistant to aging, establishing ATM and ATR as determinants of seed viability. In response to aging, ATM delays germination, whereas atm mutant seeds germinate with extensive chromosomal abnormalities. This identifies ATM as a major factor that controls germination in aged seeds, integrating progression through germination with surveillance of genome integrity. Mechanistically, ATM functions through control of DNA replication in imbibing seeds. ATM signaling is mediated by transcriptional control of the cell cycle inhibitor SIAMESE-RELATED 5, an essential factor required for the aging-induced delay to germination. In the soil seed bank, seeds exhibit increased transcript levels of ATM and ATR, with changes in dormancy and germination potential modulated by environmental signals, including temperature and soil moisture. Collectively, our findings reveal physiological functions for these sensor kinases in linking genome integrity to germination, thereby influencing seed quality, crucial for plant survival in the natural environment and sustainable crop production.

  17. Inhibition of aurora B kinase blocks chromosome segregation, overrides the spindle checkpoint, and perturbs microtubule dynamics in mitosis.

    PubMed

    Kallio, Marko J; McCleland, Mark L; Stukenberg, P Todd; Gorbsky, Gary J

    2002-06-04

    How kinetochores correct improper microtubule attachments and regulate the spindle checkpoint signal is unclear. In budding yeast, kinetochores harboring mutations in the mitotic kinase Ipl1 fail to bind chromosomes in a bipolar fashion. In C. elegans and Drosophila, inhibition of the Ipl1 homolog, Aurora B kinase, induces aberrant anaphase and cytokinesis. To study Aurora B kinase in vertebrates, we microinjected mitotic XTC cells with inhibitory antibody and found several related effects. After injection of the antibody, some chromosomes failed to congress to the metaphase plate, consistent with a conserved role for Aurora B in bipolar attachment of chromosomes. Injected cells exited mitosis with no evidence of anaphase or cytokinesis. Injection of anti-Xaurora B antibody also altered the microtubule network in mitotic cells with an extension of the astral microtubules and a reduction of kinetochore microtubules. Finally, inhibition of Aurora B in cultured cells and in cycling Xenopus egg extracts caused escape from the spindle checkpoint arrest induced by microtubule drugs. Our findings implicate Aurora B as a critical coordinator relating changes in microtubule dynamics in mitosis, chromosome movement in prometaphase and anaphase, signaling of the spindle checkpoint, and cytokinesis.

  18. Checkpoint kinase 2 (CHK2) negatively regulates androgen sensitivity and prostate cancer cell growth

    PubMed Central

    Ta, Huy Q; Ivey, Melissa L; Frierson, Henry F; Conaway, Mark R; Dziegielewski, Jaroslaw; Larner, James M; Gioeli, Daniel

    2015-01-01

    Prostate cancer (PCa) is the second leading cause of cancer death in American men, and curing metastatic disease remains a significant challenge. Nearly all patients with disseminated PCa initially respond to androgen deprivation therapy (ADT), but virtually all patient will relapse and develop incurable castration-resistant prostate cancer (CRPC). A high-throughput RNAi screen to identify signaling pathways regulating PCa cell growth led to our discovery that Checkpoint Kinase 2 (CHK2) knockdown dramatically increased PCa growth and hypersensitized cells to low androgen levels. Mechanistic investigations revealed that the effects of CHK2 were dependent on the downstream signaling proteins CDC25C and CDK1. Moreover, CHK2 depletion increased androgen receptor (AR) transcriptional activity on androgen-regulated genes, substantiating the finding that CHK2 affects PCa proliferation, partly, through the AR. Remarkably, we further show that CHK2 is a novel AR-repressed gene, suggestive of a negative feedback loop between CHK2 and AR. Additionally, we provide evidence that CHK2 physically associates with the AR, and that cell cycle inhibition increased this association. Finally, immunohistochemical analysis of CHK2 in prostate cancer patient samples demonstrated a decrease in CHK2 expression in high-grade tumors. In conclusion, we propose that CHK2 is a negative regulator of androgen sensitivity and PCa growth, and that CHK2 signaling is lost during prostate cancer progression to castration resistance. Thus, perturbing CHK2 signaling may offer a new therapeutic approach for sensitizing CRPC to ADT and radiation. PMID:26573794

  19. The cell-cycle checkpoint kinase Chk1 is required for mammalian homologous recombination repair.

    PubMed

    Sørensen, Claus Storgaard; Hansen, Lasse Tengbjerg; Dziegielewski, Jaroslaw; Syljuåsen, Randi G; Lundin, Cecilia; Bartek, Jiri; Helleday, Thomas

    2005-02-01

    The essential checkpoint kinase Chk1 is required for cell-cycle delays after DNA damage or blocked DNA replication. However, it is unclear whether Chk1 is involved in the repair of damaged DNA. Here we establish that Chk1 is a key regulator of genome maintenance by the homologous recombination repair (HRR) system. Abrogation of Chk1 function with small interfering RNA or chemical antagonists inhibits HRR, leading to persistent unrepaired DNA double-strand breaks (DSBs) and cell death after replication inhibition with hydroxyurea or DNA-damage caused by camptothecin. After hydroxyurea treatment, the essential recombination repair protein RAD51 is recruited to DNA repair foci performing a vital role in correct HRR. We demonstrate that Chk1 interacts with RAD51, and that RAD51 is phosphorylated on Thr 309 in a Chk1-dependent manner. Consistent with a functional interplay between Chk1 and RAD51, Chk1-depleted cells failed to form RAD51 nuclear foci after exposure to hydroxyurea, and cells expressing a phosphorylation-deficient mutant RAD51(T309A) were hypersensitive to hydroxyurea. These results highlight a crucial role for the Chk1 signalling pathway in protecting cells against lethal DNA lesions through regulation of HRR.

  20. X-ray structures of checkpoint kinase 2 in complex with inhibitors that target its gatekeeper-dependent hydrophobic pocket

    SciTech Connect

    Lountos, George T.; Jobson, Andrew G.; Tropea, Joseph E.; Self, Christopher R.; Zhang, Guangtao; Pommier, Yves; Shoemaker, Robert H.; Waugh, David S.

    2012-09-17

    The serine/threonine checkpoint kinase 2 (Chk2) is an attractive molecular target for the development of small molecule inhibitors to treat cancer. Here, we report the rational design of Chk2 inhibitors that target the gatekeeper-dependent hydrophobic pocket located behind the adenine-binding region of the ATP-binding site. These compounds exhibit IC{sub 50} values in the low nanomolar range and are highly selective for Chk2 over Chk1. X-ray crystallography was used to determine the structures of the inhibitors in complex with the catalytic kinase domain of Chk2 to verify their modes of binding.

  1. A High Throughput, Whole Cell Screen for Small Molecule Inhibitors of the Mitotic Spindle Checkpoint Identifies OM137, a Novel Aurora Kinase Inhibitor

    PubMed Central

    DeMoe, Joanna H.; Santaguida, Stefano; Daum, John R.; Musacchio, Andrea; Gorbsky, Gary J.

    2008-01-01

    In mitosis the kinetochores of chromosomes that lack full microtubule attachments and/or mechanical tension activate a signaling pathway called the mitotic spindle checkpoint that blocks progression into anaphase and prevents premature segregation of the chromatids until chromosomes become aligned at the metaphase plate (1). The spindle checkpoint is responsible for arresting cells in mitosis in response to chemotherapeutic spindle poisons such as paclitaxel or vinblastine. Some cancer cells show a weakened checkpoint signaling system that may contribute to chromosome instability in tumors. Since complete absence of the spindle checkpoint leads to catastrophic cell division, we reasoned that drugs targeting the checkpoint might provide a therapeutic window in which the checkpoint would be eliminated in cancer cells but sufficiently preserved in normal cells. We developed an assay to identify lead compounds that inhibit the spindle checkpoint. Most cells respond to microtubule drugs by activating the spindle checkpoint and arresting in mitosis with a rounded morphology. Our assay depended on the ability of checkpoint inhibitor compounds to drive mitotic exit and cause cells to flatten onto the substrate in the continuous presence of microtubule drugs. In this study we characterize one of the compounds, OM137, as an inhibitor of Aurora kinases. We find that this compound is growth inhibitory to cultured cells when applied at high concentration and potentiates the growth inhibitory effects of subnanomolar concentrations of paclitaxel. PMID:19190331

  2. Checkpoint Kinase 2 Negatively Regulates Androgen Sensitivity and Prostate Cancer Cell Growth.

    PubMed

    Ta, Huy Q; Ivey, Melissa L; Frierson, Henry F; Conaway, Mark R; Dziegielewski, Jaroslaw; Larner, James M; Gioeli, Daniel

    2015-12-01

    Prostate cancer is the second leading cause of cancer death in American men, and curing metastatic disease remains a significant challenge. Nearly all patients with disseminated prostate cancer initially respond to androgen deprivation therapy (ADT), but virtually all patients will relapse and develop incurable castration-resistant prostate cancer (CRPC). A high-throughput RNAi screen to identify signaling pathways regulating prostate cancer cell growth led to our discovery that checkpoint kinase 2 (CHK2) knockdown dramatically increased prostate cancer growth and hypersensitized cells to low androgen levels. Mechanistic investigations revealed that the effects of CHK2 were dependent on the downstream signaling proteins CDC25C and CDK1. Moreover, CHK2 depletion increased androgen receptor (AR) transcriptional activity on androgen-regulated genes, substantiating the finding that CHK2 affects prostate cancer proliferation, partly, through the AR. Remarkably, we further show that CHK2 is a novel AR-repressed gene, suggestive of a negative feedback loop between CHK2 and AR. In addition, we provide evidence that CHK2 physically associates with the AR and that cell-cycle inhibition increased this association. Finally, IHC analysis of CHK2 in prostate cancer patient samples demonstrated a decrease in CHK2 expression in high-grade tumors. In conclusion, we propose that CHK2 is a negative regulator of androgen sensitivity and prostate cancer growth, and that CHK2 signaling is lost during prostate cancer progression to castration resistance. Thus, perturbing CHK2 signaling may offer a new therapeutic approach for sensitizing CRPC to ADT and radiation. ©2015 American Association for Cancer Research.

  3. Fragment-Based Screening Maps Inhibitor Interactions in the ATP-Binding Site of Checkpoint Kinase 2

    PubMed Central

    Silva-Santisteban, M. Cris; Westwood, Isaac M.; Boxall, Kathy; Brown, Nathan; Peacock, Sam; McAndrew, Craig; Barrie, Elaine; Richards, Meirion; Mirza, Amin; Oliver, Antony W.; Burke, Rosemary; Hoelder, Swen; Jones, Keith; Aherne, G. Wynne; Blagg, Julian; Collins, Ian; Garrett, Michelle D.; van Montfort, Rob L. M.

    2013-01-01

    Checkpoint kinase 2 (CHK2) is an important serine/threonine kinase in the cellular response to DNA damage. A fragment-based screening campaign using a combination of a high-concentration AlphaScreen™ kinase assay and a biophysical thermal shift assay, followed by X-ray crystallography, identified a number of chemically different ligand-efficient CHK2 hinge-binding scaffolds that have not been exploited in known CHK2 inhibitors. In addition, it showed that the use of these orthogonal techniques allowed efficient discrimination between genuine hit matter and false positives from each individual assay technology. Furthermore, the CHK2 crystal structures with a quinoxaline-based fragment and its follow-up compound highlight a hydrophobic area above the hinge region not previously explored in rational CHK2 inhibitor design, but which might be exploited to enhance both potency and selectivity of CHK2 inhibitors. PMID:23776527

  4. Inhibition of the spindle assembly checkpoint kinase Mps-1 as a novel therapeutic strategy in malignant mesothelioma.

    PubMed

    Szymiczek, A; Carbone, M; Pastorino, S; Napolitano, A; Tanji, M; Minaai, M; Pagano, I; Mason, J M; Pass, H I; Bray, M R; Mak, T W; Yang, H

    2017-07-31

    Malignant mesothelioma (MM) is an aggressive malignancy, highly resistant to current medical and surgical therapies, whose tumor cells characteristically show a high level of aneuploidy and genomic instability. We tested our hypothesis that targeting chromosomal instability in MM would improve response to therapy. Thr/Tyr kinase (TTK)/monopolar spindle 1 kinase (Mps-1) is a kinase of the spindle assembly checkpoint that controls cell division and cell fate. CFI-402257 is a novel, selective inhibitor of Mps-1 with antineoplastic activity. We found that CFI-402257 suppresses MM growth. We found that Mps-1 is overexpressed in MM and that its expression correlates with poor patients' outcome. In vitro, CFI-402257-mediated inhibition of Mps-1 resulted in abrogation of the mitotic checkpoint, premature progression through mitosis, marked aneuploidy and mitotic catastrophe. In vivo, CFI-402257 reduced MM growth in an orthotopic, syngeneic model, when used as a single agent, and more so when used in combination with cisplatin+pemetrexed, the current standard of care. Our preclinical findings indicate that CFI-402257 is a promising novel therapeutic agent to improve the efficacy of the current chemotherapeutic regimens for MM patients.Oncogene advance online publication, 31 July 2017; doi:10.1038/onc.2017.266.

  5. Is activation of the intra-S checkpoint in human fibroblasts an important factor in protection against UV-induced mutagenesis?

    PubMed

    Sproul, Christopher D; Rao, Shangbang; Ibrahim, Joseph G; Kaufmann, William K; Cordeiro-Stone, Marila

    2013-11-15

    The ATR/CHK1-dependent intra-S checkpoint inhibits replicon initiation and replication fork progression in response to DNA damage caused by UV (UV) radiation. It has been proposed that this signaling cascade protects against UV-induced mutations by reducing the probability that damaged DNA will be replicated before it can be repaired. Normal human fibroblasts (NHF) were depleted of ATR or CHK1, or treated with the CHK1 kinase inhibitor TCS2312, and the UV-induced mutation frequency at the HPRT locus was measured. Despite clear evidence of S-phase checkpoint abrogation, neither ATR/CHK1 depletion nor CHK1 inhibition caused an increase in the UV-induced HPRT mutation frequency. These results question the premise that the UV-induced intra-S checkpoint plays a prominent role in protecting against UV-induced mutagenesis.

  6. Phase I Study of LY2606368, a Checkpoint Kinase 1 Inhibitor, in Patients With Advanced Cancer

    PubMed Central

    Infante, Jeffrey; Janku, Filip; Jones, Suzanne; Nguyen, Ly M.; Burris, Howard; Naing, Aung; Bauer, Todd M.; Piha-Paul, Sarina; Johnson, Faye M.; Kurzrock, Razelle; Golden, Lisa; Hynes, Scott; Lin, Ji; Lin, Aimee Bence; Bendell, Johanna

    2016-01-01

    Purpose The primary objective was to determine safety, toxicity, and a recommended phase II dose regimen of LY2606368, an inhibitor of checkpoint kinase 1, as monotherapy. Patients and Methods This phase I, nonrandomized, open-label, dose-escalation trial used a 3 + 3 dose-escalation scheme and included patients with advanced solid tumors. Intravenous LY2606368 was dose escalated from 10 to 50 mg/m2 on schedule 1 (days 1 to 3 every 14 days) or from 40 to 130 mg/m2 on schedule 2 (day 1 every 14 days). Safety measures and pharmacokinetics were assessed, and pharmacodynamics were measured in blood, hair follicles, and circulating tumor cells. Results Forty-five patients were treated; seven experienced dose-limiting toxicities (all hematologic). The maximum-tolerated doses (MTDs) were 40 mg/m2 (schedule 1) and 105 mg/m2 (schedule 2). The most common related grade 3 or 4 treatment-emergent adverse events were neutropenia, leukopenia, anemia, thrombocytopenia, and fatigue. Grade 4 neutropenia occurred in 73.3% of patients and was transient (typically < 5 days). Febrile neutropenia incidence was low (7%). The LY2606368 exposure over the first 72 hours (area under the curve from 0 to 72 hours) at the MTD for each schedule coincided with the exposure in mouse xenografts that resulted in maximal tumor responses. Minor intra- and intercycle accumulation of LY2606368 was observed at the MTDs for both schedules. Two patients (4.4%) had a partial response; one had squamous cell carcinoma (SCC) of the anus and one had SCC of the head and neck. Fifteen patients (33.3%) had a best overall response of stable disease (range, 1.2 to 6.7 months), six of whom had SCC. Conclusion An LY2606368 dose of 105 mg/m2 once every 14 days is being evaluated as the recommended phase II dose in dose-expansion cohorts for patients with SCC. PMID:27044938

  7. Lte1 promotes mitotic exit by controlling the localization of the spindle position checkpoint kinase Kin4.

    PubMed

    Falk, Jill E; Chan, Leon Y; Amon, Angelika

    2011-08-02

    For a daughter cell to receive a complete genomic complement, it is essential that the mitotic spindle be positioned accurately within the cell. In budding yeast, a signaling system known as the spindle position checkpoint (SPOC) monitors spindle position and regulates the activity of the mitotic exit network (MEN), a GTPase signaling pathway that promotes exit from mitosis. The protein kinase Kin4 is a central component of the spindle position checkpoint. Kin4 primarily localizes to the mother cell and associates with spindle pole bodies (SPBs) located in the mother cell to inhibit MEN signaling. In contrast, the kinase does not associate with the SPB in the bud. Thus, only when a MEN bearing SPB leaves the mother cell and the spindle is accurately positioned along the mother-bud axis can MEN signaling occur and cell division proceed. Here, we describe a mechanism ensuring that Kin4 only associates with mother cell-located SPBs. The bud-localized MEN regulator Lte1, whose molecular function has long been unclear, prevents Kin4 that escapes into the bud from associating with SPBs in the daughter cell.

  8. Lte1 promotes mitotic exit by controlling the localization of the spindle position checkpoint kinase Kin4

    PubMed Central

    Falk, Jill E.; Chan, Leon Y.; Amon, Angelika

    2011-01-01

    For a daughter cell to receive a complete genomic complement, it is essential that the mitotic spindle be positioned accurately within the cell. In budding yeast, a signaling system known as the spindle position checkpoint (SPOC) monitors spindle position and regulates the activity of the mitotic exit network (MEN), a GTPase signaling pathway that promotes exit from mitosis. The protein kinase Kin4 is a central component of the spindle position checkpoint. Kin4 primarily localizes to the mother cell and associates with spindle pole bodies (SPBs) located in the mother cell to inhibit MEN signaling. In contrast, the kinase does not associate with the SPB in the bud. Thus, only when a MEN bearing SPB leaves the mother cell and the spindle is accurately positioned along the mother–bud axis can MEN signaling occur and cell division proceed. Here, we describe a mechanism ensuring that Kin4 only associates with mother cell-located SPBs. The bud-localized MEN regulator Lte1, whose molecular function has long been unclear, prevents Kin4 that escapes into the bud from associating with SPBs in the daughter cell. PMID:21709215

  9. A Safety Checkpoint to Eliminate Cancer Risk of the Immune Evasive Cells Derived from Human Embryonic Stem Cells.

    PubMed

    He, Jingjin; Rong, Zhili; Fu, Xuemei; Xu, Yang

    2017-01-16

    Human embryonic stem cells (hESCs) hold great promise in the regenerative therapy of many currently untreatable human diseases. One of the key bottlenecks is the immune rejection of hESC-derived allografts by the recipient. To overcome this challenge, we have established new approaches to induce immune protection of hESC-derived allografts through the coexpression of immune suppressive molecules CTLA4-Ig and PD-L1. However, this in turn raises a safety concern of cancer risk because these hESC-derived cells can evade immune surveillance. To address this safety concern, we developed a safety checkpoint so that the immune evasive hESC-derived cells in the graft can be effectively eliminated if any cellular transformation is detected. In this context, we knock-in the suicidal gene herpes simplex virus thymidine kinase (HSVTK) into the constitutive HPRT locus of CP hESCs (knock-in hESCs expressing CTLA4-Ig and PD-L1), denoted CPTK hESCs. Employing humanized mice (Hu-mice) reconstituted with human immune system, we demonstrated that the CPTK hESC-derived cells are protected from immune rejection. In addition, CPTK hESC-derived cells can be efficiently eliminated in vitro and in vivo with FDA approved TK-targeting drug ganciclovir. Therefore, this new safety checkpoint improves the feasibility to use the immune evasive hESC-derived cells for regenerative medicine. Stem Cells 2017.

  10. Combining Immune Checkpoint Inhibitors and Kinase-Inhibiting Supramolecular Therapeutics for Enhanced Anticancer Efficacy.

    PubMed

    Kulkarni, Ashish; Natarajan, Siva Kumar; Chandrasekar, Vineethkrishna; Pandey, Prithvi Raj; Sengupta, Shiladitya

    2016-09-29

    A major limitation of immune checkpoint inhibitors is that only a small subset of patients achieve durable clinical responses. This necessitates the development of combinatorial regimens with immunotherapy. However, some combinations, such as MEK- or PI3K-inhibitors with a PD1-PDL1 checkpoint inhibitor, are pharmacologically challenging to implement. We rationalized that such combinations can be enabled using nanoscale supramolecular targeted therapeutics, which spatially home into tumors and exert temporally sustained inhibition of the target. Here we describe two case studies where nanoscale MEK- and PI3K-targeting supramolecular therapeutics were engineered using a quantum mechanical all-atomistic simulation-based approach. The combinations of nanoscale MEK- and PI3K-targeting supramolecular therapeutics with checkpoint PDL1 and PD1 inhibitors exert enhanced antitumor outcome in melanoma and breast cancers in vivo, respectively. Additionally, the temporal sequence of administration impacts the outcome. The combination of supramolecular therapeutics and immunotherapy could emerge as a paradigm shift in the treatment of cancer.

  11. Aurora B Kinase Regulates the Postmitotic Endoreduplication Checkpoint via Phosphorylation of the Retinoblastoma Protein at Serine 780

    PubMed Central

    Nair, Jayasree S.; Ho, Alan L.; Tse, Archie N.; Coward, Jesse; Cheema, Haider; Ambrosini, Grazia; Keen, Nicholas

    2009-01-01

    The phenotypic change characteristic of Aurora B inhibition is the induction of polyploidy. Utilizing specific siRNA duplexes and a selective small molecule inhibitor (AZD1152) to inhibit Aurora B activity in tumor cells, we sought to elucidate the mechanism by which Aurora B inhibition results in polyploidy. Cells treated with AZD1152 progressed through mitosis with misaligned chromosomes and exited without cytokinesis and subsequently underwent endoreduplication of DNA despite activation of a p53-dependent pseudo G1 checkpoint. Concomitant with polyploid cell formation, we observed the appearance of Rb hypophosphorylation, an event that occurred independently of cyclin-dependent kinase inhibition. We went on to discover that Aurora B directly phosphorylates Rb at serine 780 both in vitro and in vivo. This novel interaction plays a critical role in regulating the postmitotic checkpoint to prevent endoreduplication after an aberrant mitosis. Thus, we propose for the first time that Aurora B determines cellular fate after an aberrant mitosis by directly regulating the Rb tumor suppressor protein. PMID:19225156

  12. Structural analysis reveals features of the spindle checkpoint kinase Bub1–kinetochore subunit Knl1 interaction

    PubMed Central

    Krenn, Veronica; Wehenkel, Annemarie; Santaguida, Stefano

    2012-01-01

    The function of the essential checkpoint kinases Bub1 and BubR1 requires their recruitment to mitotic kinetochores. Kinetochore recruitment of Bub1 and BubR1 is proposed to rely on the interaction of the tetratricopeptide repeats (TPRs) of Bub1 and BubR1 with two KI motifs in the outer kinetochore protein Knl1. We determined the crystal structure of the Bub1 TPRs in complex with the cognate Knl1 KI motif and compared it with the structure of the equivalent BubR1TPR–KI motif complex. The interaction developed along the convex surface of the TPR assembly. Point mutations on this surface impaired the interaction of Bub1 and BubR1 with Knl1 in vitro and in vivo but did not cause significant displacement of Bub1 and BubR1 from kinetochores. Conversely, a 62-residue segment of Bub1 that includes a binding domain for the checkpoint protein Bub3 and is C terminal to the TPRs was necessary and largely sufficient for kinetochore recruitment of Bub1. These results shed light on the determinants of kinetochore recruitment of Bub1. PMID:22331848

  13. Bcl-xL phosphorylation at Ser49 by polo kinase 3 during cell cycle progression and checkpoints

    PubMed Central

    Wang, Jianfang; Beauchemin, Myriam; Bertrand, Richard

    2013-01-01

    Functional analysis of a Bcl-xL phosphorylation mutant series has revealed that cells expressing Bcl-xL(Ser49Ala) mutant are less stable at G2 checkpoint after DNA damage and enter cytokinesis more slowly after microtubule poisoning, than cells expressing wild-type Bcl-xL. These effects of Bcl-xL(Ser49Ala) mutant seem to be separable from Bcl-xL function in apoptosis. Bcl-xL(Ser49) phosphorylation is cell cycle-dependent. In synchronized cells, phospho-Bcl-xL(Ser49) appears during the S phase and G2, whereas it disappears rapidly in early mitosis during prometaphase, metaphase and early anaphase, and re-appears during telophase and cytokinesis. During DNA damage-induced G2 arrest, an important pool of phospho-Bcl-xL(Ser49) accumulates in centrosomes which act as essential decision centers for progression from G2 to mitosis. During telophase/cytokinesis, phospho-Bcl-xL (Ser49) is found with dynein motor protein. In a series of in vitro kinase assays, specific small interfering RNA and pharmacological inhibition experiments, polo kinase 3 (PLK3) was implicated in Bcl-xL(Ser49) phosphorylation. These data indicate that, during G2 checkpoint, phospho-Bcl-xL(Ser49) is another downstream target of PLK3, acting to stabilize G2 arrest. Bcl-xL phosphorylation at Ser49 also correlates with essential PLK3 activity and function, enabling cytokinesis and mitotic exit. PMID:21840391

  14. Targeting Focal Adhesion Kinase Renders Pancreatic Cancers Responsive to Checkpoint Immunotherapy

    PubMed Central

    Jiang, Hong; Hegde, Samarth; Knolhoff, Brett L.; Zhu, Yu; Herndon, John M.; Meyer, Melissa A.; Nywening, Timothy M.; Hawkins, William G.; Shapiro, Irina M.; Weaver, David T.; Pachter, Jonathan A.; Wang-Gillam, Andrea; DeNardo, David G.

    2016-01-01

    Single-agent immunotherapy has achieved limited clinical benefit to date in patients suffering from pancreatic ductal adenocarcinoma (PDAC). This may be due to the presence of a uniquely immunosuppressive tumor microenvironment (TME). Critical obstacles to immunotherapy in PDAC tumors include a high number of tumor-associated immunosuppressive cells and a uniquely desmoplastic stroma that acts as a barrier to T-cell infiltration. We have identified hyperactivated focal adhesion kinase (FAK) activity in neoplastic PDAC cells as a significant regulator of the fibrotic and immunosuppressive TME. We found that FAK activity was elevated in human PDAC tissues and correlates with high levels of fibrosis and poor CD8+ cytotoxic T-cell infiltration. Single-agent FAK inhibition using the selective FAK inhibitor VS-4718 significantly limited tumor progression, resulting in a doubling of survival in the p48-Cre/LSL-KrasG12D/p53Flox/+ (KPC) mouse model of human PDAC. This delay in tumor progression was associated with dramatically reduced tumor fibrosis, and decreased numbers of tumor-infiltrating immunosuppressive cells. We also found that FAK inhibition rendered the previously unresponsive KPC mouse model responsive to T cell immunotherapy and PD-1 antagonists. These data suggest that FAK inhibition increases immune surveillance by overcoming the fibrotic and immunosuppressive PDAC TME and renders tumors responsive to immunotherapy. PMID:27376576

  15. Polo-like kinase 1 inhibitor BI2536 causes mitotic catastrophe following activation of the spindle assembly checkpoint in non-small cell lung cancer cells.

    PubMed

    Choi, Minji; Kim, Wootae; Cheon, Min Gyeong; Lee, Chang Woo; Kim, Ja Eun

    2015-02-28

    Polo-like kinase 1 (PLK1), a critical kinase that regulates multiple steps in mitosis, is overexpressed in diverse human cancers; thus many PLK1 inhibitors have been developed as potential cancer therapeutic agents. One of these compounds, the PLK1-specific inhibitor BI2536, has been investigated as a cytotoxic drug in several cancers, including lung cancer; however, the detailed mechanism by which BI2536 induces defects in cell proliferation of non-small cell lung cancer (NSCLC) has not yet been determined. We found that BI2536 treatment resulted in mitotic arrest due to improper formation of the mitotic spindles and mitotic centrosomes. The unattached kinetochores in BI2536-treated NSCLC cells activated the spindle assembly checkpoint (SAC). The prolonged activation of the SAC led to a type of apoptotic cell death referred to as mitotic catastrophe. Finally, BI2536-treated NSCLC cells show a defect in cell proliferation. Overall, these data indicate that PLK1 inhibition via mitotic disruption represents a potential approach for the treatment of NSCLC. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  16. Mitogen-Activated Protein Kinase Hog1 Mediates Adaptation to G1 Checkpoint Arrest during Arsenite and Hyperosmotic Stress▿

    PubMed Central

    Migdal, Iwona; Ilina, Yulia; Tamás, Markus J.; Wysocki, Robert

    2008-01-01

    Cells slow down cell cycle progression in order to adapt to unfavorable stress conditions. Yeast (Saccharomyces cerevisiae) responds to osmotic stress by triggering G1 and G2 checkpoint delays that are dependent on the mitogen-activated protein kinase (MAPK) Hog1. The high-osmolarity glycerol (HOG) pathway is also activated by arsenite, and the hog1Δ mutant is highly sensitive to arsenite, partly due to increased arsenite influx into hog1Δ cells. Yeast cell cycle regulation in response to arsenite and the role of Hog1 in this process have not yet been analyzed. Here, we found that long-term exposure to arsenite led to transient G1 and G2 delays in wild-type cells, whereas cells that lack the HOG1 gene or are defective in Hog1 kinase activity displayed persistent G1 cell cycle arrest. Elevated levels of intracellular arsenite and “cross talk” between the HOG and pheromone response pathways, observed in arsenite-treated hog1Δ cells, prolonged the G1 delay but did not cause a persistent G1 arrest. In contrast, deletion of the SIC1 gene encoding a cyclin-dependent kinase inhibitor fully suppressed the observed block of G1 exit in hog1Δ cells. Moreover, the Sic1 protein was stabilized in arsenite-treated hog1Δ cells. Interestingly, Sic1-dependent persistent G1 arrest was also observed in hog1Δ cells during hyperosmotic stress. Taken together, our data point to an important role of the Hog1 kinase in adaptation to stress-induced G1 cell cycle arrest. PMID:18552285

  17. The cortical protein Lte1 promotes mitotic exit by inhibiting the spindle position checkpoint kinase Kin4.

    PubMed

    Bertazzi, Daniela Trinca; Kurtulmus, Bahtiyar; Pereira, Gislene

    2011-06-13

    The spindle position checkpoint (SPOC) is an essential surveillance mechanism that allows mitotic exit only when the spindle is correctly oriented along the cell axis. Key SPOC components are the kinase Kin4 and the Bub2-Bfa1 GAP complex that inhibit the mitotic exit-promoting GTPase Tem1. During an unperturbed cell cycle, Kin4 associates with the mother spindle pole body (mSPB), whereas Bub2-Bfa1 is at the daughter SPB (dSPB). When the spindle is mispositioned, Bub2-Bfa1 and Kin4 bind to both SPBs, which enables Kin4 to phosphorylate Bfa1 and thereby block mitotic exit. Here, we show that the daughter cell protein Lte1 physically interacts with Kin4 and inhibits Kin4 kinase activity. Specifically, Lte1 binds to catalytically active Kin4 and promotes Kin4 hyperphosphorylation, which restricts Kin4 binding to the mSPB. This Lte1-mediated exclusion of Kin4 from the dSPB is essential for proper mitotic exit of cells with a correctly aligned spindle. Therefore, Lte1 promotes mitotic exit by inhibiting Kin4 activity at the dSPB.

  18. Structural characterization of inhibitor complexes with checkpoint kinase 2 (Chk2), a drug target for cancer therapy

    SciTech Connect

    Lountos, George T.; Jobson, Andrew G.; Tropea, Joseph E.; Self, Christopher R.; Zhang, Guangtao; Pommier, Yves; Shoemaker, Robert H.; Waugh, David S.

    2012-01-20

    Chk2 (checkpoint kinase 2) is a serine/threonine kinase that participates in a series of signaling networks responsible for maintaining genomic integrity and responding to DNA damage. The development of selective Chk2 inhibitors has recently attracted much interest as a means of sensitizing cancer cells to current DNA-damaging agents used in the treatment of cancer. Additionally, selective Chk2 inhibitors may reduce p53-mediated apoptosis in normal tissues, thereby helping to mitigate adverse side effects from chemotherapy and radiation. Thus far, relatively few selective inhibitors of Chk2 have been described and none have yet progressed into clinical trials. Here, we report crystal structures of the catalytic domain of Chk2 in complex with a novel series of potent and selective small molecule inhibitors. These compounds exhibit nanomolar potencies and are selective for Chk2 over Chk1. The structures reported here elucidate the binding modes of these inhibitors to Chk2 and provide information that can be exploited for the structure-assisted design of novel chemotherapeutics.

  19. The cortical protein Lte1 promotes mitotic exit by inhibiting the spindle position checkpoint kinase Kin4

    PubMed Central

    Bertazzi, Daniela Trinca; Kurtulmus, Bahtiyar

    2011-01-01

    The spindle position checkpoint (SPOC) is an essential surveillance mechanism that allows mitotic exit only when the spindle is correctly oriented along the cell axis. Key SPOC components are the kinase Kin4 and the Bub2–Bfa1 GAP complex that inhibit the mitotic exit–promoting GTPase Tem1. During an unperturbed cell cycle, Kin4 associates with the mother spindle pole body (mSPB), whereas Bub2–Bfa1 is at the daughter SPB (dSPB). When the spindle is mispositioned, Bub2–Bfa1 and Kin4 bind to both SPBs, which enables Kin4 to phosphorylate Bfa1 and thereby block mitotic exit. Here, we show that the daughter cell protein Lte1 physically interacts with Kin4 and inhibits Kin4 kinase activity. Specifically, Lte1 binds to catalytically active Kin4 and promotes Kin4 hyperphosphorylation, which restricts Kin4 binding to the mSPB. This Lte1-mediated exclusion of Kin4 from the dSPB is essential for proper mitotic exit of cells with a correctly aligned spindle. Therefore, Lte1 promotes mitotic exit by inhibiting Kin4 activity at the dSPB. PMID:21670215

  20. Checkpoint kinase1 (CHK1) is an important biomarker in breast cancer having a role in chemotherapy response

    PubMed Central

    Al-kaabi, M M; Alshareeda, A T; Jerjees, D A; Muftah, A A; Green, A R; Alsubhi, N H; Nolan, C C; Chan, S; Cornford, E; Madhusudan, S; Ellis, I O; Rakha, E A

    2015-01-01

    Background: Checkpoint kinase1 (CHK1), which is a key component of DNA-damage-activated checkpoint signalling response, may have a role in breast cancer (BC) pathogenesis and influence response to chemotherapy. This study investigated the clinicopathological significance of phosphorylated CHK1 (pCHK1) protein in BC. Method: pCHK1 protein expression was assessed using immunohistochemistry in a large, well-characterized annotated series of early-stage primary operable invasive BC prepared as tissue microarray (n=1200). Result: pCHK1 showed nuclear and/or cytoplasmic expression. Tumours with nuclear expression showed positive associations with favourable prognostic features such as lower grade, lower mitotic activity, expression of hormone receptor and lack of expression of KI67 and PI3K (P<0.001). On the other hand, cytoplasmic expression was associated with features of poor prognosis such as higher grade, triple-negative phenotype and expression of KI67, p53, AKT and PI3K. pCHK1 expression showed an association with DNA damage response (ATM, RAD51, BRCA1, KU70/KU80, DNA-PKCα and BARD1) and sumoylation (UBC9 and PIASγ) biomarkers. Subcellular localisation of pCHK1 was associated with the expression of the nuclear transport protein KPNA2. Positive nuclear expression predicted better survival outcome in patients who did not receive chemotherapy in the whole series and in ER-positive tumours. In ER-negative and triple-negative subgroups, nuclear pCHK1 predicted shorter survival in patients who received cyclophosphamide, methotrexate and 5-florouracil chemotherapy. Conclusions: Our data suggest that pCHK1 may have prognostic and predictive significance in BC. Subcellular localisation of pCHK1 protein is related to its function. PMID:25688741

  1. Checkpointing filesystem

    DOEpatents

    Gara, Alan G.; Giampapa, Mark E.; Steinmacher-Burow, Burkhard D.

    2005-05-17

    The present in invention is directed to a checkpointing filesystem of a distributed-memory parallel supercomputer comprising a node that accesses user data on the filesystem, the filesystem comprising an interface that is associated with a disk for storing the user data. The checkpointing filesystem provides for taking and checkpoint of the filesystem and rolling back to a previously taken checkpoint, as well as for writing user data to and deleting user data from the checkpointing filesystem. The checkpointing filesystem provides a recently written file allocation table (WFAT) for maintaining information regarding the user data written since a previously taken checkpoint and a recently deleted file allocation table (DFAT) for maintaining information regarding user data deleted from since the previously taken checkpoint, both of which are utilized by the checkpointing filesystem to take a checkpoint of the filesystem and rollback the filesystem to a previously taken checkpoint, as well as to write and delete user data from the checkpointing filesystem.

  2. SCFFBXW7α modulates the intra-S-phase DNA-damage checkpoint by regulating Polo like kinase-1 stability

    PubMed Central

    Giráldez, Servando; Herrero-Ruiz, Joaquín; Mora-Santos, Mar; Japón, Miguel Á.; Tortolero, Maria; Romero, Francisco

    2014-01-01

    The intra-S-checkpoint is essential to control cell progression through S phase under normal conditions and in response to replication stress. When DNA lesions are detected, replication fork progression is blocked allowing time for repair to avoid genomic instability and the risk of cancer. DNA replication initiates at many origins of replication in eukaryotic cells, where a series of proteins form pre-replicative complexes (pre-RCs) that are activated to become pre-initiation complexes and ensure a single round of replication in each cell cycle. PLK1 plays an important role in the regulation of DNA replication, contributing to the regulation of pre-RCs formation by phosphorylating several proteins, under both normal and stress conditions. Here we report that PLK1 is ubiquitinated and degraded by SCFFBXW7α/proteasome. Moreover, we identified a new Cdc4 phosphodegron in PLK1, conserved from yeast to humans, whose mutation prevents PLK1 destruction. We established that endogenous SCFFBXW7α degrades PLK1 in the G1 and S phases of an unperturbed cell cycle and in S phase following UV irradiation. Furthermore, we showed that FBXW7α overexpression or UV irradiation prevented the loading of proteins onto chromatin to form pre-RCs and, accordingly, reduced cell proliferation. We conclude that PLK1 degradation mediated by SCFFBXW7α modulates the intra-S-phase checkpoint. PMID:24970797

  3. The Schizosaccharomyces pombe rad3 checkpoint gene.

    PubMed Central

    Bentley, N J; Holtzman, D A; Flaggs, G; Keegan, K S; DeMaggio, A; Ford, J C; Hoekstra, M; Carr, A M

    1996-01-01

    The rad3 gene of Schizosaccharomyces pombe is required for checkpoint pathways that respond to DNA damage and replication blocks. We report the complete rad3 gene sequence and show that rad3 is the homologue of Saccharomyces cerevisiae ESR1 (MEC1/SAD3) and Drosophila melanogaster mei-41 checkpoint genes. This establishes Rad3/Mec1 as the only conserved protein which is required for all the DNA structure checkpoints in both yeast model systems. Rad3 is an inessential member of the 'lipid kinase' subclass of kinases which includes the ATM protein defective in ataxia telangiectasia patients. Mutational analysis indicates that the kinase domain is required for Rad3 function, and immunoprecipitation of overexpressed Rad3 demonstrates an associated protein kinase activity. The previous observation that rad3 mutations can be rescued by a truncated clone lacking the kinase domain may be due to intragenic complementation. Consistent with this, biochemical data suggest that Rad3 exists in a complex containing multiple copies of Rad3. We have identified a novel human gene (ATR) whose product is closely related to Rad3/Esr1p/Mei-41. ATR can functionally complement esr1-1 radiation sensitivity in S. cerevisiae. Together, the structural conservation and functional complementation suggest strongly that the mechanisms underlying the DNA structure checkpoints are conserved throughout evolution. Images PMID:8978690

  4. RACH2, a novel human gene that complements a fission yeast cell cycle checkpoint mutation.

    PubMed Central

    Davey, S; Beach, D

    1995-01-01

    We have identified a novel human gene by virtue of its ability to complement the rad1-1 checkpoint mutant of Schizosaccharomyces pombe. This gene, called RACH2, rescues the temperature-sensitive lethality of a rad1-1 wee1-50 double mutant of S. pombe. Expression of RACH2 in S. pombe rad1-1 strains partially restores UV resistance to the rad1-1 mutant strain. Expression of RACH2 in a rad1-1 cdc25-22 double mutant partially restores the dose-dependent delay in mitotic entry after irradiation that is lost in rad1-1 checkpoint-deficient mutants. Overexpression of RACH2 in human tissue culture cells induces apoptosis. Images PMID:8573795

  5. Inhibition of checkpoint kinase 1 sensitizes lung cancer brain metastases to radiotherapy

    SciTech Connect

    Yang, Heekyoung; Yoon, Su Jin; Jin, Juyoun; Choi, Seung Ho; Seol, Ho Jun; Lee, Jung-Il; and others

    2011-03-04

    Research highlights: {yields} The most important therapeutic tool in brain metastasis is radiation therapy. {yields} Radiosensitivity of cancer cells was enhanced with treatment of Chk1 inhibitor. {yields} Depletion of Chk1 in cancer cells showed an enhancement of sensitivity to radiation. {yields} Chk1 can be a good target for enhancement of radiosensitivity. -- Abstract: The most important therapeutic tool in brain metastasis is radiation therapy. However, resistance to radiation is a possible cause of recurrence or treatment failure. Recently, signal pathways about DNA damage checkpoints after irradiation have been noticed. We investigated the radiosensitivity can be enhanced with treatment of Chk1 inhibitor, AZD7762 in lung cancer cell lines and xenograft models of lung cancer brain metastasis. Clonogenic survival assays showed enhancement of radiosensitivity with AZD7762 after irradiation of various doses. AZD7762 increased ATR/ATM-mediated Chk1 phosphorylation and stabilized Cdc25A, suppressed cyclin A expression in lung cancer cell lines. In xenograft models of lung cancer (PC14PE6) brain metastasis, AZD7762 significantly prolonged the median survival time in response to radiation. Depletion of Chk1 using shRNA also showed an enhancement of sensitivity to radiation in PC14PE6 cells. The results of this study support that Chk1 can be a good target for enhancement of radiosensitivity.

  6. Coupling of human DNA excision repair and the DNA damage checkpoint in a defined in vitro system.

    PubMed

    Lindsey-Boltz, Laura A; Kemp, Michael G; Reardon, Joyce T; DeRocco, Vanessa; Iyer, Ravi R; Modrich, Paul; Sancar, Aziz

    2014-02-21

    DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5' to 3' exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5' to 3' exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.

  7. Variation in the checkpoint kinase 2 gene is associated with type 2 diabetes in multiple populations

    PubMed Central

    Franceschini, Nora; Avery, Christy L.; Baird, Lisa; Graff, Mariaelisa; Leppert, Mark; Chung, Jay H.; Zhang, Jinghui; Hanis, Craig; Boerwinkle, Eric; Volcik, Kelly A.; Grove, Megan L.; Mosley, Thomas H.; Gu, Charles; Heiss, Gerardo; Pankow, James S.; Couper, David J.; Ballantyne, Christie M.; Linda Kao, W. H.; Weder, Alan B.; Cooper, Richard S.; Ehret, Georg B.; O'Connor, Ashley A.; Chakravarti, Aravinda; Hunt, Steven C.

    2010-01-01

    Identification and characterization of the genetic variants underlying type 2 diabetes susceptibility can provide important understanding of the etiology and pathogenesis of type 2 diabetes. We previously identified strong evidence of linkage for type 2 diabetes on chromosome 22 among 3,383 Hypertension Genetic Epidemiology Network (HyperGEN) participants from 1,124 families. The checkpoint 2 (CHEK2) gene, an important mediator of cellular responses to DNA damage, is located 0.22 Mb from this linkage peak. In this study, we tested the hypothesis that the CHEK2 gene contains one or more polymorphic variants that are associated with type 2 diabetes in HyperGEN individuals. In addition, we replicated our findings in two other Family Blood Pressure Program (FBPP) populations and in the population-based Atherosclerosis Risk in Communities (ARIC) study. We genotyped 1,584 African-American and 1,531 white HyperGEN participants, 1,843 African-American and 1,569 white GENOA participants, 871 African-American and 1,009 white GenNet participants, and 4,266 African-American and 11,478 white ARIC participants for four single nucleotide polymorphisms (SNPs) in CHEK2. Using additive models, we evaluated the association of CHEK2 SNPs with type 2 diabetes in participants within each study population stratified by race, and in a meta-analysis, adjusting for age, age2, sex, sex-by-age interaction, study center, and relatedness. One CHEK2 variant, rs4035540, was associated with an increased risk of type 2 diabetes in HyperGEN participants, two replication samples, and in the meta-analysis. These results may suggest a new pathway in the pathogenesis of type 2 diabetes that involves pancreatic beta-cell damage and apoptosis. PMID:19855918

  8. Interaction between human MCM7 and Rad17 proteins is required for replication checkpoint signaling

    PubMed Central

    Tsao, Cheng-Chung; Geisen, Christoph; Abraham, Robert T

    2004-01-01

    Human Rad17 (hRad17) is centrally involved in the activation of cell-cycle checkpoints by genotoxic agents or replication stress. Here we identify hMCM7, a core component of the DNA replication apparatus, as a novel hRad17-interacting protein. In HeLa cells, depletion of either hRad17 or hMCM7 with small-interfering RNA suppressed ultraviolet (UV) light- or aphidicolin-induced hChk1 phosphorylation, and abolished UV-induced S-phase checkpoint activation. Similar results were obtained after transfection of these cells with a fusion protein containing the hMCM7-binding region of hRad17. The hMCM7-depleted cells were also defective for the formation of ATR-containing nuclear foci after UV irradiation, suggesting that hMCM7 is required for stable recruitment of ATR to damaged DNA. These results demonstrate that hMCM7 plays a direct role in the transmission of DNA damage signals from active replication forks to the S-phase checkpoint machinery in human cells. PMID:15538388

  9. Checkpoint kinase 1 expression is an adverse prognostic marker and therapeutic target in MYC-driven medulloblastoma

    PubMed Central

    Shah, Monil; Mulcahy Levy, Jean M.; Griesinger, Andrea M.; Alimova, Irina; Harris, Peter S.; Birks, Diane K.; Donson, Andrew M.; Davidson, Nathan; Remke, Marc; Taylor, Michael D.; Handler, Michael H.; Foreman, Nicholas K.; Venkataraman, Sujatha; Vibhakar, Rajeev

    2016-01-01

    Checkpoint kinase 1 (CHK1) is an integral component of the cell cycle as well as the DNA Damage Response (DDR) pathway. Previous work has demonstrated the effectiveness of inhibiting CHK1 with small-molecule inhibitors, but the role of CHK1 mediated DDR in medulloblastoma is unknown. CHK1, both at the mRNA and protein level, is highly expressed in medulloblastoma and elevated CHK1 expression in Group3 medulloblastoma is an adverse prognostic marker. CHK1 inhibition with the small-molecule drug AZD7762, results in decreased cell growth, increased DNA damage and cell apoptosis. Furthermore, AZD7762 acts in synergy with cisplatin in reducing cell proliferation in medulloblastoma. Similar phenotypic changes were observed with another CHK1 inhibitor, PF477736, as well as genetic knockdown using siRNA against CHK1. Treatments with small-molecule inhibitors of CHK1 profoundly modulated the expression of both upstream and downstream target proteins within the CHK1 signaling pathways. This suggests the presence of a feedback loop in activating CHK1. Overall, our results demonstrate that small-molecule inhibition of CHK1 in combination with, cisplatin, is more advantageous than either treatment alone, especially for Group 3 medulloblastoma, and therefore this combined therapeutic approach serves as an avenue for further investigation. PMID:27449089

  10. The checkpoint kinase ATM protects against stress-induced elevation of cyclin D1 and potential cell death in neurons.

    PubMed

    Hitomi, Masahiro; Stacey, Dennis W

    2010-06-01

    Quantitative cytometric studies show that cyclin D1 levels must decline during S phase for proper cell cycle progression, and that cyclin D1 decline follows phosphorylation induced by the checkpoint kinases ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR). ATM is mutated in ataxia telangiectasia (AT), a disease characterized by progressive neurodegeneration. Importantly, neurodegeneration in many cases has been linked to the increased expression of cyclin D1 in neurons leading to inappropriate cell cycle entry. These facts prompted us to test the possibility that ATM normally protects against neural degeneration by suppressing cyclin D1 levels, particularly following genotoxic stress. For this purpose, neural stem cells were induced to differentiate into mature neural cells, including neurons. ATM activity in these cultures was inhibited with a specific chemical inhibitor in the presence or absence of hydrogen peroxide treatment, and the effect on cyclin D1 expression was determined by quantitative, single cell cytometric analyses. As predicted, inhibition of ATM did promote elevation of cyclin D1 in differentiated neurons, particularly under conditions of oxidative stress. The survival of differentiated neurons and of neural stem cells was reduced by such treatments. These data support our suggestion that ATM functions to maintain low levels of cyclin D1 expression in differentiated neurons; and may provide important clues in understanding neural degeneration in general. Copyright 2010 International Society for Advancement of Cytometry.

  11. The Dimeric Architecture of Checkpoint Kinases Mec1ATR and Tel1ATM Reveal a Common Structural Organization*

    PubMed Central

    Sawicka, Marta; Wanrooij, Paulina H.; Darbari, Vidya C.; Tannous, Elias; Hailemariam, Sarem; Bose, Daniel; Makarova, Alena V.; Burgers, Peter M.; Zhang, Xiaodong

    2016-01-01

    The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1·Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT (named after Huntingtin, elongation factor 3, protein phosphatase 2A, and yeast kinase TOR1) repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1·Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form. PMID:27129217

  12. The Dimeric Architecture of Checkpoint Kinases Mec1ATR and Tel1ATM Reveal a Common Structural Organization.

    PubMed

    Sawicka, Marta; Wanrooij, Paulina H; Darbari, Vidya C; Tannous, Elias; Hailemariam, Sarem; Bose, Daniel; Makarova, Alena V; Burgers, Peter M; Zhang, Xiaodong

    2016-06-24

    The phosphatidylinositol 3-kinase-related protein kinases are key regulators controlling a wide range of cellular events. The yeast Tel1 and Mec1·Ddc2 complex (ATM and ATR-ATRIP in humans) play pivotal roles in DNA replication, DNA damage signaling, and repair. Here, we present the first structural insight for dimers of Mec1·Ddc2 and Tel1 using single-particle electron microscopy. Both kinases reveal a head to head dimer with one major dimeric interface through the N-terminal HEAT (named after Huntingtin, elongation factor 3, protein phosphatase 2A, and yeast kinase TOR1) repeat. Their dimeric interface is significantly distinct from the interface of mTOR complex 1 dimer, which oligomerizes through two spatially separate interfaces. We also observe different structural organizations of kinase domains of Mec1 and Tel1. The kinase domains in the Mec1·Ddc2 dimer are located in close proximity to each other. However, in the Tel1 dimer they are fully separated, providing potential access of substrates to this kinase, even in its dimeric form.

  13. Identification of Residues in Fission Yeast and Human P34(cdc2) Required for S-M Checkpoint Control

    PubMed Central

    Basi, G.; Enoch, T.

    1996-01-01

    In fission yeast, regulation of p34(cdc2) plays an important role in the checkpoint coupling mitosis to completion of DNA replication. The cdc2 mutations cdc2-3w (C67Y) and cdc2-4w (C67F) abolish checkpoint control without seriously affecting normal cell proliferation. However the molecular basis of this phenotype is not known. To better understand the role of p34(cdc2) in checkpoint control, we have screened for more mutations in Schizosaccharomyces pombe cdc2 with this phenotype. We have isolated cdc2-3w and cdc2-4w, as well as three new cdc2 alleles: cdc2-6w (N66I), cdc2-7w (E8V) and cdc2-8w (K9E). The altered residues map to two different regions on opposite faces of the protein, suggesting that the interaction between p34(cdc2) and components of the checkpoint pathway may be complex. In contrast to cdc2-3w and cdc2-4w, the new mutations alter residues that are conserved between the fission yeast cdc2(+) and other cdks, including the human CDC2 protein. Expression of the equivalent human CDC2 mutants in fission yeast abolishes checkpoint control, suggesting that these residues could be involved in checkpoint-dependent regulation of other eukaryotic cdks. PMID:8978030

  14. Sensor and effector kinases in DNA damage checkpoint regulate capacity for homologous recombination repair of fission yeast in G2 phase.

    PubMed

    Yasuhira, Shinji; Saito, Takeshi; Maesawa, Chihaya; Masuda, Tomoyuki

    2012-08-01

    Although the G2/M DNA damage checkpoint is currently viewed as a set of coordinated cellular responses affecting both cell cycle progression and non-cell cycle targets, the relative contributions of the two target categories to DNA repair and cell survival after exposure to ionizing radiation have not been clearly addressed. We investigated how rad3 (ATR ortholog) or chk1/cds1 (CHK1/CHK2 orthologs) null mutations change the kinetics of double-strand break (DSB) repair in Schizosaccharomyces pombe cells under conditions of forced G2 arrest. After 200-Gy γ-ray irradiation, DSBs were repaired in rad3Δ cdc25-22 or chk1Δ cds1Δ cdc25-22 cells, almost as efficiently as in cdc25-22 cells at the restrictive temperature. In contrast, little repair was observed in the checkpoint-deficient cells up to 4h after higher-dose (500Gy) irradiation, whereas repair was still efficient in the control cdc25-22 cells. Immediate loss of viability appeared not be responsible for the repair defect after the higher dose, since both checkpoint-proficient and deficient cells with cdc25-22 allele synchronously resumed cycling with a similar time course when released to the permissive temperature 4h after irradiation. Recruitment of repair proteins Rad11 (Rpa1 ortholog), Rad22 (Rad52 ortholog), and Rhp54 (Rad54 ortholog) to the damage sites was not significantly impaired in the checkpoint-deficient cells, whereas their release was profoundly delayed. Our results suggest that sensor and effector kinases in the damage checkpoint machinery affect the efficiency of repair downstream of, or in parallel with the core repair reaction. Copyright © 2012 Elsevier B.V. All rights reserved.

  15. Discovery of 1,4-dihydroindeno[1,2-c]pyrazoles as a novel class of potent and selective checkpoint kinase 1 inhibitors.

    PubMed

    Tong, Yunsong; Claiborne, Akiyo; Stewart, Kent D; Park, Chang; Kovar, Peter; Chen, Zehan; Credo, Robert B; Gu, Wen-Zhen; Gwaltney, Stephen L; Judge, Russell A; Zhang, Haiying; Rosenberg, Saul H; Sham, Hing L; Sowin, Thomas J; Lin, Nan-Horng

    2007-04-01

    A new class of checkpoint kinase 1 (CHK-1) inhibitors bearing a 1,4-dihydroindeno[1,2-c]pyrazole core was developed after initial hits from high throughput screening. The efficient hit-to-lead process was facilitated by X-ray crystallography and led to potent inhibitors (<10nM) against CHK-1. X-ray co-crystal structures of bound inhibitors demonstrated that two sub-series of this class of compounds, exemplified by 21 and 41, exhibit distinctive hydrogen bonding patterns in the specificity pocket of the active site. Two compounds, 41 and 43, were capable of potentiating doxorubicin and camptothecin, both DNA-damaging agents, in cell proliferation assays (MTS and soft agar assays) and abrogating G2/M checkpoint in a mechanism-based FACS assay.

  16. A Bioinformatics Approach Identifies Signal Transducer and Activator of Transcription-3 and Checkpoint Kinase 1 as Upstream Regulators of Kidney Injury Molecule-1 after Kidney Injury

    PubMed Central

    Ajay, Amrendra Kumar; Kim, Tae-Min; Ramirez-Gonzalez, Victoria; Park, Peter J.; Frank, David A.

    2014-01-01

    Kidney injury molecule-1 (KIM-1)/T cell Ig and mucin domain-containing protein-1 (TIM-1) is upregulated more than other proteins after AKI, and it is highly expressed in renal damage of various etiologies. In this capacity, KIM-1/TIM-1 acts as a phosphatidylserine receptor on the surface of injured proximal tubular epithelial cells, mediating phagocytosis of apoptotic cells, and it may also act as a costimulatory molecule for immune cells. Despite recognition of KIM-1 as an important therapeutic target for kidney disease, the regulators of KIM-1 transcription in the kidney remain unknown. Using a bioinformatics approach, we identified upstream regulators of KIM-1 after AKI. In response to tubular injury in rat and human kidneys or oxidant stress in human proximal tubular epithelial cells (HPTECs), KIM-1 expression increased significantly in a manner that corresponded temporally and regionally with increased phosphorylation of checkpoint kinase 1 (Chk1) and STAT3. Both ischemic and oxidant stress resulted in a dramatic increase in reactive oxygen species that phosphorylated and activated Chk1, which subsequently bound to STAT3, phosphorylating it at S727. Furthermore, STAT3 bound to the KIM-1 promoter after ischemic and oxidant stress, and pharmacological or genetic induction of STAT3 in HPTECs increased KIM-1 mRNA and protein levels. Conversely, inhibition of STAT3 using siRNAs or dominant negative mutants reduced KIM-1 expression in a kidney cancer cell line (769-P) that expresses high basal levels of KIM-1. These observations highlight Chk1 and STAT3 as critical upstream regulators of KIM-1 expression after AKI and may suggest novel approaches for therapeutic intervention. PMID:24158981

  17. A bioinformatics approach identifies signal transducer and activator of transcription-3 and checkpoint kinase 1 as upstream regulators of kidney injury molecule-1 after kidney injury.

    PubMed

    Ajay, Amrendra Kumar; Kim, Tae-Min; Ramirez-Gonzalez, Victoria; Park, Peter J; Frank, David A; Vaidya, Vishal S

    2014-01-01

    Kidney injury molecule-1 (KIM-1)/T cell Ig and mucin domain-containing protein-1 (TIM-1) is upregulated more than other proteins after AKI, and it is highly expressed in renal damage of various etiologies. In this capacity, KIM-1/TIM-1 acts as a phosphatidylserine receptor on the surface of injured proximal tubular epithelial cells, mediating phagocytosis of apoptotic cells, and it may also act as a costimulatory molecule for immune cells. Despite recognition of KIM-1 as an important therapeutic target for kidney disease, the regulators of KIM-1 transcription in the kidney remain unknown. Using a bioinformatics approach, we identified upstream regulators of KIM-1 after AKI. In response to tubular injury in rat and human kidneys or oxidant stress in human proximal tubular epithelial cells (HPTECs), KIM-1 expression increased significantly in a manner that corresponded temporally and regionally with increased phosphorylation of checkpoint kinase 1 (Chk1) and STAT3. Both ischemic and oxidant stress resulted in a dramatic increase in reactive oxygen species that phosphorylated and activated Chk1, which subsequently bound to STAT3, phosphorylating it at S727. Furthermore, STAT3 bound to the KIM-1 promoter after ischemic and oxidant stress, and pharmacological or genetic induction of STAT3 in HPTECs increased KIM-1 mRNA and protein levels. Conversely, inhibition of STAT3 using siRNAs or dominant negative mutants reduced KIM-1 expression in a kidney cancer cell line (769-P) that expresses high basal levels of KIM-1. These observations highlight Chk1 and STAT3 as critical upstream regulators of KIM-1 expression after AKI and may suggest novel approaches for therapeutic intervention.

  18. Checkpoint Kinase 1 Activation Enhances Intestinal Epithelial Barrier Function via Regulation of Claudin-5 Expression.

    PubMed

    Watari, Akihiro; Hasegawa, Maki; Yagi, Kiyohito; Kondoh, Masuo

    2016-01-01

    Several stressors are known to influence epithelial tight junction (TJ) integrity, but the association between DNA damage and TJ integrity remains unclear. Here we examined the effects of daunorubicin and rebeccamycin, two anti-tumor chemicals that induce DNA damage, on TJ integrity in human intestinal epithelial cells. Daunorubicin and rebeccamycin dose-dependently enhanced transepithelial electrical resistance (TER) and decreased flux of the 4 kDa FITC-dextran in Caco-2 cell monolayer. Daunorubicin- or rebeccamycin-induced enhancement of the TJ barrier function partly rescued attenuation of the barrier function by the inflammatory cytokines TNF-α and IFN-γ. Daunorubicin and rebeccamycin increased claudin-5 expression and the product was distributed in the actin cytoskeleton fraction, which was enriched with TJ proteins. Caffeine, which is an inhibitor of ataxia telangiectasia mutated protein (ATM) and ataxia telangiectasia mutated and Rad3-related protein (ATR), and the Chk1 inhibitor inhibited the TER increases induced by daunorubicin and rebeccamycin, whereas a Chk2 inhibitor did not. Treatment with Chk1 siRNA also significantly inhibited the TER increases. Induction of claudin-5 expression was inhibited by Chk1 inhibitor and by siRNA treatment. Our results suggest that Chk1 activation by daunorubicin and rebeccamycin induced claudin-5 expression and enhanced TJ barrier function in Caco-2 cell monolayer, which suggests a link between DNA damage and TJ integrity in the human intestine.

  19. Checkpoint Kinase 1 Activation Enhances Intestinal Epithelial Barrier Function via Regulation of Claudin-5 Expression

    PubMed Central

    Watari, Akihiro; Hasegawa, Maki; Yagi, Kiyohito; Kondoh, Masuo

    2016-01-01

    Several stressors are known to influence epithelial tight junction (TJ) integrity, but the association between DNA damage and TJ integrity remains unclear. Here we examined the effects of daunorubicin and rebeccamycin, two anti-tumor chemicals that induce DNA damage, on TJ integrity in human intestinal epithelial cells. Daunorubicin and rebeccamycin dose-dependently enhanced transepithelial electrical resistance (TER) and decreased flux of the 4 kDa FITC-dextran in Caco-2 cell monolayer. Daunorubicin- or rebeccamycin-induced enhancement of the TJ barrier function partly rescued attenuation of the barrier function by the inflammatory cytokines TNF-α and IFN-γ. Daunorubicin and rebeccamycin increased claudin-5 expression and the product was distributed in the actin cytoskeleton fraction, which was enriched with TJ proteins. Caffeine, which is an inhibitor of ataxia telangiectasia mutated protein (ATM) and ataxia telangiectasia mutated and Rad3-related protein (ATR), and the Chk1 inhibitor inhibited the TER increases induced by daunorubicin and rebeccamycin, whereas a Chk2 inhibitor did not. Treatment with Chk1 siRNA also significantly inhibited the TER increases. Induction of claudin-5 expression was inhibited by Chk1 inhibitor and by siRNA treatment. Our results suggest that Chk1 activation by daunorubicin and rebeccamycin induced claudin-5 expression and enhanced TJ barrier function in Caco-2 cell monolayer, which suggests a link between DNA damage and TJ integrity in the human intestine. PMID:26727128

  20. Identification of CD112R as a novel checkpoint for human T cells

    PubMed Central

    Paniccia, Alessandro; Schulick, Alexander C.; Chen, Wei; Koenig, Michelle R.; Byers, Joshua T.; Yao, Sheng; Bevers, Shaun

    2016-01-01

    T cell immunoglobulin and ITIM domain (TIGIT) and CD226 emerge as a novel T cell cosignaling pathway in which CD226 and TIGIT serve as costimulatory and coinhibitory receptors, respectively, for the ligands CD155 and CD112. In this study, we describe CD112R, a member of poliovirus receptor–like proteins, as a new coinhibitory receptor for human T cells. CD112R is preferentially expressed on T cells and inhibits T cell receptor–mediated signals. We further identify that CD112, widely expressed on antigen-presenting cells and tumor cells, is the ligand for CD112R with high affinity. CD112R competes with CD226 to bind to CD112. Disrupting the CD112R–CD112 interaction enhances human T cell response. Our experiments identify CD112R as a novel checkpoint for human T cells via interaction with CD112. PMID:26755705

  1. Design of targeted libraries against the human Chk1 kinase using PGVL Hub.

    PubMed

    Peng, Zhengwei; Hu, Qiyue

    2011-01-01

    PGVL Hub is a Pfizer internal desktop tool for chemical library and singleton design. In this chapter, we give a short introduction to PGVL Hub, the core workflow it supports, and the rich design capabilities it provides. By re-creating two legacy targeted libraries against the human checkpoint kinase 1 (Chk1) as a showcase, we illustrate how PGVL Hub could be used to help library designers carry out the steps in library design and realize design objectives such as SAR expansion and improvement in both kinase selectivity and compound aqueous solubility. Finally we share several tips about library design and usage of PGVL Hub.

  2. The checkpoint for agonist selection precedes conventional selection in human thymus.

    PubMed

    Verstichel, Greet; Vermijlen, David; Martens, Liesbet; Goetgeluk, Glenn; Brouwer, Margreet; Thiault, Nicolas; Van Caeneghem, Yasmine; De Munter, Stijn; Weening, Karin; Bonte, Sarah; Leclercq, Georges; Taghon, Tom; Kerre, Tessa; Saeys, Yvan; Van Dorpe, Jo; Cheroutre, Hilde; Vandekerckhove, Bart

    2017-02-24

    The thymus plays a central role in self-tolerance, partly by eliminating precursors with a T cell receptor (TCR) that binds strongly to self-antigens. However, the generation of self-agonist-selected lineages also relies on strong TCR signaling. How thymocytes discriminate between these opposite outcomes remains elusive. Here, we identified a human agonist-selected PD-1(+) CD8αα(+) subset of mature CD8αβ(+) T cells that displays an effector phenotype associated with agonist selection. TCR stimulation of immature post-β-selection thymocyte blasts specifically gives rise to this innate subset and fixes early T cell receptor alpha variable (TRAV) and T cell receptor alpha joining (TRAJ) rearrangements in the TCR repertoire. These findings suggest that the checkpoint for agonist selection precedes conventional selection in the human thymus. Copyright © 2017, American Association for the Advancement of Science.

  3. Budding yeast dma proteins control septin dynamics and the spindle position checkpoint by promoting the recruitment of the Elm1 kinase to the bud neck.

    PubMed

    Merlini, Laura; Fraschini, Roberta; Boettcher, Barbara; Barral, Yves; Lucchini, Giovanna; Piatti, Simonetta

    2012-01-01

    The first step towards cytokinesis in budding yeast is the assembly of a septin ring at the future site of bud emergence. Integrity of this ring is crucial for cytokinesis, proper spindle positioning, and the spindle position checkpoint (SPOC). This checkpoint delays mitotic exit and cytokinesis as long as the anaphase spindle does not properly align with the division axis. SPOC signalling requires the Kin4 protein kinase and the Kin4-regulating Elm1 kinase, which also controls septin dynamics. Here, we show that the two redundant ubiquitin-ligases Dma1 and Dma2 control septin dynamics and the SPOC by promoting the efficient recruitment of Elm1 to the bud neck. Indeed, dma1 dma2 mutant cells show reduced levels of Elm1 at the bud neck and Elm1-dependent activation of Kin4. Artificial recruitment of Elm1 to the bud neck of the same cells is sufficient to re-establish a normal septin ring, proper spindle positioning, and a proficient SPOC response in dma1 dma2 cells. Altogether, our data indicate that septin dynamics and SPOC function are intimately linked and support the idea that integrity of the bud neck is crucial for SPOC signalling.

  4. Positive regulation of meiotic DNA double-strand break formation by activation of the DNA damage checkpoint kinase Mec1(ATR).

    PubMed

    Gray, Stephen; Allison, Rachal M; Garcia, Valerie; Goldman, Alastair S H; Neale, Matthew J

    2013-07-31

    During meiosis, formation and repair of programmed DNA double-strand breaks (DSBs) create genetic exchange between homologous chromosomes-a process that is critical for reductional meiotic chromosome segregation and the production of genetically diverse sexually reproducing populations. Meiotic DSB formation is a complex process, requiring numerous proteins, of which Spo11 is the evolutionarily conserved catalytic subunit. Precisely how Spo11 and its accessory proteins function or are regulated is unclear. Here, we use Saccharomyces cerevisiae to reveal that meiotic DSB formation is modulated by the Mec1(ATR) branch of the DNA damage signalling cascade, promoting DSB formation when Spo11-mediated catalysis is compromised. Activation of the positive feedback pathway correlates with the formation of single-stranded DNA (ssDNA) recombination intermediates and activation of the downstream kinase, Mek1. We show that the requirement for checkpoint activation can be rescued by prolonging meiotic prophase by deleting the NDT80 transcription factor, and that even transient prophase arrest caused by Ndt80 depletion is sufficient to restore meiotic spore viability in checkpoint mutants. Our observations are unexpected given recent reports that the complementary kinase pathway Tel1(ATM) acts to inhibit DSB formation. We propose that such antagonistic regulation of DSB formation by Mec1 and Tel1 creates a regulatory mechanism, where the absolute frequency of DSBs is maintained at a level optimal for genetic exchange and efficient chromosome segregation.

  5. Full activation of p34CDC28 histone H1 kinase activity is unable to promote entry into mitosis in checkpoint-arrested cells of the yeast Saccharomyces cerevisiae.

    PubMed Central

    Stueland, C S; Lew, D J; Cismowski, M J; Reed, S I

    1993-01-01

    In most cells, mitosis is dependent upon completion of DNA replication. The feedback mechanisms that prevent entry into mitosis by cells with damaged or incompletely replicated DNA have been termed checkpoint controls. Studies with the fission yeast Schizosaccharomyces pombe and Xenopus egg extracts have shown that checkpoint controls prevent activation of the master regulatory protein kinase, p34cdc2, that normally triggers entry into mitosis. This is achieved through inhibitory phosphorylation of the Tyr-15 residue of p34cdc2. However, studies with the budding yeast Saccharomyces cerevisiae have shown that phosphorylation of this residue is not essential for checkpoint controls to prevent mitosis. We have investigated the basis for checkpoint controls in this organism and show that these controls can prevent entry into mitosis even in cells which have fully activated the cyclin B (Clb)-associated forms of the budding yeast homolog of p34cdc2, p34CDC28, as assayed by histone H1 kinase activity. However, the active complexes in checkpoint-arrested cells are smaller than those in cycling cells, suggesting that assembly of mitosis-inducing complexes requires additional steps following histone H1 kinase activation. Images PMID:8388545

  6. Kaempferol Activates G₂-Checkpoint of the Cell Cycle Resulting in G₂-Arrest and Mitochondria-Dependent Apoptosis in Human Acute Leukemia Jurkat T Cells.

    PubMed

    Kim, Ki Yun; Jang, Won Young; Lee, Ji Young; Jun, Do Youn; Ko, Jee Youn; Yun, Young Ho; Kim, Young Ho

    2016-02-01

    The effect of kaempferol (3,5,7,4-tetrahydroxyflavone), a flavonoid compound that was identified in barnyard millet (Echinochloa crus-galli var. frumentacea) grains, on G2-checkpoint and apoptotic pathways was investigated in human acute leukemia Jurkat T cell clones stably transfected with an empty vector (J/Neo) or a Bcl-xL expression vector (J/Bcl-xL). Exposure of J/Neo cells to kaempeferol caused cytotoxicity and activation of the ATM/ATR-Chk1/Chk2 pathway, activating the phosphorylation of p53 (Ser-15), inhibitory phosphorylation of Cdc25C (Ser-216), and inactivation of cyclin-dependent kinase 1 (Cdk1), with resultant G2- arrest of the cell cycle. Under these conditions, apoptotic events, including upregulation of Bak and PUMA levels, Bak activation, mitochondrial membrane potential (Δψm) loss, activation of caspase-9, -8, and -3, anti-poly (ADP-ribose) polymerase (PARP) cleavage, and accumulation of apoptotic sub-G1 cells, were induced without accompanying necrosis. However, these apoptotic events, except for upregulation of Bak and PUMA levels, were completely abrogated in J/Bcl-xL cells overexpressing Bcl-xL, suggesting that the G2-arrest and the Bcl-xL-sensitive mitochondrial apoptotic events were induced, in parallel, as downstream events of the DNA-damage-mediated G2-checkpoint activation. Together these results demonstrate that kaempferol-mediated antitumor activity toward Jurkat T cells was attributable to G2-checkpoint activation, which caused not only G2-arrest of the cell cycle but also activating phosphorylation of p53 (Ser-15) and subsequent induction of mitochondriadependent apoptotic events, including Bak and PUMA upregulation, Bak activation, Δpsim loss, and caspase cascade activation.

  7. Coordinate action of distinct sequence elements localizes checkpoint kinase Hsl1 to the septin collar at the bud neck in Saccharomyces cerevisiae

    PubMed Central

    Finnigan, Gregory C.; Sterling, Sarah M.; Duvalyan, Angela; Liao, Elizabeth N.; Sargsyan, Aspram; Garcia, Galo; Nogales, Eva; Thorner, Jeremy

    2016-01-01

    Passage through the eukaryotic cell cycle requires processes that are tightly regulated both spatially and temporally. Surveillance mechanisms (checkpoints) exert quality control and impose order on the timing and organization of downstream events by impeding cell cycle progression until the necessary components are available and undamaged and have acted in the proper sequence. In budding yeast, a checkpoint exists that does not allow timely execution of the G2/M transition unless and until a collar of septin filaments has properly assembled at the bud neck, which is the site where subsequent cytokinesis will occur. An essential component of this checkpoint is the large (1518-residue) protein kinase Hsl1, which localizes to the bud neck only if the septin collar has been correctly formed. Hsl1 reportedly interacts with particular septins; however, the precise molecular determinants in Hsl1 responsible for its recruitment to this cellular location during G2 have not been elucidated. We performed a comprehensive mutational dissection and accompanying image analysis to identify the sequence elements within Hsl1 responsible for its localization to the septins at the bud neck. Unexpectedly, we found that this targeting is multipartite. A segment of the central region of Hsl1 (residues 611–950), composed of two tandem, semiredundant but distinct septin-associating elements, is necessary and sufficient for binding to septin filaments both in vitro and in vivo. However, in addition to 611–950, efficient localization of Hsl1 to the septin collar in the cell obligatorily requires generalized targeting to the cytosolic face of the plasma membrane, a function normally provided by the C-terminal phosphatidylserine-binding KA1 domain (residues 1379–1518) in Hsl1 but that can be replaced by other, heterologous phosphatidylserine-binding sequences. PMID:27193302

  8. Coordinate action of distinct sequence elements localizes checkpoint kinase Hsl1 to the septin collar at the bud neck in Saccharomyces cerevisiae.

    PubMed

    Finnigan, Gregory C; Sterling, Sarah M; Duvalyan, Angela; Liao, Elizabeth N; Sargsyan, Aspram; Garcia, Galo; Nogales, Eva; Thorner, Jeremy

    2016-07-15

    Passage through the eukaryotic cell cycle requires processes that are tightly regulated both spatially and temporally. Surveillance mechanisms (checkpoints) exert quality control and impose order on the timing and organization of downstream events by impeding cell cycle progression until the necessary components are available and undamaged and have acted in the proper sequence. In budding yeast, a checkpoint exists that does not allow timely execution of the G2/M transition unless and until a collar of septin filaments has properly assembled at the bud neck, which is the site where subsequent cytokinesis will occur. An essential component of this checkpoint is the large (1518-residue) protein kinase Hsl1, which localizes to the bud neck only if the septin collar has been correctly formed. Hsl1 reportedly interacts with particular septins; however, the precise molecular determinants in Hsl1 responsible for its recruitment to this cellular location during G2 have not been elucidated. We performed a comprehensive mutational dissection and accompanying image analysis to identify the sequence elements within Hsl1 responsible for its localization to the septins at the bud neck. Unexpectedly, we found that this targeting is multipartite. A segment of the central region of Hsl1 (residues 611-950), composed of two tandem, semiredundant but distinct septin-associating elements, is necessary and sufficient for binding to septin filaments both in vitro and in vivo. However, in addition to 611-950, efficient localization of Hsl1 to the septin collar in the cell obligatorily requires generalized targeting to the cytosolic face of the plasma membrane, a function normally provided by the C-terminal phosphatidylserine-binding KA1 domain (residues 1379-1518) in Hsl1 but that can be replaced by other, heterologous phosphatidylserine-binding sequences.

  9. P38 Mitogen-activated Protein Kinase Activity Is Required during Mitosis for Timely Satisfaction of the Mitotic Checkpoint But Not for the Fidelity of Chromosome Segregation

    PubMed Central

    Lee, Kyunghee; Kenny, Alison E.

    2010-01-01

    Although p38 activity is reported to be required as cells enter mitosis for proper spindle assembly and checkpoint function, its role during the division process remains controversial in lieu of direct data. We therefore conducted live cell studies to determine the effect on mitosis of inhibiting or depleting p38. We found that in the absence of p38 activity the duration of mitosis is prolonged by ∼40% in nontransformed human RPE-1, ∼80% in PtK2 (rat kangaroo), and ∼25% in mouse cells, and this prolongation leads to an elevated mitotic index. However, under this condition chromatid segregation and cytokinesis are normal. Using Mad2/YFP-expressing cells, we show the prolongation of mitosis in the absence of p38 activity is directly due to a delay in satisfying the mitotic checkpoint. Inhibiting p38 did not affect the rate of chromosome motion; however, it did lead to the formation of significantly (10%) longer metaphase spindles. From these data we conclude that normal p38 activity is required for the timely stable attachment of all kinetochores to spindle microtubules, but not for the fidelity of the mitotic process. We speculate that p38 activity promotes timely checkpoint satisfaction by indirectly influencing those motor proteins (e.g., Klp10, Klp67A) involved in regulating the dynamics of kinetochore microtubule ends. PMID:20462950

  10. P38 mitogen-activated protein kinase activity is required during mitosis for timely satisfaction of the mitotic checkpoint but not for the fidelity of chromosome segregation.

    PubMed

    Lee, Kyunghee; Kenny, Alison E; Rieder, Conly L

    2010-07-01

    Although p38 activity is reported to be required as cells enter mitosis for proper spindle assembly and checkpoint function, its role during the division process remains controversial in lieu of direct data. We therefore conducted live cell studies to determine the effect on mitosis of inhibiting or depleting p38. We found that in the absence of p38 activity the duration of mitosis is prolonged by approximately 40% in nontransformed human RPE-1, approximately 80% in PtK2 (rat kangaroo), and approximately 25% in mouse cells, and this prolongation leads to an elevated mitotic index. However, under this condition chromatid segregation and cytokinesis are normal. Using Mad2/YFP-expressing cells, we show the prolongation of mitosis in the absence of p38 activity is directly due to a delay in satisfying the mitotic checkpoint. Inhibiting p38 did not affect the rate of chromosome motion; however, it did lead to the formation of significantly (10%) longer metaphase spindles. From these data we conclude that normal p38 activity is required for the timely stable attachment of all kinetochores to spindle microtubules, but not for the fidelity of the mitotic process. We speculate that p38 activity promotes timely checkpoint satisfaction by indirectly influencing those motor proteins (e.g., Klp10, Klp67A) involved in regulating the dynamics of kinetochore microtubule ends.

  11. A humanized antibody for imaging immune checkpoint ligand PD-L1 expression in tumors.

    PubMed

    Chatterjee, Samit; Lesniak, Wojciech G; Gabrielson, Matthew; Lisok, Ala; Wharram, Bryan; Sysa-Shah, Polina; Azad, Babak Behnam; Pomper, Martin G; Nimmagadda, Sridhar

    2016-03-01

    Antibodies targeting the PD-1/PD-L1 immune checkpoint lead to tumor regression and improved survival in several cancers. PD-L1 expression in tumors may be predictive of response to checkpoint blockade therapy. Because tissue samples might not always be available to guide therapy, we developed and evaluated a humanized antibody for non-invasive imaging of PD-L1 expression in tumors. Radiolabeled [111In]PD-L1-mAb and near-infrared dye conjugated NIR-PD-L1-mAb imaging agents were developed using the mouse and human cross-reactive PD-L1 antibody MPDL3280A. We tested specificity of [111In]PD-L1-mAb and NIR-PD-L1-mAb in cell lines and in tumors with varying levels of PD-L1 expression. We performed SPECT/CT imaging, biodistribution and blocking studies in NSG mice bearing tumors with constitutive PD-L1 expression (CHO-PDL1) and in controls (CHO). Results were confirmed in triple negative breast cancer (TNBC) (MDAMB231 and SUM149) and non-small cell lung cancer (NSCLC) (H2444 and H1155) xenografts with varying levels of PD-L1 expression. There was specific binding of [111In]PD-L1-mAb and NIR-PD-L1-mAb to tumor cells in vitro, correlating with PD-L1 expression levels. In mice bearing subcutaneous and orthotopic tumors, there was specific and persistent high accumulation of signal intensity in PD-L1 positive tumors (CHO-PDL1, MDAMB231, H2444) but not in controls. These results demonstrate that [111In]PD-L1-mAb and NIR-PD-L1-mAb can detect graded levels of PD-L1 expression in human tumor xenografts in vivo. As a humanized antibody, these findings suggest clinical translation of radiolabeled versions of MPDL3280A for imaging. Specificity of NIR-PD-L1-mAb indicates the potential for optical imaging of PD-L1 expression in tumors in relevant pre-clinical as well as clinical settings.

  12. Sterigmatocystin-induced checkpoint adaptation depends on Chk1 in immortalized human gastric epithelial cells in vitro.

    PubMed

    Jiang, Xiujuan; Wang, Juan; Xing, Lingxiao; Shen, Haitao; Lian, Weiguang; Yi, Li; Zhang, Donghui; Yang, Haiyan; Liu, Jianghui; Zhang, Xianghong

    2017-01-01

    Sterigmatocystin (ST) is a common contaminant detected in food and animal feed that has been recognized as a possible human carcinogen. Our previous studies demonstrate that ST causes DNA damage and subsequently triggers cell cycle arrest in G2 and apoptosis in immortalized human gastric epithelial cells (GES-1). Recently, studies have shown that in certain contexts, cells with DNA damage may escape checkpoint arrest and enter mitosis without repairing the damage. The term for this process is "checkpoint adaptation," and it increases the risk of unstable genome propagation, which may contribute to carcinogenesis. Thus, we aimed to investigate whether checkpoint adaptation occurs in GES-1 cells treated with ST and explored the underlying molecular mechanisms that contribute to this phenotype. In this study, we found that ST treatment for 24 h in GES-1 cells led to an initial G2 arrest; however, a fraction of GES-1 cells became large and rounded, and the number of p-H3-positive cells increased sharply after ST treatment for 48 h. Moreover, collection of the large and rounded cells by mechanical shake-off revealed that the majority of these large cells were found in the mitotic phase of the cell cycle. Importantly, we found that these rounded cells entered mitosis despite damaged DNA and that a small subset of this cell population survived and continued to propagate. These results suggest that ST induces an initial G2 arrest that is subsequently followed by G2 phase checkpoint adaptation, which may potentially promote genomic instability and result in tumorigenesis. Furthermore, we showed that activation of Chk1 contributes to the G2 arrest in GES-1 cells that are treated with ST for 24 h and that prolonged treatment of cells with ST for 48 h led to a decrease in the total protein and phosphorylation levels of Chk1 in mitotic cells, indicating that checkpoint adaptation may be driven by inactivation of Chk1. Knockdown studies confirmed that cells entered mitosis

  13. M2, a novel anthracenedione, elicits a potent DNA damage response that can be subverted through checkpoint kinase inhibition to generate mitotic catastrophe.

    PubMed

    Evison, Benny J; Pastuovic, Mile; Bilardi, Rebecca A; Forrest, Robert A; Pumuye, Paul P; Sleebs, Brad E; Watson, Keith G; Phillips, Don R; Cutts, Suzanne M

    2011-12-01

    Pixantrone is a promising anti-cancer aza-anthracenedione that has prompted the development of new anthracenediones incorporating symmetrical side-chains of increasing length varying from two to five methylene units in each pair of drug side-chains. A striking relationship has emerged in which anthracenedione-induced growth inhibition and apoptosis was inversely associated with side-chain length, a relationship that was attributable to a differential ability to stabilise the topoisomerase II (TOP2) cleavage complex. Processing of the complex to a DNA double strand break (DSB) flanked by γH2AX in nuclear foci is likely to occur, as the generation of the primary lesion was antecedent to γH2AX induction. M2, bearing the shortest pair of side-chains, induced TOP2-mediated DSBs efficiently and activated cell cycle checkpoints via Chk1 and Chk2 phosphorylation, implicating the involvement of ATM and ATR, and induced a protracted S phase and subsequent G2/M arrest. The inactive analogue M5, containing the longest pair of side-chains, only weakly stimulated any of these responses, suggesting that efficient stabilisation of the TOP2 cleavage complex was crucial for eliciting a strong DNA damage response (DDR). An M2 induced DDR in p53-defective MDA-MB-231 cells was abrogated by UCN-01, a ubiquitous inhibitor of kinases including Chk1, in a response associated with substantial mitotic catastrophe and strong synergy. The rational selection of checkpoint kinase inhibitors may significantly enhance the therapeutic benefit of anthracenediones that efficiently stabilise the TOP2 cleavage complex.

  14. Genes involved in cell cycle G1 checkpoint control are frequently mutated in human melanoma metastases.

    PubMed Central

    Platz, A.; Sevigny, P.; Norberg, T.; Ring, P.; Lagerlöf, B.; Ringborg, U.

    1996-01-01

    A common characteristic of cancer cells is unrestrained cell division. This may be caused by mutational changes in genes coding for components of cell cycle-controlling networks. Alterations in genes involved in G1 checkpoint control have been registered in many human tumours, and investigations from several laboratories show that such alterations, taken together, are the most frequent changes detected in cancer cells. The present paper describes mutational analysis by polymerase chain reaction-single-strand conformation polymorphism (PCR/SSCP) and nucleotide sequence analysis of the genes coding for the p15, p53 and N-ras proteins in 26 metastases from 25 melanoma patients. The registered mutation frequencies add together with previously registered mutations in p16 in the same patient samples to a substantial total frequency of 44% of patients with mutation in at least one of the investigated genes. These results show the occurrence of heterogeneous defects among components of the cell cycle controlling machinery in a human melanoma tumour sample collection and demonstrate that the total frequency of detected alterations increases with the number of cell cycle controlling genes included in the screening panel. Images Figure 1 PMID:8826861

  15. Caspase activity is not required for the mitotic checkpoint or mitotic slippage in human cells

    PubMed Central

    Lee, Kyunghee; Kenny, Alison E.; Rieder, Conly L.

    2011-01-01

     Biochemical studies suggest that caspase activity is required for a functional mitotic checkpoint (MC) and mitotic slippage. To test this directly, we followed nontransformed human telomerase immortalized human retinal pigment epithelia (RPE-1) cells through mitosis after inhibiting or depleting selected caspases. We found that inhibiting caspases individually, in combination, or in toto did not affect the duration or fidelity of mitosis in otherwise untreated cells. When satisfaction of the MC was prevented with 500 nM nocodazole or 2.5 μM dimethylenastron (an Eg5 inhibitor), 92–100% of RPE-1 cells slipped from mitosis in the presence of pan-caspase inhibitors or after simultaneously depleting caspase-3 and -9, and they did so with the same kinetics (∼21–22 h) as after treatment with nocodazole or Eg5 inhibitors alone. Surprisingly, inhibiting or depleting caspase-9 alone doubled the number of nocodazole-treated, but not Eg5-inhibited, cells that died in mitosis. In addition, inhibiting or depleting caspase-9 and -3 together accelerated the rate of slippage ∼40% (to ∼13–15 h). Finally, nocodazole-treated cells that recently slipped through mitosis in the presence or absence of pan-caspase inhibitors contained numerous BubR1 foci in their nuclei. From these data, we conclude that caspase activity is not required for a functional MC or for mitotic slippage. PMID:21613548

  16. An atlas of human kinase regulation.

    PubMed

    Ochoa, David; Jonikas, Mindaugas; Lawrence, Robert T; El Debs, Bachir; Selkrig, Joel; Typas, Athanasios; Villén, Judit; Santos, Silvia Dm; Beltrao, Pedro

    2016-12-01

    The coordinated regulation of protein kinases is a rapid mechanism that integrates diverse cues and swiftly determines appropriate cellular responses. However, our understanding of cellular decision-making has been limited by the small number of simultaneously monitored phospho-regulatory events. Here, we have estimated changes in activity in 215 human kinases in 399 conditions derived from a large compilation of phosphopeptide quantifications. This atlas identifies commonly regulated kinases as those that are central in the signaling network and defines the logic relationships between kinase pairs. Co-regulation along the conditions predicts kinase-complex and kinase-substrate associations. Additionally, the kinase regulation profile acts as a molecular fingerprint to identify related and opposing signaling states. Using this atlas, we identified essential mediators of stem cell differentiation, modulators of Salmonella infection, and new targets of AKT1. This provides a global view of human phosphorylation-based signaling and the necessary context to better understand kinase-driven decision-making. © 2016 The Authors. Published under the terms of the CC BY 4.0 license.

  17. Kinetochore–microtubule attachment is sufficient to satisfy the human spindle assembly checkpoint

    PubMed Central

    Etemad, Banafsheh; Kuijt, Timo E. F.; Kops, Geert J. P. L.

    2015-01-01

    The spindle assembly checkpoint (SAC) is a genome surveillance mechanism that protects against aneuploidization. Despite profound progress on understanding mechanisms of its activation, it remains unknown what aspect of chromosome–spindle interactions is monitored by the SAC: kinetochore–microtubule attachment or the force generated by dynamic microtubules that signals stable biorientation of chromosomes? To answer this, we uncoupled these two processes by expressing a non-phosphorylatable version of the main microtubule-binding protein at kinetochores (HEC1-9A), causing stabilization of incorrect kinetochore–microtubule attachments despite persistent activity of the error-correction machinery. The SAC is fully functional in HEC1-9A-expressing cells, yet cells in which chromosomes cannot biorient but are stably attached to microtubules satisfy the SAC and exit mitosis. SAC satisfaction requires neither intra-kinetochore stretching nor dynamic microtubules. Our findings support the hypothesis that in human cells the end-on interactions of microtubules with kinetochores are sufficient to satisfy the SAC without the need for microtubule-based pulling forces. PMID:26621779

  18. Cellular Inhibition of Checkpoint Kinase 2 (Chk2) and Potentiation of Camptothecins and Radiation by the Novel Chk2 Inhibitor PV1019 [7-Nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide

    PubMed Central

    Jobson, Andrew G.; Lountos, George T.; Lorenzi, Philip L.; Llamas, Jenny; Connelly, John; Cerna, David; Tropea, Joseph E.; Onda, Akikazu; Zoppoli, Gabriele; Kondapaka, Sudhir; Zhang, Guangtao; Caplen, Natasha J.; Cardellina, John H.; Yoo, Stephen S.; Monks, Anne; Self, Christopher; Waugh, David S.; Shoemaker, Robert H.

    2009-01-01

    Chk2 is a checkpoint kinase involved in the ataxia telangiectasia mutated pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported 4,4′-diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555) as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039) [7-nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide], which is a selective submicromolar inhibitor of Chk2 in vitro. The cocrystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against ionizing radiation-induced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 and Chk2 small interfering RNAs can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI-60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential. PMID:19741151

  19. Surviving the breakup: the DNA damage checkpoint.

    PubMed

    Harrison, Jacob C; Haber, James E

    2006-01-01

    In response to even a single chromosomal double-strand DNA break, cells enact the DNA damage checkpoint. This checkpoint triggers cell cycle arrest, providing time for the cell to repair damaged chromosomes before entering mitosis. This mechanism helps prevent the segregation of damaged or mutated chromosomes and thus promotes genomic stability. Recent work has elucidated the molecular mechanisms underlying several critical steps in checkpoint activation, notably the recruitment of the upstream checkpoint kinases of the ATM and ATR families to different damaged DNA structures and the molecular events through which these kinases activate their effectors. Chromatin modification has emerged as one important component of checkpoint activation and maintenance. Following DNA repair, the checkpoint pathway is inactivated in a process termed recovery. A related but genetically distinct process, adaptation, controls cell cycle re-entry in the face of unrepairable damage.

  20. Cell cycle control, checkpoint mechanisms, and genotoxic stress.

    PubMed Central

    Shackelford, R E; Kaufmann, W K; Paules, R S

    1999-01-01

    The ability of cells to maintain genomic integrity is vital for cell survival and proliferation. Lack of fidelity in DNA replication and maintenance can result in deleterious mutations leading to cell death or, in multicellular organisms, cancer. The purpose of this review is to discuss the known signal transduction pathways that regulate cell cycle progression and the mechanisms cells employ to insure DNA stability in the face of genotoxic stress. In particular, we focus on mammalian cell cycle checkpoint functions, their role in maintaining DNA stability during the cell cycle following exposure to genotoxic agents, and the gene products that act in checkpoint function signal transduction cascades. Key transitions in the cell cycle are regulated by the activities of various protein kinase complexes composed of cyclin and cyclin-dependent kinase (Cdk) molecules. Surveillance control mechanisms that check to ensure proper completion of early events and cellular integrity before initiation of subsequent events in cell cycle progression are referred to as cell cycle checkpoints and can generate a transient delay that provides the cell more time to repair damage before progressing to the next phase of the cycle. A variety of cellular responses are elicited that function in checkpoint signaling to inhibit cyclin/Cdk activities. These responses include the p53-dependent and p53-independent induction of Cdk inhibitors and the p53-independent inhibitory phosphorylation of Cdk molecules themselves. Eliciting proper G1, S, and G2 checkpoint responses to double-strand DNA breaks requires the function of the Ataxia telangiectasia mutated gene product. Several human heritable cancer-prone syndromes known to alter DNA stability have been found to have defects in checkpoint surveillance pathways. Exposures to several common sources of genotoxic stress, including oxidative stress, ionizing radiation, UV radiation, and the genotoxic compound benzo[a]pyrene, elicit cell cycle

  1. Ki-67 Contributes to Normal Cell Cycle Progression and Inactive X Heterochromatin in p21 Checkpoint-Proficient Human Cells.

    PubMed

    Sun, Xiaoming; Bizhanova, Aizhan; Matheson, Timothy D; Yu, Jun; Zhu, Lihua Julie; Kaufman, Paul D

    2017-09-01

    The Ki-67 protein is widely used as a tumor proliferation marker. However, whether Ki-67 affects cell cycle progression has been controversial. Here we demonstrate that depletion of Ki-67 in human hTERT-RPE1, WI-38, IMR90, and hTERT-BJ cell lines and primary fibroblast cells slowed entry into S phase and coordinately downregulated genes related to DNA replication. Some gene expression changes were partially relieved in Ki-67-depleted hTERT-RPE1 cells by codepletion of the Rb checkpoint protein, but more thorough suppression of the transcriptional and cell cycle defects was observed upon depletion of the cell cycle inhibitor p21. Notably, induction of p21 upon depletion of Ki-67 was a consistent hallmark of cell types in which transcription and cell cycle distribution were sensitive to Ki-67; these responses were absent in cells that did not induce p21. Furthermore, upon Ki-67 depletion, a subset of inactive X (Xi) chromosomes in female hTERT-RPE1 cells displayed several features of compromised heterochromatin maintenance, including decreased H3K27me3 and H4K20me1 labeling. These chromatin alterations were limited to Xi chromosomes localized away from the nuclear lamina and were not observed in checkpoint-deficient 293T cells. Altogether, our results indicate that Ki-67 integrates normal S-phase progression and Xi heterochromatin maintenance in p21 checkpoint-proficient human cells. Copyright © 2017 American Society for Microbiology.

  2. In Silico Exploration of 1,7-Diazacarbazole Analogs as Checkpoint Kinase 1 Inhibitors by Using 3D QSAR, Molecular Docking Study, and Molecular Dynamics Simulations.

    PubMed

    Gao, Xiaodong; Han, Liping; Ren, Yujie

    2016-05-05

    Checkpoint kinase 1 (Chk1) is an important serine/threonine kinase with a self-protection function. The combination of Chk1 inhibitors and anti-cancer drugs can enhance the selectivity of tumor therapy. In this work, a set of 1,7-diazacarbazole analogs were identified as potent Chk1 inhibitors through a series of computer-aided drug design processes, including three-dimensional quantitative structure-activity relationship (3D-QSAR) modeling, molecular docking, and molecular dynamics simulations. The optimal QSAR models showed significant cross-validated correlation q² values (0.531, 0.726), fitted correlation r² coefficients (higher than 0.90), and standard error of prediction (less than 0.250). These results suggested that the developed models possess good predictive ability. Moreover, molecular docking and molecular dynamics simulations were applied to highlight the important interactions between the ligand and the Chk1 receptor protein. This study shows that hydrogen bonding and electrostatic forces are key interactions that confer bioactivity.

  3. Effective intra-S checkpoint responses to UVC in primary human melanocytes and melanoma cell lines.

    PubMed

    Cordeiro-Stone, Marila; McNulty, John J; Sproul, Christopher D; Chastain, Paul D; Gibbs-Flournoy, Eugene; Zhou, Yingchun; Carson, Craig; Rao, Shangbang; Mitchell, David L; Simpson, Dennis A; Thomas, Nancy E; Ibrahim, Joseph G; Kaufmann, William K

    2016-01-01

    The objective of this study was to assess potential functional attenuation or inactivation of the intra-S checkpoint during melanoma development. Proliferating cultures of skin melanocytes, fibroblasts, and melanoma cell lines were exposed to increasing fluences of UVC and intra-S checkpoint responses were quantified. Melanocytes displayed stereotypic intra-S checkpoint responses to UVC qualitatively and quantitatively equivalent to those previously demonstrated in skin fibroblasts. In comparison with fibroblasts, primary melanocytes displayed reduced UVC-induced inhibition of DNA strand growth and enhanced degradation of p21Waf1 after UVC, suggestive of enhanced bypass of UVC-induced DNA photoproducts. All nine melanoma cell lines examined, including those with activating mutations in BRAF or NRAS oncogenes, also displayed proficiency in activation of the intra-S checkpoint in response to UVC irradiation. The results indicate that bypass of oncogene-induced senescence during melanoma development was not associated with inactivation of the intra-S checkpoint response to UVC-induced DNA replication stress.

  4. A sequential multi-target Mps1 phosphorylation cascade promotes spindle checkpoint signaling

    PubMed Central

    Ji, Zhejian; Gao, Haishan; Jia, Luying; Li, Bing; Yu, Hongtao

    2017-01-01

    The master spindle checkpoint kinase Mps1 senses kinetochore-microtubule attachment and promotes checkpoint signaling to ensure accurate chromosome segregation. The kinetochore scaffold Knl1, when phosphorylated by Mps1, recruits checkpoint complexes Bub1–Bub3 and BubR1–Bub3 to unattached kinetochores. Active checkpoint signaling ultimately enhances the assembly of the mitotic checkpoint complex (MCC) consisting of BubR1–Bub3, Mad2, and Cdc20, which inhibits the anaphase-promoting complex or cyclosome bound to Cdc20 (APC/CCdc20) to delay anaphase onset. Using in vitro reconstitution, we show that Mps1 promotes APC/C inhibition by MCC components through phosphorylating Bub1 and Mad1. Phosphorylated Bub1 binds to Mad1–Mad2. Phosphorylated Mad1 directly interacts with Cdc20. Mutations of Mps1 phosphorylation sites in Bub1 or Mad1 abrogate the spindle checkpoint in human cells. Therefore, Mps1 promotes checkpoint activation through sequentially phosphorylating Knl1, Bub1, and Mad1. This sequential multi-target phosphorylation cascade makes the checkpoint highly responsive to Mps1 and to kinetochore-microtubule attachment. DOI: http://dx.doi.org/10.7554/eLife.22513.001 PMID:28072388

  5. SCF(FBXW7α) modulates the intra-S-phase DNA-damage checkpoint by regulating Polo like kinase-1 stability.

    PubMed

    Giráldez, Servando; Herrero-Ruiz, Joaquín; Mora-Santos, Mar; Japón, Miguel Á; Tortolero, Maria; Romero, Francisco

    2014-06-30

    The intra-S-checkpoint is essential to control cell progression through S phase under normal conditions and in response to replication stress. When DNA lesions are detected, replication fork progression is blocked allowing time for repair to avoid genomic instability and the risk of cancer. DNA replication initiates at many origins of replication in eukaryotic cells, where a series of proteins form pre-replicative complexes (pre-RCs) that are activated to become pre-initiation complexes and ensure a single round of replication in each cell cycle. PLK1 plays an important role in the regulation of DNA replication, contributing to the regulation of pre-RCs formation by phosphorylating several proteins, under both normal and stress conditions. Here we report that PLK1 is ubiquitinated and degraded by SCFFBXW7α/proteasome. Moreover, we identified a new Cdc4 phosphodegron in PLK1, conserved from yeast to humans, whose mutation prevents PLK1 destruction. We established that endogenous SCFFBXW7α degrades PLK1 in the G1 and S phases of an unperturbed cell cycle and in S phase following UV irradiation. Furthermore, we showed that FBXW7α overexpression or UV irradiation prevented the loading of proteins onto chromatin to form pre-RCs and, accordingly, reduced cell proliferation. We conclude that PLK1 degradation mediated by SCFFBXW7α modulates the intra-S-phase checkpoint.

  6. Glycolate kinase activity in human red cells.

    PubMed

    Fujii, S; Beutler, E

    1985-02-01

    Human red cells manifest glycolate kinase activity. This activity copurifies with pyruvate kinase and is decreased in the red cells of subjects with hereditary pyruvate kinase deficiency. Glycolate kinase activity was detected in the presence of FDP or glucose-1,6-P2. In the presence of 1 mmol/L FDP, the Km for adenosine triphosphate (ATP) was 0.28 mmol/L and a half maximum velocity for glycolate was obtained at 40 mmol/L. The pH optimum of the reaction was over 10.5 With 10 mumol/L FDP, 500 mumol/L glucose-1,6-P2, 2 mmol/L ATP, 5 mmol/L MgCl2, and 50 mmol/L glycolate at pH 7.5, glycolate kinase activity was calculated to be approximately 0.0013 U/mL RBC. In view of this low activity even in the presence of massive amounts of glycolate, the glycolate kinase reaction cannot account for the maintenance of the reported phosphoglycolate level in human red cells.

  7. Antiproliferation potential of withaferin A on human osteosarcoma cells via the inhibition of G2/M checkpoint proteins

    PubMed Central

    LV, TING-ZHUO; WANG, GUANG-SHUN

    2015-01-01

    Withaferin A (WA) is a well-known steroidal lactone of the medicinally important plant, Withania somnifera. This secondary metabolite has been noted for its anticancer effects against a number of human cancer cell lines. However, there are a limited number of studies investigating the growth inhibitory potential of WA against human osteosarcoma cells and the underlying molecular mechanisms. Thus, in the present study, the antiproliferative activities of WA, along with the underlying mechanisms of action, were investigated using flow cytometry for cell cycle distribution and western blot analysis for the assessment of various checkpoint proteins. In addition, the antiproliferative activity was evaluated using a sulforhodamine B assay, where MG-63 and U2OS human osteosarcoma cell lines were treated with different concentrations of WA. Furthermore, the mRNA expression levels of the checkpoint proteins in the WA-treated MG-63 and U2OS cells were examined. The results obtained corresponded with the western blot analysis results. Furthermore, WA was shown to significantly inhibit the proliferation of the two types of treated cell lines (MG-63 and U2OS). Flow cytometric analysis revealed that WA induced cell cycle arrest at the G2/M phase, which was associated with the inhibition of cyclin B1, cyclin A, Cdk2 and p-Cdc2 (Tyr15) expression and an increase in the levels of p-Chk1 (Ser345) and p-Chk2 (Thr68). In conclusion, the present study found that the antiproliferative potential of WA was associated with the induction of cell cycle arrest at the G2/M phase, which was a result of the attenuation of the expression levels of G2/M checkpoint proteins. PMID:26170956

  8. Transforming growth factor beta-activated kinase 1 (TAK1)-dependent checkpoint in the survival of dendritic cells promotes immune homeostasis and function.

    PubMed

    Wang, Yanyan; Huang, Gonghua; Vogel, Peter; Neale, Geoffrey; Reizis, Boris; Chi, Hongbo

    2012-02-07

    Homeostatic control of dendritic cell (DC) survival is crucial for adaptive immunity, but the molecular mechanism is not well defined. Moreover, how DCs influence immune homeostasis under steady state remains unclear. Combining DC-specific and -inducible deletion systems, we report that transforming growth factor beta-activated kinase 1 (TAK1) is an essential regulator of DC survival and immune system homeostasis and function. Deficiency of TAK1 in CD11c(+) cells induced markedly elevated apoptosis, leading to the depletion of DC populations, especially the CD8(+) and CD103(+) DC subsets in lymphoid and nonlymphoid tissues, respectively. TAK1 also contributed to DC development by promoting the generation of DC precursors. Prosurvival signals from Toll-like receptors, CD40 and receptor activator of nuclear factor-κB (RANK) are integrated by TAK1 in DCs, which in turn mediated activation of downstream NF-κB and AKT-Foxo pathways and established a gene-expression program. TAK1 deficiency in DCs caused a myeloid proliferative disorder characterized by expansion of neutrophils and inflammatory monocytes, disrupted T-cell homeostasis, and prevented effective T-cell priming and generation of regulatory T cells. Moreover, TAK1 signaling in DCs was required to prevent myeloid proliferation even in the absence of lymphocytes, indicating a previously unappreciated regulatory mechanism of DC-mediated control of myeloid cell-dependent inflammation. Therefore, TAK1 orchestrates a prosurvival checkpoint in DCs that affects the homeostasis and function of the immune system.

  9. SUMO-interacting motifs (SIMs) in Polo-like kinase 1-interacting checkpoint helicase (PICH) ensure proper chromosome segregation during mitosis.

    PubMed

    Sridharan, Vinidhra; Azuma, Yoshiaki

    2016-08-17

    Polo-like kinase 1 (Plk1)-interacting checkpoint helicase (PICH) localizes at the centromere and is critical for proper chromosome segregation during mitosis. However, the precise molecular mechanism of PICH's centromeric localization and function at the centromere is not yet fully understood. Recently, using Xenopus egg extract assays, we showed that PICH is a promiscuous SUMO binding protein. To further determine the molecular consequence of PICH/SUMO interaction on PICH function, we identified 3 SUMO-interacting motifs (SIMs) on PICH and generated a SIM-deficient PICH mutant. Using the conditional expression of PICH in cells, we found distinct roles of PICH SIMs during mitosis. Although all SIMs are dispensable for PICH's localization on ultrafine anaphase DNA bridges, only SIM3 (third SIM, close to the C-terminus end of PICH) is critical for its centromeric localization. Intriguingly, the other 2 SIMs function in chromatin bridge prevention. With these results, we propose a novel SUMO-dependent regulation of PICH's function on mitotic centromeres.

  10. SUMO-interacting motifs (SIMs) in Polo-like kinase 1-interacting checkpoint helicase (PICH) ensure proper chromosome segregation during mitosis

    PubMed Central

    Sridharan, Vinidhra; Azuma, Yoshiaki

    2016-01-01

    ABSTRACT Polo-like kinase 1 (Plk1)-interacting checkpoint helicase (PICH) localizes at the centromere and is critical for proper chromosome segregation during mitosis. However, the precise molecular mechanism of PICH's centromeric localization and function at the centromere is not yet fully understood. Recently, using Xenopus egg extract assays, we showed that PICH is a promiscuous SUMO binding protein. To further determine the molecular consequence of PICH/SUMO interaction on PICH function, we identified 3 SUMO-interacting motifs (SIMs) on PICH and generated a SIM-deficient PICH mutant. Using the conditional expression of PICH in cells, we found distinct roles of PICH SIMs during mitosis. Although all SIMs are dispensable for PICH's localization on ultrafine anaphase DNA bridges, only SIM3 (third SIM, close to the C-terminus end of PICH) is critical for its centromeric localization. Intriguingly, the other 2 SIMs function in chromatin bridge prevention. With these results, we propose a novel SUMO-dependent regulation of PICH's function on mitotic centromeres. PMID:27230136

  11. Mediator kinase module and human tumorigenesis

    PubMed Central

    Clark, Alison D.; Oldenbroek, Marieke; Boyer, Thomas G.

    2016-01-01

    Mediator is a conserved multi-subunit signal processor through which regulatory informatiosn conveyed by gene-specific transcription factors is transduced to RNA Polymerase II (Pol II). In humans, MED13, MED12, CDK8 and Cyclin C (CycC) comprise a four-subunit “kinase” module that exists in variable association with a 26-subunit Mediator core. Genetic and biochemical studies have established the Mediator kinase module as a major ingress of developmental and oncogenic signaling through Mediator, and much of its function in signal-dependent gene regulation derives from its resident CDK8 kinase activity. For example, CDK8-targeted substrate phosphorylation impacts transcription factor half-life, Pol II activity and chromatin chemistry and functional status. Recent structural and biochemical studies have revealed a precise network of physical and functional subunit interactions required for proper kinase module activity. Accordingly, pathologic change in this activity through altered expression or mutation of constituent kinase module subunits can have profound consequences for altered signaling and tumor formation. Herein, we review the structural organization, biological function and oncogenic potential of the Mediator kinase module. We focus principally on tumor-associated alterations in kinase module subunits for which mechanistic relationships as opposed to strictly correlative associations are established. These considerations point to an emerging picture of the Mediator kinase module as an oncogenic unit, one in which pathogenic activation/deactivation through component change drives tumor formation through perturbation of signal-dependent gene regulation. It follows that therapeutic strategies to combat CDK8-driven tumors will involve targeted modulation of CDK8 activity or pharmacologic manipulation of dysregulated CDK8-dependent signaling pathways. PMID:26182352

  12. Crystal structure of human nicotinamide riboside kinase.

    PubMed

    Khan, Javed A; Xiang, Song; Tong, Liang

    2007-08-01

    Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD(+) as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 A resolution and in a ternary complex with ADP and tiazofurin at 2.7 A resolution. The active site is located in a groove between the central parallel beta sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.

  13. Crystal Structure of Human Nicotinamide Riboside Kinase

    SciTech Connect

    Khan,J.; Xiang, S.; Tong, L.

    2007-01-01

    Nicotinamide riboside kinase (NRK) has an important role in the biosynthesis of NAD{sup +} as well as the activation of tiazofurin and other NR analogs for anticancer therapy. NRK belongs to the deoxynucleoside kinase and nucleoside monophosphate (NMP) kinase superfamily, although the degree of sequence conservation is very low. We report here the crystal structures of human NRK1 in a binary complex with the reaction product nicotinamide mononucleotide (NMN) at 1.5 {angstrom} resolution and in a ternary complex with ADP and tiazofurin at 2.7 {angstrom} resolution. The active site is located in a groove between the central parallel {beta} sheet core and the LID and NMP-binding domains. The hydroxyl groups on the ribose of NR are recognized by Asp56 and Arg129, and Asp36 is the general base of the enzyme. Mutation of residues in the active site can abolish the catalytic activity of the enzyme, confirming the structural observations.

  14. Cellular Inhibition of Checkpoint Kinase 2 (Chk2) and Potentiation of Camptothecins and Radiation by the Novel Chk2 Inhibitor PV1019 [7-Nitro-1H-indole-2-carboxylic acid {4-[1-(guanidinohydrazone)-ethyl]-phenyl}-amide

    SciTech Connect

    Jobson, Andrew G.; Lountos, George T.; Lorenzi, Philip L.; Llamas, Jenny; Connelly, John; Cerna, David; Tropea, Joseph E.; Onda, Akikazu; Zoppoli, Gabriele; Kondapaka, Sudhir; Zhang, Guangtao; Caplen, Natasha J.; Cardellina, II, John H.; Yoo, Stephen S.; Monks, Anne; Self, Christopher; Waugh, David S.; Shoemaker, Robert H.; Pommier, Yves

    2010-04-05

    Chk2 is a checkpoint kinase involved in the ataxia telangiectasia mutated pathway, which is activated by genomic instability and DNA damage, leading to either cell death (apoptosis) or cell cycle arrest. Chk2 provides an unexplored therapeutic target against cancer cells. We recently reported 4,4'-diacetyldiphenylurea-bis(guanylhydrazone) (NSC 109555) as a novel chemotype Chk2 inhibitor. We have now synthesized a derivative of NSC 109555, PV1019 (NSC 744039) [7-nitro-1H-indole-2-carboxylic acid {l_brace}4-[1-(guanidinohydrazone)-ethyl]-phenyl{r_brace}-amide], which is a selective submicromolar inhibitor of Chk2 in vitro. The cocrystal structure of PV1019 bound in the ATP binding pocket of Chk2 confirmed enzymatic/biochemical observations that PV1019 acts as a competitive inhibitor of Chk2 with respect to ATP. PV1019 was found to inhibit Chk2 in cells. It inhibits Chk2 autophosphorylation (which represents the cellular kinase activation of Chk2), Cdc25C phosphorylation, and HDMX degradation in response to DNA damage. PV1019 also protects normal mouse thymocytes against ionizing radiation-induced apoptosis, and it shows synergistic antiproliferative activity with topotecan, camptothecin, and radiation in human tumor cell lines. We also show that PV1019 and Chk2 small interfering RNAs can exert antiproliferative activity themselves in the cancer cells with high Chk2 expression in the NCI-60 screen. These data indicate that PV1019 is a potent and selective inhibitor of Chk2 with chemotherapeutic and radiosensitization potential.

  15. Minichromosome Maintenance Complex is Required for Checkpoint Kinase 2 Chromatin Loading and its Phosphorylation to DNA Damage Response in SCC-4 Cells.

    PubMed

    Li, Liang; Feng, Yi; Luo, Rui

    2017-01-01

    Checkpoint kinase 2 (Chk2) is a significant mediator of diverse responses to DNA damage. The present study was aimed to identify possible interactive proteins of Chk2 and try to clarify the underlying mechanism regarding Chk2 chromatin loading and its phosphorylation to DNA damage response in oral squamous cell carcinoma (OSCC). Differently tagged Chk2 and minichromosome maintenance (MCM) complex (MCM2, MCM3, MCM5, and MCM6) were overexpressed into SCC-4 cells. After 48 h of transfection cell fractionation was performed to localize proteins. In addition, immunoreactive species were detected by immunoprecipitation (IP) and immunoblot (IB) analysis, and protein-protein interaction between Chk2 and MCM complex was ensured by glutathione S-transferase (GST) pull-down assay. Expression of MCM2 and MCM6 was downregulated by small interfering RNA (siRNA), and the chromatin and non-chromatin fraction were analyzed. The expression of Chk2 phosphorylation (pT68-Chk2) was measured after administration of different dosages of siMCM2 (0.5 μg, 1 μg, and 2.5 μg) and camptothecin (CPT). Our results showed that Chk2 directly interacts with MCM2, MCM3, MCM5, and MCM6 in SCC-4 cells. Downregulation of MCM2 and MCM6 markedly reduced Chk2 chromatin fraction, and downregulation of MCM2 decreased the expression of pT68-Chk2 to DNA damage response in a dose manner. Our results suggest that the interaction between Chk2 and MCM complex is required for Chk2 chromatin loading and its phosphorylation to DNA damage response in SCC-4 cells. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  16. The human papillomavirus type 58 E7 oncoprotein modulates cell cycle regulatory proteins and abrogates cell cycle checkpoints

    SciTech Connect

    Zhang Weifang; Li Jing; Kanginakudru, Sriramana; Zhao Weiming; Yu Xiuping; Chen, Jason J.

    2010-02-05

    HPV type 58 (HPV-58) is the third most common HPV type in cervical cancer from Eastern Asia, yet little is known about how it promotes carcinogenesis. In this study, we demonstrate that HPV-58 E7 significantly promoted the proliferation and extended the lifespan of primary human keratinocytes (PHKs). HPV-58 E7 abrogated the G1 and the postmitotic checkpoints, although less efficiently than HPV-16 E7. Consistent with these observations, HPV-58 E7 down-regulated the cellular tumor suppressor pRb to a lesser extent than HPV-16 E7. Similar to HPV-16 E7 expressing PHKs, Cdk2 remained active in HPV-58 E7 expressing PHKs despite the presence of elevated levels of p53 and p21. Interestingly, HPV-58 E7 down-regulated p130 more efficiently than HPV-16 E7. Our study demonstrates a correlation between the ability of down-regulating pRb/p130 and abrogating cell cycle checkpoints by HPV-58 E7, which also correlates with the biological risks of cervical cancer progression associated with HPV-58 infection.

  17. A novel role of farnesylation in targeting a mitotic checkpoint protein, human Spindly, to kinetochores

    PubMed Central

    Moudgil, Devinderjit K.; Westcott, Nathan; Famulski, Jakub K.; Patel, Kinjal; Macdonald, Dawn; Hang, Howard

    2015-01-01

    Kinetochore (KT) localization of mitotic checkpoint proteins is essential for their function during mitosis. hSpindly KT localization is dependent on the RZZ complex and hSpindly recruits the dynein–dynactin complex to KTs during mitosis, but the mechanism of hSpindly KT recruitment is unknown. Through domain-mapping studies we characterized the KT localization domain of hSpindly and discovered it undergoes farnesylation at the C-terminal cysteine residue. The N-terminal 293 residues of hSpindly are dispensable for its KT localization. Inhibition of farnesylation using a farnesyl transferase inhibitor (FTI) abrogated hSpindly KT localization without affecting RZZ complex, CENP-E, and CENP-F KT localization. We showed that hSpindly is farnesylated in vivo and farnesylation is essential for its interaction with the RZZ complex and hence KT localization. FTI treatment and hSpindly knockdown displayed the same mitotic phenotypes, indicating that hSpindly is a key FTI target in mitosis. Our data show a novel role of lipidation in targeting a checkpoint protein to KTs through protein–protein interaction. PMID:25825516

  18. Receptor tyrosine kinase EphA5 is a functional molecular target in human lung cancer

    DOE PAGES

    Staquicini, Fernanda I.; Qian, Ming D.; Salameh, Ahmad; ...

    2015-03-20

    Lung cancer is often refractory to radiotherapy, but molecular mechanisms of tumor resistance remain poorly defined. Here we show that the receptor tyrosine kinase EphA5 is specifically overexpressed in lung cancer and is involved in regulating cellular responses to genotoxic insult. In the absence of EphA5, lung cancer cells displayed a defective G1/S cell cycle checkpoint, were unable to resolve DNA damage, and became radiosensitive. Upon irradiation, EphA5 was transported into the nucleus where it interacted with activated ATM (ataxia-telangiectasia mutated) at sites of DNA repair. In conclusion, we demonstrate that a new monoclonal antibody against human EphA5 sensitized lungmore » cancer cells and human lung cancer xenografts to radiotherapy and significantly prolonged survival, thus suggesting the likelihood of translational applications.« less

  19. Receptor tyrosine kinase EphA5 is a functional molecular target in human lung cancer

    SciTech Connect

    Staquicini, Fernanda I.; Qian, Ming D.; Salameh, Ahmad; Dobroff, Andrey S.; Edwards, Julianna K.; Cimino, Daniel F.; Moeller, Benjamin J.; Kelly, Patrick; Nunez, Maria I.; Tang, Ximing; Liu, Diane D.; Lee, J. Jack; Hong, Waun Ki; Ferrara, Fortunato; Bradbury, Andrew R. M.; Lobb, Roy R.; Edelman, Martin J.; Sidman, Richard L.; Wistuba, Ignacio I.; Arap, Wadih; Pasqualini, Renata

    2015-03-20

    Lung cancer is often refractory to radiotherapy, but molecular mechanisms of tumor resistance remain poorly defined. Here we show that the receptor tyrosine kinase EphA5 is specifically overexpressed in lung cancer and is involved in regulating cellular responses to genotoxic insult. In the absence of EphA5, lung cancer cells displayed a defective G1/S cell cycle checkpoint, were unable to resolve DNA damage, and became radiosensitive. Upon irradiation, EphA5 was transported into the nucleus where it interacted with activated ATM (ataxia-telangiectasia mutated) at sites of DNA repair. In conclusion, we demonstrate that a new monoclonal antibody against human EphA5 sensitized lung cancer cells and human lung cancer xenografts to radiotherapy and significantly prolonged survival, thus suggesting the likelihood of translational applications.

  20. Immune checkpoint inhibitors enhance cytotoxicity of cytokine-induced killer cells against human myeloid leukaemic blasts.

    PubMed

    Poh, Su Li; Linn, Yeh Ching

    2016-05-01

    We studied whether blockade of inhibitory receptors on cytokine-induced killer (CIK) cells by immune checkpoint inhibitors could increase its anti-tumour potency against haematological malignancies. CIK cultures were generated from seven normal donors and nine patients with acute myeloid leukaemia (AML), acute lymphoblastic leukaemia (ALL) or multiple myeloma (MM). The inhibitory receptors B and T lymphocyte attenuator, CD200 receptor, lymphocyte activation gene-3 (LAG-3) and T cell immunoglobulin and mucin-domain-containing-3 (TIM-3) were present at variable percentages in most CIK cultures, while cytotoxic T lymphocyte-associated protein 4 (CTLA-4), programmed death-1 (PD-1) and killer cell immunoglobulin-like receptors (KIR2DL1/2/3) were expressed at low level in most cultures. Without blockade, myeloid leukaemia cells were susceptible to autologous and allogeneic CIK-mediated cytotoxicity. Blockade of KIR, LAG-3, PD-1 and TIM-3 but not CTLA-4 resulted in remarkable increase in killing against these targets, even in those with poor baseline cytotoxicity. ALL and MM targets were resistant to CIK-mediated cytotoxicity, and blockade of receptors did not increase cytotoxicity to a meaningful extent. Combination of inhibitors against two receptors did not further increase cytotoxicity. Interestingly, potentiation of CIK killing by blocking antibodies was not predicted by expression of receptors on CIK and their respective ligands on the targets. Compared to un-activated T and NK cells, blockade potentiated the cytotoxicity of CIK cells to a greater degree and at a lower E:T ratio, but without significant increase in cytotoxicity against normal white cell. Our findings provide the basis for clinical trial combining autologous CIK cells with checkpoint inhibitors for patients with AML.

  1. DNA damage checkpoint recovery and cancer development

    SciTech Connect

    Wang, Haiyong; Zhang, Xiaoshan; Teng, Lisong; Legerski, Randy J.

    2015-06-10

    Cell cycle checkpoints were initially presumed to function as a regulator of cell cycle machinery in response to different genotoxic stresses, and later found to play an important role in the process of tumorigenesis by acting as a guard against DNA over-replication. As a counterpart of checkpoint activation, the checkpoint recovery machinery is working in opposition, aiming to reverse the checkpoint activation and resume the normal cell cycle. The DNA damage response (DDR) and oncogene induced senescence (OIS) are frequently found in precancerous lesions, and believed to constitute a barrier to tumorigenesis, however, the DDR and OIS have been observed to be diminished in advanced cancers of most tissue origins. These findings suggest that when progressing from pre-neoplastic lesions to cancer, DNA damage checkpoint barriers are overridden. How the DDR checkpoint is bypassed in this process remains largely unknown. Activated cytokine and growth factor-signaling pathways were very recently shown to suppress the DDR and to promote uncontrolled cell proliferation in the context of oncovirus infection. In recent decades, data from cell line and tumor models showed that a group of checkpoint recovery proteins function in promoting tumor progression; data from patient samples also showed overexpression of checkpoint recovery proteins in human cancer tissues and a correlation with patients' poor prognosis. In this review, the known cell cycle checkpoint recovery proteins and their roles in DNA damage checkpoint recovery are reviewed, as well as their implications in cancer development. This review also provides insight into the mechanism by which the DDR suppresses oncogene-driven tumorigenesis and tumor progression. - Highlights: • DNA damage checkpoint works as a barrier to cancer initiation. • DDR machinary response to genotoxic and oncogenic stress in similar way. • Checkpoint recovery pathways provide active signaling in cell cycle control. • Checkpoint

  2. Polychlorinated Biphenyl Quinone Metabolite Promotes p53-Dependent DNA Damage Checkpoint Activation, S-Phase Cycle Arrest and Extrinsic Apoptosis in Human Liver Hepatocellular Carcinoma HepG2 Cells.

    PubMed

    Song, Xiufang; Li, Lingrui; Shi, Qiong; Lehmler, Hans-Joachim; Fu, Juanli; Su, Chuanyang; Xia, Xiaomin; Song, Erqun; Song, Yang

    2015-11-16

    Polychlorinated biphenyls (PCBs) are a group of persistent organic pollutants. The toxic behavior and mechanism of PCBs individuals and congeners have been extensively investigated. However, there is only limited information on their metabolites. Our previous studies have shown that a synthetic PCB metabolite, PCB29-pQ, causes oxidative damage with the evidence of cytotoxicity, genotoxicity, and mitochondrial-derived intrinsic apoptosis. Here, we investigate the effects of PCB29-pQ on DNA damage checkpoint activation, cell cycle arrest, and death receptor-related extrinsic apoptosis in human liver hepatocellular carcinoma HepG2 cells. Our results illustrate that PCB29-pQ increases the S-phase cell population by down-regulating cyclins A/D1/E, cyclin-dependent kinases (CDK 2/4/6), and cell division cycle 25A (CDC25A) and up-regulating p21/p27 protein expressions. PCB29-pQ also induces apoptosis via the up-regulation of Fas/FasL and the activation of caspase 8/3. Moreover, p53 plays a pivotal role in PCB29-pQ-induced cell cycle arrest and apoptosis via the activation of ATM/Chk2 and ATR/Chk1 checkpoints. Cell cycle arrest and apoptotic cell death were attenuated by the pretreatment with antioxidant N-acetyl-cysteine (NAC). Taken together, these results demonstrate that PCB29-pQ induces oxidative stress and promotes p53-dependent DNA damage checkpoint activation, S-phase cycle arrest, and extrinsic apoptosis in HepG2 cells.

  3. Obtusilactone A and (-)-sesamin induce apoptosis in human lung cancer cells by inhibiting mitochondrial Lon protease and activating DNA damage checkpoints.

    PubMed

    Wang, Hui-Min; Cheng, Kuo-Chen; Lin, Cheng-Jung; Hsu, Shu-Wei; Fang, Wei-Cheng; Hsu, Tai-Feng; Chiu, Chien-Chih; Chang, Hsueh-Wei; Hsu, Chun-Hua; Lee, Alan Yueh-Luen

    2010-12-01

    Several compounds from Cinnamomum kotoense show anticancer activities. However, the detailed mechanisms of most compounds from C. kotoense remain unknown. In this study, we investigated the anticancer activity of obtusilactone A (OA) and (-)-sesamin in lung cancer. Our results show that human Lon is upregulated in non-small-cell lung cancer (NSCLC) cell lines, and downregulation of Lon triggers caspase-3 mediated apoptosis. Through enzyme-based screening, we identified two small-molecule compounds, obtusilactone A (OA) and (-)-sesamin from C. kotoense, as potent Lon protease inhibitors. Obtusilactone A and (-)-sesamin interact with Ser855 and Lys898 residues in the active site of the Lon protease according to molecular docking analysis. Thus, we suggest that cancer cytotoxicity of the compounds is partly due to the inhibitory effects on Lon protease. In addition, the compounds are able to cause DNA double-strand breaks and activate checkpoints. Treatment with OA and (-)-sesamin induced p53-independent DNA damage responses in NSCLC cells, including G(1) /S checkpoint activation and apoptosis, as evidenced by phosphorylation of checkpoint proteins (H2AX, Nbs1, and Chk2), caspase-3 cleavage, and sub-G(1) accumulation. In conclusion, OA and (-)-sesamin act as both inhibitors of human mitochondrial Lon protease and DNA damage agents to activate the DNA damage checkpoints as well induce apoptosis in NSCLC cells. These dual functions open a bright avenue to develop more selective chemotherapy agents to overcome chemoresistance and sensitize cancer cells to other chemotherapeutics.

  4. The energy sensor AMPK regulates Hedgehog signaling in human cells through a unique Gli1 metabolic checkpoint

    PubMed Central

    Di Magno, Laura; Basile, Alessio; Coni, Sonia; Manni, Simona; Sdruscia, Giulia; D'Amico, Davide; Antonucci, Laura; Infante, Paola; De Smaele, Enrico; Cucchi, Danilo; Ferretti, Elisabetta; Di Marcotullio, Lucia; Screpanti, Isabella; Canettieri, Gianluca

    2016-01-01

    Hedgehog signaling controls proliferation of cerebellar granule cell precursors (GCPs) and its aberrant activation is a leading cause of Medulloblastoma, the most frequent pediatric brain tumor. We show here that the energy sensor AMPK inhibits Hh signaling by phosphorylating a single residue of human Gli1 that is not conserved in other species. Studies with selective agonists and genetic deletion have revealed that AMPK activation inhibits canonical Hh signaling in human, but not in mouse cells. Indeed we show that AMPK phosphorylates Gli1 at the unique residue Ser408, which is conserved only in primates but not in other species. Once phosphorylated, Gli1 is targeted for proteasomal degradation. Notably, we show that selective AMPK activation inhibits Gli1-driven proliferation and that this effect is linked to Ser408 phosphorylation, which represents a key metabolic checkpoint for Hh signaling. Collectively, this data unveil a novel mechanism of inhibition of Gli1 function, which is exclusive for human cells and may be exploited for the treatment of Medulloblastoma or other Gli1 driven tumors. PMID:26843621

  5. Prediction of 492 human protein kinase substrate specificities.

    PubMed

    Safaei, Javad; Maňuch, Ján; Gupta, Arvind; Stacho, Ladislav; Pelech, Steven

    2011-10-14

    Complex intracellular signaling networks monitor diverse environmental inputs to evoke appropriate and coordinated effector responses. Defective signal transduction underlies many pathologies, including cancer, diabetes, autoimmunity and about 400 other human diseases. Therefore, there is high impetus to define the composition and architecture of cellular communications networks in humans. The major components of intracellular signaling networks are protein kinases and protein phosphatases, which catalyze the reversible phosphorylation of proteins. Here, we have focused on identification of kinase-substrate interactions through prediction of the phosphorylation site specificity from knowledge of the primary amino acid sequence of the catalytic domain of each kinase. The presented method predicts 488 different kinase catalytic domain substrate specificity matrices in 478 typical and 4 atypical human kinases that rely on both positive and negative determinants for scoring individual phosphosites for their suitability as kinase substrates. This represents a marked advancement over existing methods such as those used in NetPhorest (179 kinases in 76 groups) and NetworKIN (123 kinases), which consider only positive determinants for kinase substrate prediction. Comparison of our predicted matrices with experimentally-derived matrices from about 9,000 known kinase-phosphosite substrate pairs revealed a high degree of concordance with the established preferences of about 150 well studied protein kinases. Furthermore for many of the better known kinases, the predicted optimal phosphosite sequences were more accurate than the consensus phosphosite sequences inferred by simple alignment of the phosphosites of known kinase substrates. Application of this improved kinase substrate prediction algorithm to the primary structures of over 23, 000 proteins encoded by the human genome has permitted the identification of about 650, 000 putative phosphosites, which are posted on the

  6. A role for the spindle assembly checkpoint in the DNA damage response.

    PubMed

    Palou, Roger; Palou, Gloria; Quintana, David G

    2017-05-01

    Spontaneous DNA damage poses a continuous threat to genomic integrity. If unchecked, genotoxic insults result in genomic instability, a hallmark of cancer cells. In eukaryotic cells a DNA Damage Response (DDR) detects and responds to genotoxic stress, acting as an anti-cancer barrier in humans. Among other actions, the DDR blocks the segregation of incompletely replicated or damaged chromosomes, thus preventing aneuploidy. In a work aimed at better understanding such S-M control, we recently showed that cells block anaphase through different control pathways. The S phase checkpoint kinase Mec1/ATR inhibits mitotic Cyclin Dependent Kinase activity through effector kinases Swe1/Wee1 and Rad53/Chk2. Cells also stabilize the levels of Pds1/securin to block sister chromatid segregation in response to DNA damage. We show here that Pds1/securin abundance is still secured when the S phase checkpoint response is fully abrogated in mec1/ATR tel1/ATM double null mutants. When such cells are exposed to genotoxic stress, Pds1/securin is stabilized in a spindle assembly checkpoint (SAC) dependent manner. Disruption of the SAC and the S phase checkpoint together, allows chromosome segregation in the presence of DNA damage or replication stress. Our results place the SAC as a part of the DDR, which appears to count on different, independent control layers to preserve genomic integrity when chromosome replication is challenged.

  7. Human Gastric Cancer Kinase Profile and Prognostic Significance of MKK4 Kinase

    PubMed Central

    Wu, Chew-Wun; Li, Anna F.-Y.; Chi, Chin-Wen; Huang, Chen Lung; Shen, King-Han; Liu, Wing-Yiu; Lin, Wen-chang

    2000-01-01

    Alterations of protein tyrosine kinase are often associated with uncontrolled cell growth and tumor progression. Knowledge of the overall expression pattern of tyrosine kinases should prove beneficial in understanding the signaling pathways involved in gastric cancer oncogenesis and in providing possible biomarkers for gastric cancer progression. To establish a general tyrosine-kinase expression profile, degenerated polymerase chain reaction primers designed from the consensus catalytic kinase motifs were used to amplify protein tyrosine kinase molecules from gastric cancer tissues. We observed more than 50 tyrosine and serine/threonine kinases from matching pairs of gastric cancer tissue and normal mucosa. Based on this new kinase profile information, we selected the MKK4 gene for further immunohistochemical studies. Statistical analysis of MKK4 protein expression and clinicopathological features indicated that MKK4 kinase expression could serve as a significant prognostic factor for relapse-free survival and for overall survival. We demonstrated a simple and sensitive method for establishing protein tyrosine-kinase expression profiles of human gastric cancer tissues as well as for discovering novel and useful clinical biomarkers from such kinase expression profiles. PMID:10854223

  8. Taxifolin Enhances Andrographolide-Induced Mitotic Arrest and Apoptosis in Human Prostate Cancer Cells via Spindle Assembly Checkpoint Activation

    PubMed Central

    Wong, Matthew Man-Kin; Chiu, Sung-Kay; Cheung, Hon-Yeung

    2013-01-01

    Andrographolide (Andro) suppresses proliferation and triggers apoptosis in many types of cancer cells. Taxifolin (Taxi) has been proposed to prevent cancer development similar to other dietary flavonoids. In the present study, the cytotoxic and apoptotic effects of the addition of Andro alone and Andro and Taxi together on human prostate carcinoma DU145 cells were assessed. Andro inhibited prostate cancer cell proliferation by mitotic arrest and activation of the intrinsic apoptotic pathway. Although the effect of Taxi alone on DU145 cell proliferation was not significant, the combined use of Taxi with Andro significantly potentiated the anti-proliferative effect of increased mitotic arrest and apoptosis by enhancing the cleavage of poly(ADP-ribose) polymerase, and caspases-7 and -9. Andro together with Taxi enhanced microtubule polymerization in vitro, and they induced the formation of twisted and elongated spindles in the cancer cells, thus leading to mitotic arrest. In addition, we showed that depletion of MAD2, a component in the spindle assembly checkpoint (SAC), alleviated the mitotic block induced by the two compounds, suggesting that they trigger mitotic arrest by SAC activation. This study suggests that the anti-cancer activity of Andro can be significantly enhanced in combination with Taxi by disrupting microtubule dynamics and activating the SAC. PMID:23382917

  9. Taxifolin enhances andrographolide-induced mitotic arrest and apoptosis in human prostate cancer cells via spindle assembly checkpoint activation.

    PubMed

    Zhang, Zhong Rong; Al Zaharna, Mazen; Wong, Matthew Man-Kin; Chiu, Sung-Kay; Cheung, Hon-Yeung

    2013-01-01

    Andrographolide (Andro) suppresses proliferation and triggers apoptosis in many types of cancer cells. Taxifolin (Taxi) has been proposed to prevent cancer development similar to other dietary flavonoids. In the present study, the cytotoxic and apoptotic effects of the addition of Andro alone and Andro and Taxi together on human prostate carcinoma DU145 cells were assessed. Andro inhibited prostate cancer cell proliferation by mitotic arrest and activation of the intrinsic apoptotic pathway. Although the effect of Taxi alone on DU145 cell proliferation was not significant, the combined use of Taxi with Andro significantly potentiated the anti-proliferative effect of increased mitotic arrest and apoptosis by enhancing the cleavage of poly(ADP-ribose) polymerase, and caspases-7 and -9. Andro together with Taxi enhanced microtubule polymerization in vitro, and they induced the formation of twisted and elongated spindles in the cancer cells, thus leading to mitotic arrest. In addition, we showed that depletion of MAD2, a component in the spindle assembly checkpoint (SAC), alleviated the mitotic block induced by the two compounds, suggesting that they trigger mitotic arrest by SAC activation. This study suggests that the anti-cancer activity of Andro can be significantly enhanced in combination with Taxi by disrupting microtubule dynamics and activating the SAC.

  10. LIM kinases: function, regulation and association with human disease.

    PubMed

    Scott, Rebecca W; Olson, Michael F

    2007-06-01

    The LIM kinase family consists of just two members: LIM kinase 1 (LIMK1) and LIM kinase 2 (LIMK2). With uniquely organised signalling domains, LIM kinases are regulated by several upstream signalling pathways, principally acting downstream of Rho GTPases to influence the architecture of the actin cytoskeleton by regulating the activity of the cofilin family proteins cofilin1, cofilin2 and destrin. Although the LIM kinases are very homologous, particularly when comparing kinase domains, there is emerging evidence that each may be subject to different regulatory pathways and may contribute to both distinct and overlapping cellular and developmental functions. Normal central nervous system development is reliant upon the presence of LIMK1, and its deletion has been implicated in the development of the human genetic disorder Williams syndrome. Normal testis development, on the other hand, is disrupted by the deletion of LIMK2. In addition, the possible involvement of each kinase in cardiovascular disorders as well as cancer has recently emerged. The LIM kinases have been proposed to play an important role in tumour-cell invasion and metastasis; fine-tuning the balance between phosphorylated and non-phosphorylated cofilin may be a significant determinant of tumour-cell metastatic potential. In this review, we outline the structure, regulation and function of LIM kinases and their functions at cellular and organismal levels, as well as their possible contributions to human disease.

  11. Anaphase-Promoting Complex/Cyclosome–Dependent Proteolysis of Human Cyclin a Starts at the Beginning of Mitosis and Is Not Subject to the Spindle Assembly Checkpoint

    PubMed Central

    Geley, Stephan; Kramer, Edgar; Gieffers, Christian; Gannon, Julian; Peters, Jan-Michael; Hunt, Tim

    2001-01-01

    Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome (APC/C) or against human Cdc20 (fizzy) arrested cells at metaphase and stabilized both cyclins A and B1. Cyclin A was efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A was not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 was accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The “destruction box” (D-box) of cyclin A is 10–20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delayed the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrested cells in anaphase. PMID:11285280

  12. Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition

    PubMed Central

    Cherkassky, Leonid; Morello, Aurore; Villena-Vargas, Jonathan; Feng, Yang; Dimitrov, Dimiter S.; Jones, David R.; Sadelain, Michel; Adusumilli, Prasad S.

    2016-01-01

    Following immune attack, solid tumors upregulate coinhibitory ligands that bind to inhibitory receptors on T cells. This adaptive resistance compromises the efficacy of chimeric antigen receptor (CAR) T cell therapies, which redirect T cells to solid tumors. Here, we investigated whether programmed death-1–mediated (PD-1–mediated) T cell exhaustion affects mesothelin-targeted CAR T cells and explored cell-intrinsic strategies to overcome inhibition of CAR T cells. Using an orthotopic mouse model of pleural mesothelioma, we determined that relatively high doses of both CD28- and 4-1BB–based second-generation CAR T cells achieved tumor eradication. CAR-mediated CD28 and 4-1BB costimulation resulted in similar levels of T cell persistence in animals treated with low T cell doses; however, PD-1 upregulation within the tumor microenvironment inhibited T cell function. At lower doses, 4-1BB CAR T cells retained their cytotoxic and cytokine secretion functions longer than CD28 CAR T cells. The prolonged function of 4-1BB CAR T cells correlated with improved survival. PD-1/PD-1 ligand [PD-L1] pathway interference, through PD-1 antibody checkpoint blockade, cell-intrinsic PD-1 shRNA blockade, or a PD-1 dominant negative receptor, restored the effector function of CD28 CAR T cells. These findings provide mechanistic insights into human CAR T cell exhaustion in solid tumors and suggest that PD-1/PD-L1 blockade may be an effective strategy for improving the potency of CAR T cell therapies. PMID:27454297

  13. Human CAR T cells with cell-intrinsic PD-1 checkpoint blockade resist tumor-mediated inhibition.

    PubMed

    Cherkassky, Leonid; Morello, Aurore; Villena-Vargas, Jonathan; Feng, Yang; Dimitrov, Dimiter S; Jones, David R; Sadelain, Michel; Adusumilli, Prasad S

    2016-08-01

    Following immune attack, solid tumors upregulate coinhibitory ligands that bind to inhibitory receptors on T cells. This adaptive resistance compromises the efficacy of chimeric antigen receptor (CAR) T cell therapies, which redirect T cells to solid tumors. Here, we investigated whether programmed death-1-mediated (PD-1-mediated) T cell exhaustion affects mesothelin-targeted CAR T cells and explored cell-intrinsic strategies to overcome inhibition of CAR T cells. Using an orthotopic mouse model of pleural mesothelioma, we determined that relatively high doses of both CD28- and 4-1BB-based second-generation CAR T cells achieved tumor eradication. CAR-mediated CD28 and 4-1BB costimulation resulted in similar levels of T cell persistence in animals treated with low T cell doses; however, PD-1 upregulation within the tumor microenvironment inhibited T cell function. At lower doses, 4-1BB CAR T cells retained their cytotoxic and cytokine secretion functions longer than CD28 CAR T cells. The prolonged function of 4-1BB CAR T cells correlated with improved survival. PD-1/PD-1 ligand [PD-L1] pathway interference, through PD-1 antibody checkpoint blockade, cell-intrinsic PD-1 shRNA blockade, or a PD-1 dominant negative receptor, restored the effector function of CD28 CAR T cells. These findings provide mechanistic insights into human CAR T cell exhaustion in solid tumors and suggest that PD-1/PD-L1 blockade may be an effective strategy for improving the potency of CAR T cell therapies.

  14. A family of human cdc2-related protein kinases.

    PubMed Central

    Meyerson, M; Enders, G H; Wu, C L; Su, L K; Gorka, C; Nelson, C; Harlow, E; Tsai, L H

    1992-01-01

    The p34cdc2 protein kinase is known to regulate important transitions in the eukaryotic cell cycle. We have identified 10 human protein kinases based on their structural relation to p34cdc2. Seven of these kinases are novel and the products of five share greater than 50% amino acid sequence identity with p34cdc2. The seven novel genes are broadly expressed in human cell lines and tissues with each displaying some cell type or tissue specificity. The cdk3 gene, like cdc2 and cdk2, can complement cdc28 mutants of Saccharomyces cerevisiae, suggesting that all three of these protein kinases can play roles in the regulation of the mammalian cell cycle. The identification of a large family of cdc2-related kinases opens the possibility of combinatorial regulation of the cell cycle together with the emerging large family of cyclins. Images PMID:1639063

  15. Diverse functions of spindle assembly checkpoint genes in Saccharomyces cerevisiae.

    PubMed

    Daniel, Jewel A; Keyes, Brice E; Ng, Yvonne P Y; Freeman, C Onyi; Burke, Daniel J

    2006-01-01

    The spindle assembly checkpoint regulates the metaphase-to-anaphase transition from yeast to humans. We examined the genetic interactions with four spindle assembly checkpoint genes to identify nonessential genes involved in chromosome segregation, to identify the individual roles of the spindle assembly checkpoint genes within the checkpoint, and to reveal potential complexity that may exist. We used synthetic genetic array (SGA) analysis using spindle assembly checkpoint mutants mad1, mad2, mad3, and bub3. We found 228 synthetic interactions with the four spindle assembly checkpoint mutants with substantial overlap in the spectrum of interactions between mad1, mad2, and bub3. In contrast, there were many synthetic interactions that were common to mad1, mad2, and bub3 that were not shared by mad3. We found shared interactions between pairs of spindle assembly checkpoint mutants, suggesting additional complexity within the checkpoint and unique interactions for all of the spindle assembly checkpoint genes. We show that most genes in the interaction network, including ones with unique interactions, affect chromosome transmission or microtubule function, suggesting that the complexity of interactions reflects diverse roles for the checkpoint genes within the checkpoint. Our analysis expands our understanding of the spindle assembly checkpoint and identifies new candidate genes with possible roles in chromosome transmission and mitotic spindle function.

  16. The mysterious human epidermal cell cycle, or an oncogene-induced differentiation checkpoint

    PubMed Central

    Gandarillas, Alberto

    2012-01-01

    Fifteen years ago, we reported that proto-oncogene MYC promoted differentiation of human epidermal stem cells, a finding that was surprising to the MYC and the skin research communities. MYC was one of the first human oncogenes identified, and it had been strongly associated with proliferation. However, it was later shown that MYC could induce apoptosis under low survival conditions. Currently, the notion that MYC promotes epidermal differentiation is widely accepted, but the cell cycle mechanisms that elicit this function remain unresolved. We have recently reported that keratinocytes respond to cell cycle deregulation and DNA damage by triggering terminal differentiation. This mechanism might constitute a homeostatic protection face to cell cycle insults. Here, I discuss recent and not-so-recent evidence suggesting the existence of a largely unexplored oncogene-induced differentiation response (OID) analogous to oncogene-induced apoptosis (OIA) or senescence (OIS). In addition, I propose a model for the role of the cell cycle in skin homeostasis maintenance and for the dual role of MYC in differentiation. PMID:23114621

  17. Contrasting roles of checkpoint proteins as recombination modulators at Fob1-Ter complexes with or without fork arrest.

    PubMed

    Mohanty, Bidyut K; Bairwa, Narendra K; Bastia, Deepak

    2009-04-01

    The replication terminator protein Fob1 of Saccharomyces cerevisiae specifically interacts with two tandem Ter sites (replication fork barriers) located in the nontranscribed spacer of ribosomal DNA (rDNA) to cause polar fork arrest. The Fob1-Ter complex is multifunctional and controls other DNA transactions such as recombination by multiple mechanisms. Here, we report on the regulatory roles of the checkpoint proteins in the initiation and progression of recombination at Fob1-Ter complexes. The checkpoint adapter proteins Tof1 and Csm3 either positively or negatively controlled recombination depending on whether it was provoked by polar fork arrest or by transcription, respectively. The absolute requirements for these proteins for inducing recombination at an active replication terminus most likely masked their negative modulatory role at a later step of the process. Other checkpoint proteins of the checkpoint adapter/mediator class such as Mrc1 and Rad9, which channel signals from the sensor to the effector kinase, tended to suppress recombination at Fob1-Ter complexes regardless of how it was initiated. We have also discovered that the checkpoint sensor kinase Mec1 and the effector Rad53 were positive modulators of recombination initiated by transcription but had little effect on recombination at Ter. The work also showed that the two pathways were Rad52 dependent but Rad51 independent. Since Ter sites occur in the intergenic spacer of rDNA from yeast to humans, the mechanism is likely to be of widespread occurrence.

  18. Alteration of cell cycle in cervical tumor associated with human papillomavirus: cyclin-dependent kinase inhibitors.

    PubMed

    Cho, Nam Hoon; Kim, Young Tae; Kim, Jae Wook

    2002-12-01

    The ability of viral oncoproteins to subvert cell cycle checkpoints may constitute a mechanism by which viral oncoproteins induce genetic instability. HPV 16 E6 and E7 disrupt cell cycle checkpoints, particularly affecting nearly all cyclin-dependent kinase inhibitors linked to the G1- and G2- checkpoints, in each case by means of a different mechanism. HPV 16 E7 shows homology with the pRb binding sites of cyclin D1, which consequently releases E2F. In addition, E7 directly binds to p21, and releases PCNA and other S-phase promoting genes. In turn, released E2F activates cyclin E, and cyclin E accelerates p27 proteolysis as a function of the antagonistic reaction of its own inhibitor. The induction of p16 expression is assumed to be indirectly associated with E7, which is upregulated only after prolonged inactivation of Rb. HPV 16 E6 decreased the fidelity of multiple checkpoints controlling both entry into and exit from mitosis, with the mechanism of p53 inactivation. In addition, HPV 16 E6 increased the sensitivity to chemically induced S-phase premature mitosis and decreased mitotic spindle assembly checkpoint function. Alongside the impressive advances made in the understanding of the molecular mechanisms, which HPV disrupts, the validity of these conclusions should be evaluated in the diagnostic and prognostic fields.

  19. Structure of the human dimeric ATM kinase

    PubMed Central

    Lau, Wilson C. Y.; Li, Yinyin; Liu, Zhe; Gao, Yuanzhu; Zhang, Qinfen; Huen, Michael S. Y.

    2016-01-01

    ABSTRACT DNA-double strand breaks activate the serine/threonine protein kinase ataxia-telangiectasia mutated (ATM) to initiate DNA damage signal transduction. This activation process involves autophosphorylation and dissociation of inert ATM dimers into monomers that are catalytically active. Using single-particle electron microscopy (EM), we determined the structure of dimeric ATM in its resting state. The EM map could accommodate the crystal structure of the N-terminal truncated mammalian target of rapamycin (mTOR), a closely related enzyme of the phosphatidylinositol 3-kinase-related protein kinase (PIKK) family, allowing for the localization of the N- and the C-terminal regions of ATM. In the dimeric structure, the actives sites are buried, restricting the access of the substrates to these sites. The unanticipated domain organization of ATM provides a basis for understanding its mechanism of inhibition. PMID:27097373

  20. Prevention of DNA Rereplication Through a Meiotic Recombination Checkpoint Response

    PubMed Central

    Najor, Nicole A.; Weatherford, Layne; Brush, George S.

    2016-01-01

    In the budding yeast Saccharomyces cerevisiae, unnatural stabilization of the cyclin-dependent kinase inhibitor Sic1 during meiosis can trigger extra rounds of DNA replication. When programmed DNA double-strand breaks (DSBs) are generated but not repaired due to absence of DMC1, a pathway involving the checkpoint gene RAD17 prevents this DNA rereplication. Further genetic analysis has now revealed that prevention of DNA rereplication also requires MEC1, which encodes a protein kinase that serves as a central checkpoint regulator in several pathways including the meiotic recombination checkpoint response. Downstream of MEC1, MEK1 is required through its function to inhibit repair between sister chromatids. By contrast, meiotic recombination checkpoint effectors that regulate gene expression and cyclin-dependent kinase activity are not necessary. Phosphorylation of histone H2A, which is catalyzed by Mec1 and the related Tel1 protein kinase in response to DSBs, and can help coordinate activation of the Rad53 checkpoint protein kinase in the mitotic cell cycle, is required for the full checkpoint response. Phosphorylation sites that are targeted by Rad53 in a mitotic S phase checkpoint response are also involved, based on the behavior of cells containing mutations in the DBF4 and SLD3 DNA replication genes. However, RAD53 does not appear to be required, nor does RAD9, which encodes a mediator of Rad53, consistent with their lack of function in the recombination checkpoint pathway that prevents meiotic progression. While this response is similar to a checkpoint mechanism that inhibits initiation of DNA replication in the mitotic cell cycle, the evidence points to a new variation on DNA replication control. PMID:27678521

  1. Radio-sensitization of human leukaemic molt-4 cells by DNA-dependent protein kinase inhibitor, NU7026.

    PubMed

    Tichý, Ales; Novotná, Eva; Durisová, Kamila; Salovská, Barbora; Sedlaríková, Radka; Pejchal, Jaroslav; Zárybnická, Lenka; Vávrová, Jirina; Sinkorová, Zuzana; Rezácová, Martina

    2012-01-01

    In this paper we describe the influence of NU7026, a specific inhibitor of DNA-dependent protein kinase, phosphoinositide 3-kinase, and ATM-kinase on molecular and cellular mechanisms triggered by ionising irradiation in human T-lymphocyte leukaemic MOLT-4 cells. We studied the effect of this inhibitor (10 1microM) combined with gamma-radiation (1 Gy) leading to DNA damage response and induction of apoptosis. We used methods for apoptosis assessment (cell viability count and flow-cytometric analysis) and cell cycle analysis (DNA content measurement) and we detected expression and post-translational modifications (Western blotting) of proteins involved in DNA repair signalling pathways. Pre-treatment with NU7026 resulted into decreased activation of checkpoint kinase-2 (Thr68), p53 (Ser15 and Ser392), and histone H2A.X (Ser139) 2 hours after irradiation. Subsequently, combination of radiation and inhibitor led to decreased amount of cells in G2-phase arrest and into increased apoptosis after 72 hours. Our results indicate that in leukaemic cells the pre-incubation with inhibitor NU7026 followed by low doses of ionising radiation results in radio-sensitising of MOLT-4 cells via diminished DNA repair and delayed but pronounced apoptosis. This novel approach might offer new strategies in combined treatment of leukaemia diseases.

  2. The spindle checkpoint and chromosome segregation in meiosis.

    PubMed

    Gorbsky, Gary J

    2015-07-01

    The spindle checkpoint is a key regulator of chromosome segregation in mitosis and meiosis. Its function is to prevent precocious anaphase onset before chromosomes have achieved bipolar attachment to the spindle. The spindle checkpoint comprises a complex set of signaling pathways that integrate microtubule dynamics, biomechanical forces at the kinetochores, and intricate regulation of protein interactions and post-translational modifications. Historically, many key observations that gave rise to the initial concepts of the spindle checkpoint were made in meiotic systems. In contrast with mitosis, the two distinct chromosome segregation events of meiosis present a special challenge for the regulation of checkpoint signaling. Preservation of fidelity in chromosome segregation in meiosis, controlled by the spindle checkpoint, also has a significant impact in human health. This review highlights the contributions from meiotic systems in understanding the spindle checkpoint as well as the role of checkpoint signaling in controlling the complex divisions of meiosis.

  3. Effects of butyltins on mitogen-activated-protein kinase kinase kinase and Ras activity in human natural killer cells.

    PubMed

    Celada, Lindsay J; Whalen, Margaret M

    2014-09-01

    Butyltins (BTs) contaminate the environment and are found in human blood. BTs, tributyltin (TBT) and dibutyltin (DBT) diminish the cytotoxic function and levels of key proteins of human natural killer (NK) cells. NK cells are an initial immune defense against tumors, virally infected cells and antibody-coated cells and thus critical to human health. The signaling pathways that regulate NK cell functions include mitogen-activated protein kinases (MAPKs). Studies have shown that exposure to BTs leads to activation of specific MAPKs and MAPK kinases (MAP2Ks) in human NK cells. MAP2K kinases (MAP3Ks) are upstream activators of MAP2Ks, which then activate MAPKs. The current study examined if BT-induced activation of MAP3Ks was responsible for MAP2K and thus, MAPK activation. This study examines the effects of TBT and DBT on the total levels of two MAP3Ks, c-Raf and ASK1, as well as activating and inhibitory phosphorylation sites on these MAP3Ks. In addition, the immediate upstream activator of c-Raf, Ras, was examined for BT-induced alterations. Our results show significant activation of the MAP3K, c-Raf, in human NK cells within 10 min of TBT exposure and the MAP3K, ASK1, after 1 h exposures to TBT. In addition, our results suggest that both TBT and DBT affect the regulation of c-Raf.

  4. Design and evaluation of 3,6-di(hetero)aryl imidazo[1,2-a]pyrazines as inhibitors of checkpoint and other kinases.

    PubMed

    Matthews, Thomas P; McHardy, Tatiana; Klair, Suki; Boxall, Kathy; Fisher, Martin; Cherry, Michael; Allen, Charlotte E; Addison, Glynn J; Ellard, John; Aherne, G Wynne; Westwood, Isaac M; van Montfort, Rob; Garrett, Michelle D; Reader, John C; Collins, Ian

    2010-07-15

    A range of 3,6-di(hetero)arylimidazo[1,2-a]pyrazine ATP-competitive inhibitors of CHK1 were developed by scaffold hopping from a weakly active screening hit. Efficient synthetic routes for parallel synthesis were developed to prepare analogues with improved potency and ligand efficiency against CHK1. Kinase profiling showed that the imidazo[1,2-a]pyrazines could inhibit other kinases, including CHK2 and ABL, with equivalent or better potency depending on the pendant substitution. These 3,6-di(hetero)aryl imidazo[1,2-a]pyrazines appear to represent a general kinase inhibitor scaffold. 2010 Elsevier Ltd. All rights reserved.

  5. eIF2 kinases mediate β-lapachone toxicity in yeast and human cancer cells

    PubMed Central

    Menacho-Márquez, Mauricio; Rodríguez-Hernández, Carlos J; Villaronga, M Ángeles; Pérez-Valle, Jorge; Gadea, José; Belandia, Borja; Murguía, José R

    2015-01-01

    β-lapachone (β-lap) is a novel anticancer agent that selectively induces cell death in human cancer cells, by activation of the NQO1 NAD(P)H dehydrogenase and radical oxygen species (ROS) generation. We characterized the gene expression profile of budding yeast cells treated with β-lap using cDNA microarrays. Genes involved in tolerance to oxidative stress were differentially expressed in β-lap treated cells. β-lap treatment generated reactive oxygen species (ROS), which were efficiently blocked by dicoumarol, an inhibitor of NADH dehydrogenases. A yeast mutant in the mitocondrial NADH dehydrogenase Nde2p was found to be resistant to β-lap treatment, despite inducing ROS production in a WT manner. Most interestingly, DNA damage responses triggered by β-lap were abolished in the nde2Δ mutant. Amino acid biosynthesis genes were also induced in β-lap treated cells, suggesting that β-lap exposure somehow triggered the General Control of Nutrients (GCN) pathway. Accordingly, β-lap treatment increased phosphorylation of eIF2α subunit in a manner dependent on the Gcn2p kinase. eIF2α phosphorylation required Gcn1p, Gcn20p and Nde2p. Gcn2p was also required for cell survival upon exposure to β-lap and to elicit checkpoint responses. Remarkably, β-lap treatment increased phosphorylation of eIF2α in breast tumor cells, in a manner dependent on the Nde2p ortholog AIF, and the eIF2 kinase PERK. These findings uncover a new target pathway of β-lap in yeast and human cells and highlight a previously unknown functional connection between Nde2p, Gcn2p and DNA damage responses. PMID:25590579

  6. Repurposing of Human Kinase Inhibitors in Neglected Protozoan Diseases.

    PubMed

    Dichiara, Maria; Marrazzo, Agostino; Prezzavento, Orazio; Collina, Simona; Rescifina, Antonio; Amata, Emanuele

    2017-08-22

    Human African trypanosomiasis (HAT), Chagas disease, and leishmaniasis belong to a group of infectious diseases known as neglected tropical diseases and are induced by infection with protozoan parasites named trypanosomatids. Drugs in current use have several limitations, and therefore new candidate drugs are required. The majority of current therapeutic trypanosomatid targets are enzymes or cell-surface receptors. Among these, eukaryotic protein kinases are a major group of protein targets whose modulation may be beneficial for the treatment of neglected tropical protozoan diseases. This review summarizes the finding of new hit compounds for neglected tropical protozoan diseases, by repurposing known human kinase inhibitors on trypanosomatids. Kinase inhibitors are grouped by human kinase family and discussed according to the screening (target-based or phenotypic) reported for these compounds on trypanosomatids. This collection aims to provide insight into repurposed human kinase inhibitors and their importance in the development of new chemical entities with potential beneficial effects on the diseases caused by trypanosomatids. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Tactical Checkpoint: Hail/Warn Suppress/Stop (Poster)

    DTIC Science & Technology

    2010-11-15

    distractor , optical suppression , human behavior, checkpoint, ambient light, driver suppression , human experimentation, light, paintball, obscuration...HAIL/WARN AND - SUPPRESS /STOP Poster Presented at the 2010 Directed Energies Professional Society Meeting, 15-19 November 2010. 5a. CONTRACT NUMBER...warning to a driver that is approaching a checkpoint. The laser, MCNC light, and the windshield obscuration were evaluated for their suppression

  8. Protein kinase Cι: human oncogene, prognostic marker and therapeutic target

    PubMed Central

    Fields, Alan P.; Regala, Roderick P.

    2009-01-01

    The Protein kinase C (PKC) family of serine/threonine kinases has been the subject of intensive study in the field of cancer since their initial discovery as major cellular receptors for the tumor promoting phorbol esters nearly thirty years ago. However, despite these efforts, the search for a direct genetic link between members of the PKC family and human cancer has yielded only circumstantial evidence that any PKC isozyme is a true cancer gene. This situation changed in the past year with the discovery that atypical protein kinase C iota (PKCι) is a bonafide human oncogene. PKCι is required for the transformed growth of human cancer cells and the PKCι gene is the target of tumor-specific gene amplification in multiple forms of human cancer. PKCι participates in multiple aspects of the transformed phenotype of human cancer cells including transformed growth, invasion and survival. Herein, we review pertinent aspects of atypical PKC structure, function and regulation that relate to the role of these enzymes in oncogenesis. We discuss the evidence that PKCι is a human oncogene, review mechanisms controlling PKCι expression in human cancers, and describe the molecular details of PKCι-mediated oncogenic signaling. We conclude with a discussion of how oncogenic PKCι signaling has been successfully targeted to identify a novel, mechanism-based therapeutic drug currently entering clinical trials for treatment of human lung cancer. Throughout, we identify key unanswered questions and exciting future avenues of investigation regarding this important oncogenic molecule. PMID:17570678

  9. 4-Hydroxynonenal Induces G2/M Phase Cell Cycle Arrest by Activation of the Ataxia Telangiectasia Mutated and Rad3-related Protein (ATR)/Checkpoint Kinase 1 (Chk1) Signaling Pathway*

    PubMed Central

    Chaudhary, Pankaj; Sharma, Rajendra; Sahu, Mukesh; Vishwanatha, Jamboor K.; Awasthi, Sanjay; Awasthi, Yogesh C.

    2013-01-01

    4-Hydroxynonenal (HNE) has been widely implicated in the mechanisms of oxidant-induced toxicity, but the detrimental effects of HNE associated with DNA damage or cell cycle arrest have not been thoroughly studied. Here we demonstrate for the first time that HNE caused G2/M cell cycle arrest of hepatocellular carcinoma HepG2 (p53 wild type) and Hep3B (p53 null) cells that was accompanied with decreased expression of CDK1 and cyclin B1 and activation of p21 in a p53-independent manner. HNE treatment suppressed the Cdc25C level, which led to inactivation of CDK1. HNE-induced phosphorylation of Cdc25C at Ser-216 resulted in its translocation from nucleus to cytoplasm, thereby facilitating its degradation via the ubiquitin-mediated proteasomal pathway. This phosphorylation of Cdc25C was regulated by activation of the ataxia telangiectasia and Rad3-related protein (ATR)/checkpoint kinase 1 (Chk1) pathway. The role of HNE in the DNA double strand break was strongly suggested by a remarkable increase in comet tail formation and H2A.X phosphorylation in HNE-treated cells in vitro. This was supported by increased in vivo phosphorylation of H2A.X in mGsta4 null mice that have impaired HNE metabolism and increased HNE levels in tissues. HNE-mediated ATR/Chk1 signaling was inhibited by ATR kinase inhibitor (caffeine). Additionally, most of the signaling effects of HNE on cell cycle arrest were attenuated in hGSTA4 transfected cells, thereby indicating the involvement of HNE in these events. A novel role of GSTA4-4 in the maintenance of genomic integrity is also suggested. PMID:23733185

  10. Epigenetics meets immune checkpoints.

    PubMed

    Covre, Alessia; Coral, Sandra; Di Giacomo, Anna Maria; Taverna, Pietro; Azab, Mohammad; Maio, Michele

    2015-06-01

    Epigenetic alterations play a pivotal role in cancer development and progression. Pharmacologic reversion of such alterations is feasible, and second generation "epigenetic drugs" are in development and have been demonstrated to possess significant immunomodulatory properties. This knowledge, together with the availability of new and highly effective immunotherapeutic agents including immune checkpoint(s) blocking monoclonal antibodies, allows us to plan for highly innovative proof-of-principle combination studies that will likely open the path to more effective anticancer therapies.

  11. Spindle checkpoint protein Bub1 corrects mitotic aberrancy induced by human T-cell leukemia virus type I Tax.

    PubMed

    Sasaki, M; Sugimoto, K; Tamayose, K; Ando, M; Tanaka, Y; Oshimi, K

    2006-06-22

    Bub1 is a component of the mitotic spindle checkpoint apparatus. Abnormality of this apparatus is known to cause multinuclei formation, a hallmark of chromosomal instability (CIN). A549, aneuploid cell line, aberrantly passed through the mitotic phase and became multinuclei morphology in the presence of nocodazole. Time-lapse videomicroscopy showed unreported bizarre morphology, which we named 'mitotic lobulation' in A549 cells just before the exit from mitosis and multinuclei formation. External expression of wild-type Bub1-EGFP clearly suppressed the multinuclei formation by retaining A549 cells at the mitotic phase during 48 h of time-lapse observation. This suppressive effect on mitotic aberrancy should not be mere restoration of normal Bub1 function, because A549 cells express proper amount of Bub1, which distributed cytoplasm during interphase and concentrated at kinetochore in metaphase. Furthermore, external expression of wild-type Bub1-EGFP suppressed multinuclei formation induced by Tax both in A549 and HeLa cells. Tax is known to induce mitotic abnormality by binding and inactivating Mad1. These observations, therefore, suggest functional redundancy between Bub1 and other mitotic checkpoint protein(s) and a possibility of correction of mitotic aberrancy by external Bub1 expression.

  12. Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine.

    PubMed

    Santaguida, Stefano; Tighe, Anthony; D'Alise, Anna Morena; Taylor, Stephen S; Musacchio, Andrea

    2010-07-12

    The catalytic activity of the MPS1 kinase is crucial for the spindle assembly checkpoint and for chromosome biorientation on the mitotic spindle. We report that the small molecule reversine is a potent mitotic inhibitor of MPS1. Reversine inhibits the spindle assembly checkpoint in a dose-dependent manner. Its addition to mitotic HeLa cells causes the ejection of Mad1 and the ROD-ZWILCH-ZW10 complex, both of which are important for the spindle checkpoint, from unattached kinetochores. By using reversine, we also demonstrate that MPS1 is required for the correction of improper chromosome-microtubule attachments. We provide evidence that MPS1 acts downstream from the AURORA B kinase, another crucial component of the error correction pathway. Our experiments describe a very useful tool to interfere with MPS1 activity in human cells. They also shed light on the relationship between the error correction pathway and the spindle checkpoint and suggest that these processes are coregulated and are likely to share at least a subset of their catalytic machinery.

  13. Dissecting the role of MPS1 in chromosome biorientation and the spindle checkpoint through the small molecule inhibitor reversine

    PubMed Central

    Santaguida, Stefano; Tighe, Anthony; D'Alise, Anna Morena; Taylor, Stephen S.

    2010-01-01

    The catalytic activity of the MPS1 kinase is crucial for the spindle assembly checkpoint and for chromosome biorientation on the mitotic spindle. We report that the small molecule reversine is a potent mitotic inhibitor of MPS1. Reversine inhibits the spindle assembly checkpoint in a dose-dependent manner. Its addition to mitotic HeLa cells causes the ejection of Mad1 and the ROD–ZWILCH–ZW10 complex, both of which are important for the spindle checkpoint, from unattached kinetochores. By using reversine, we also demonstrate that MPS1 is required for the correction of improper chromosome–microtubule attachments. We provide evidence that MPS1 acts downstream from the AURORA B kinase, another crucial component of the error correction pathway. Our experiments describe a very useful tool to interfere with MPS1 activity in human cells. They also shed light on the relationship between the error correction pathway and the spindle checkpoint and suggest that these processes are coregulated and are likely to share at least a subset of their catalytic machinery. PMID:20624901

  14. Human papillomavirus type 16 E7 oncoprotein engages but does not abrogate the mitotic spindle assembly checkpoint

    SciTech Connect

    Yu, Yueyang; Munger, Karl

    2012-10-10

    The mitotic spindle assembly checkpoint (SAC) ensures faithful chromosome segregation during mitosis by censoring kinetochore-microtubule interactions. It is frequently rendered dysfunctional during carcinogenesis causing chromosome missegregation and genomic instability. There are conflicting reports whether the HPV16 E7 oncoprotein drives chromosomal instability by abolishing the SAC. Here we report that degradation of mitotic cyclins is impaired in cells with HPV16 E7 expression. RNAi-mediated depletion of Mad2 or BubR1 indicated the involvement of the SAC, suggesting that HPV16 E7 expression causes sustained SAC engagement. Mutational analyses revealed that HPV16 E7 sequences that are necessary for retinoblastoma tumor suppressor protein binding as well as sequences previously implicated in binding the nuclear and mitotic apparatus (NuMA) protein and in delocalizing dynein from the mitotic spindle contribute to SAC engagement. Importantly, however, HPV16 E7 does not markedly compromise the SAC response to microtubule poisons.

  15. Human pre-B cell receptor signal transduction: evidence for distinct roles of PI3kinase and MAP-kinase signalling pathways

    PubMed Central

    Anbazhagan, Kolandaswamy; Rabbind Singh, Amrathlal; Isabelle, Piec; Stella, Ibata; Céline, Alleaume-De Martel; Bissac, Eliane; Bertrand, Brassart; Rémy, Nyga; Naomi, Taylor; Vincent, Fuentes; Rochette, Jacques; Lassoued, Kaïss

    2013-01-01

    Pre-BCR acts as a critical checkpoint in B cell development. However, its signalling cascade still remains indistinctly characterised in human. We investigated pre-BCR signalling pathway to examine its regulation in normal primary pre-B lymphocytes and pre-B cell lines. In cell lines, early signalling events occurring after pre-BCR stimulation include phosphorylation of Lyn, Blk and Syk together with ZAP70, Btk, Vav, PLC-γ2 and various adaptor proteins, such as BLNK, LAB, LAT and SLP-76. Further downstream, these molecules induced activation of the PI3K/AKT and MAP-kinase resulting in an augmentation of canonical NF-κB pathways and cFos/AP1 activation. PI3K and MAPK exerted opposing effects on the pre-BCR-induced activation of the canonical NF-κB and c-Fos/AP1 pathways. Immediate nuclear export of FoxO3A and delayed import of IRF4 were additional events observed after pre-BCR crosslinking in primary cells. Pre-BCR-induced down-regulation of Rag1, Rag2, E2A and Pax5 transcripts occurred in a PI3K-dependent manner. Finally we bring evidence that pre-BCR stimulation or co stimulation with CD19 enhances cell cycle signal. PMID:25400915

  16. Human pre-B cell receptor signal transduction: evidence for distinct roles of PI3kinase and MAP-kinase signalling pathways.

    PubMed

    Anbazhagan, Kolandaswamy; Rabbind Singh, Amrathlal; Isabelle, Piec; Stella, Ibata; Céline, Alleaume-De Martel; Bissac, Eliane; Bertrand, Brassart; Rémy, Nyga; Naomi, Taylor; Vincent, Fuentes; Rochette, Jacques; Lassoued, Kaïss

    2013-10-01

    Pre-BCR acts as a critical checkpoint in B cell development. However, its signalling cascade still remains indistinctly characterised in human. We investigated pre-BCR signalling pathway to examine its regulation in normal primary pre-B lymphocytes and pre-B cell lines. In cell lines, early signalling events occurring after pre-BCR stimulation include phosphorylation of Lyn, Blk and Syk together with ZAP70, Btk, Vav, PLC-γ2 and various adaptor proteins, such as BLNK, LAB, LAT and SLP-76. Further downstream, these molecules induced activation of the PI3K/AKT and MAP-kinase resulting in an augmentation of canonical NF-κB pathways and cFos/AP1 activation. PI3K and MAPK exerted opposing effects on the pre-BCR-induced activation of the canonical NF-κB and c-Fos/AP1 pathways. Immediate nuclear export of FoxO3A and delayed import of IRF4 were additional events observed after pre-BCR crosslinking in primary cells. Pre-BCR-induced down-regulation of Rag1, Rag2, E2A and Pax5 transcripts occurred in a PI3K-dependent manner. Finally we bring evidence that pre-BCR stimulation or co stimulation with CD19 enhances cell cycle signal.

  17. ARID1A Deficiency Impairs the DNA Damage Checkpoint and Sensitizes Cells to PARP Inhibitors

    PubMed Central

    Shen, Jianfeng; Peng, Yang; Wei, Leizhen; Zhang, Wei; Yang, Lin; Lan, Li; Kapoor, Prabodh; Ju, Zhenlin; Mo, Qianxing; Shih, Ie-Ming; Uray, Ivan P.; Wu, Xiangwei; Brown, Powel H.; Shen, Xuetong; Mills, Gordon B.; Peng, Guang

    2015-01-01

    ARID1A, a chromatin remodeler of the SWI/SNF family, is a recently identified tumor suppressor that is mutated in a broad spectrum of human cancers. Thus, it is of fundamental clinical importance to understand its molecular functions and determine whether ARID1A deficiency can be exploited therapeutically. In this manuscript, we report a key function of ARID1A in regulating the DNA damage checkpoint. ARID1A is recruited to DNA double strand breaks (DSBs) via its interaction with the upstream DNA damage checkpoint kinase ATR. At the molecular level, ARID1A facilitates efficient processing of DSB to single strand ends, and sustains DNA damage signaling. Importantly, ARID1A deficiency sensitizes cancer cells to PARP inhibitors in vitro and in vivo providing a potential therapeutic strategy for patients with ARID1A-mutant tumors. PMID:26069190

  18. Structural Comparison of Human Mammalian Ste20-Like Kinases

    PubMed Central

    Record, Christopher J.; Chaikuad, Apirat; Rellos, Peter; Das, Sanjan; Pike, Ashley C. W.; Fedorov, Oleg; Marsden, Brian D.; Knapp, Stefan; Lee, Wen Hwa

    2010-01-01

    Background The serine/threonine mammalian Ste-20 like kinases (MSTs) are key regulators of apoptosis, cellular proliferation as well as polarization. Deregulation of MSTs has been associated with disease progression in prostate and colorectal cancer. The four human MSTs are regulated differently by C-terminal regions flanking the catalytic domains. Principal Findings We have determined the crystal structure of kinase domain of MST4 in complex with an ATP-mimetic inhibitor. This is the first structure of an inactive conformation of a member of the MST kinase family. Comparison with active structures of MST3 and MST1 revealed a dimeric association of MST4 suggesting an activation loop exchanged mechanism of MST4 auto-activation. Together with a homology model of MST2 we provide a comparative analysis of the kinase domains for all four members of the human MST family. Significance The comparative analysis identified new structural features in the MST ATP binding pocket and has also defined the mechanism for autophosphorylation. Both structural features may be further explored for inhibitors design. Enhanced version This article can also be viewed as an enhanced version in which the text of the article is integrated with interactive 3D representations and animated transitions. Please note that a web plugin is required to access this enhanced functionality. Instructions for the installation and use of the web plugin are available in Text S1. PMID:20730082

  19. Phosphorylation of Human Choline Kinase Beta by Protein Kinase A: Its Impact on Activity and Inhibition

    PubMed Central

    Chang, Ching Ching; Few, Ling Ling; Konrad, Manfred; See Too, Wei Cun

    2016-01-01

    Choline kinase beta (CKβ) is one of the CK isozymes involved in the biosynthesis of phosphatidylcholine. CKβ is important for normal mitochondrial function and muscle development as the lack of the ckβ gene in human and mice results in the development of muscular dystrophy. In contrast, CKα is implicated in tumorigenesis and has been extensively studied as an anticancer target. Phosphorylation of human CKα was found to regulate the enzyme’s activity and its subcellular location. This study provides evidence for CKβ phosphorylation by protein kinase A (PKA). In vitro phosphorylation of CKβ by PKA was first detected by phosphoprotein staining, as well as by in-gel kinase assays. The phosphorylating kinase was identified as PKA by Western blotting. CKβ phosphorylation by MCF-7 cell lysate was inhibited by a PKA-specific inhibitor peptide, and the intracellular phosphorylation of CKβ was shown to be regulated by the level of cyclic adenosine monophosphate (cAMP), a PKA activator. Phosphorylation sites were located on CKβ residues serine-39 and serine-40 as determined by mass spectrometry and site-directed mutagenesis. Phosphorylation increased the catalytic efficiencies for the substrates choline and ATP about 2-fold, without affecting ethanolamine phosphorylation, and the S39D/S40D CKβ phosphorylation mimic behaved kinetically very similar. Remarkably, phosphorylation drastically increased the sensitivity of CKβ to hemicholinium-3 (HC-3) inhibition by about 30-fold. These findings suggest that CKβ, in concert with CKα, and depending on its phosphorylation status, might play a critical role as a druggable target in carcinogenesis. PMID:27149373

  20. Structural and functional characterization of human NAD kinase.

    PubMed

    Lerner, F; Niere, M; Ludwig, A; Ziegler, M

    2001-10-19

    NADP is essential for biosynthetic pathways, energy, and signal transduction. Its synthesis is catalyzed by NAD kinase. Very little is known about the structure, function, and regulation of this enzyme from multicellular organisms. We identified a human NAD kinase cDNA and the corresponding gene using available database information. A cDNA was amplified from a human fibroblast cDNA library and functionally overexpressed in Escherichia coli. The obtained cDNA, slightly different from that deposited in the database, encodes a protein of 49 kDa. The gene is expressed in most human tissues, but not in skeletal muscle. Human NAD kinase differs considerably from that of prokaryotes by subunit molecular mass (49 kDa vs 30-35 kDa). The catalytically active homotetramer is highly selective for its substrates, NAD and ATP. It did not phosphorylate the nicotinic acid derivative of NAD (NAAD) suggesting that the potent calcium-mobilizing pyridine nucleotide NAADP is synthesized by an alternative route.

  1. Ionizing Radiation Activates AMP-Activated Kinase (AMPK): A Target for Radiosensitization of Human Cancer Cells

    SciTech Connect

    Sanli, Toran; Rashid, Ayesha; Liu Caiqiong

    2010-09-01

    Purpose: Adenosine monophosphate (AMP)-activated kinase (AMPK) is a molecular energy sensor regulated by the tumor suppressor LKB1. Starvation and growth factors activate AMPK through the DNA damage sensor ataxia-telangiectasia mutated (ATM). We explored the regulation of AMPK by ionizing radiation (IR) and its role as a target for radiosensitization of human cancer cells. Methods and Materials: Lung, prostate, and breast cancer cells were treated with IR (2-8 Gy) after incubation with either ATM or AMPK inhibitors or the AMPK activator metformin. Then, cells were subjected to either lysis and immunoblotting, immunofluorescence microscopy, clonogenic survival assays, or cell cycle analysis. Results: IR induced a robust phosphorylation and activation of AMPK in all tumor cells, independent of LKB1. IR activated AMPK first in the nucleus, and this extended later into cytoplasm. The ATM inhibitor KU-55933 blocked IR activation of AMPK. AMPK inhibition with Compound C or anti-AMPK {alpha} subunit small interfering RNA (siRNA) blocked IR induction of the cell cycle regulators p53 and p21{sup waf/cip} as well as the IR-induced G2/M arrest. Compound C caused resistance to IR, increasing the surviving fraction after 2 Gy, but the anti-diabetic drug metformin enhanced IR activation of AMPK and lowered the surviving fraction after 2 Gy further. Conclusions: We provide evidence that IR activates AMPK in human cancer cells in an LKB1-independent manner, leading to induction of p21{sup waf/cip} and regulation of the cell cycle and survival. AMPK appears to (1) participate in an ATM-AMPK-p21{sup waf/cip} pathway, (2) be involved in regulation of the IR-induced G2/M checkpoint, and (3) may be targeted by metformin to enhance IR responses.

  2. Ral A, via activating the mitotic checkpoint, sensitizes cells lacking a functional Nf1 to apoptosis in the absence of protein kinase C.

    PubMed

    Ganapathy, Suthakar; Fagman, Johan B; Shen, Ling; Yu, Tianqi; Zhou, Xiaodong; Dai, Wei; Makriyannis, Alexandros; Chen, Changyan

    2016-12-20

    Nf1 mutations or deletions are suggested to underlie the tumor predisposition of NF1 (neurofibromatosis type 1) and few treatments are available for treating NF1 patients with advanced malignant tumors. Aberrant activation of Ras in Nf1-deficient conditions is responsible for the promotion of tumorigenesis in NF1. PKC is proven to be an important factor in supporting the viability of Nf1-defected cells, but the molecular mechanisms are not fully understood. In this study, we demonstrate that the inhibition of protein kinase C (PKC) by 1-O-Hexadecyl-2-O-methyl-rac-glycerol (HMG, a PKC inhibitor) preferentially sensitizes Nf1-defected cells to apoptosis, via triggering a persistent mitotic arrest. In this process, Ral A is activated. Subsequently, Chk1 is phosphorylated and translocated to the nucleus. Silencing Ral A significantly blocks Chk1 nuclear translocation and releases HMG-treated Nf1-deficient cells from mitotic arrest, resulting in the reduction of the magnitude of apoptosis. Thus, our study reveals that PKC is able to maintain the homeostasis or viability of Nf1-defected cells and may serve as a potential target for developing new therapeutic strategies.

  3. Targeting the DNA replication checkpoint by pharmacologic inhibition of Chk1 kinase: a strategy to sensitize APC mutant colon cancer cells to 5-fluorouracil chemotherapy.

    PubMed

    Martino-Echarri, Estefania; Henderson, Beric R; Brocardo, Mariana G

    2014-10-30

    5-fluorouracil (5-FU) is the first line component used in colorectal cancer (CRC) therapy however even in combination with other chemotherapeutic drugs recurrence is common. Mutations of the adenomatous polyposis coli (APC) gene are considered as the initiating step of transformation in familial and sporadic CRCs. We have previously shown that APC regulates the cellular response to DNA replication stress and recently hypothesized that APC mutations might therefore influence 5-FU resistance. To test this, we compared CRC cell lines and show that those expressing truncated APC exhibit a limited response to 5-FU and arrest in G1/S-phase without undergoing lethal damage, unlike cells expressing wild-type APC. In SW480 APC-mutant CRC cells, 5-FU-dependent apoptosis was restored after transient expression of full length APC, indicating a direct link between APC and drug response. Furthermore, we could increase sensitivity of APC truncated cells to 5-FU by inactivating the Chk1 kinase using drug treatment or siRNA-mediated knockdown. Our findings identify mutant APC as a potential tumor biomarker of resistance to 5-FU, and importantly we show that APC-mutant CRC cells can be made more sensitive to 5-FU by use of Chk1 inhibitors.

  4. Role of NADPH oxidases in inducing a selective increase of oxidant stress and cyclin D1 and checkpoint 1 over-expression during progression to human gastric adenocarcinoma.

    PubMed

    Montalvo-Javé, Eduardo E; Olguín-Martínez, Marisela; Hernández-Espinosa, Diego R; Sánchez-Sevilla, Lourdes; Mendieta-Condado, Edgar; Contreras-Zentella, Martha L; Oñate-Ocaña, Luis F; Escalante-Tatersfield, Tomás; Echegaray-Donde, Agustín; Ruiz-Molina, Juan M; Herrera, Miguel F; Morán, Julio; Hernández-Muñoz, Rolando

    2016-04-01

    Gastric cancer is one of the main causes of global mortality. Here, reactive oxygen species (ROS) could largely contribute to gastric carcinogenesis. Hence, the present work was aimed to assess the role of ROS, oxidant status, NADPH oxidases (NOXs) expression, during human gastric adenocarcinoma. We obtained subcellular fraction from samples of gastric mucosa taken from control subjects (n = 20), and from 40 patients with gastric adenocarcinoma, as well as samples of distant areas (tumour-free gastric mucosa). Parameters indicative of lipid peroxidation and cell proliferation were selectively increased in both tumour-free and in cancerous gastric mucosa, despite of glutathione (GSH) content, glutathione reductase (GR) and superoxide dismutase (SOD) activities were increased in the adenocarcinoma. These high levels of antioxidant defences inversely correlated with down-regulated expression for NOX2 and 4; however, over-expression of NOX1 occurred with increased caspase-3 activity and overexpressed checkpoint 1 (MDC1) and cyclin D1 proteins. In the tumour-free mucosa an oxidant stress took place, without changing total GSH but with decreased activities for GR and mitochondrial SOD; moreover, over-expression of checkpoint 1 (MDC1) correlated with lower NOX2 and 4 expression in this mucosa. Chronically injured gastric mucosa increases lipoperoxidative events and cell proliferation. In the adenocarcinoma, cell proliferation was further enhanced, oxidant stress decreased which seemed to be linked to NOX1, MDC1 and cyclin D1 over-expression, but with a lower NOXs activity leading a 'low tone' of ROS formation. Therefore, our results could be useful for early detection and treatment of gastric adenocarcinoma. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Deregulated Ras signaling compromises DNA damage checkpoint recovery in S. cerevisiae

    PubMed Central

    Wood, Matthew D

    2010-01-01

    The DNA damage checkpoint maintains genome stability by arresting the cell cycle and promoting DNA repair under genotoxic stress. Cells must downregulate the checkpoint signaling pathways in order to resume cell division after completing DNA repair. While the mechanisms of checkpoint activation have been well-characterized, the process of checkpoint recovery, and the signals regulating it, has only recently been investigated. We have identified a new role for the Ras signaling pathway as a regulator of DNA damage checkpoint recovery. Here we report that in budding yeast, deletion of the IRA1 and IRA2 genes encoding negative regulators of Ras prevents cellular recovery from a DNA damage induced arrest. the checkpoint kinase Rad53 is dephosphorylated in an IRA-deficient strain, indicating that recovery failure is not caused by constitutive checkpoint pathway activation. the ira1Δ ira2Δ recovery defect requires the checkpoint kinase Chk1 and the cAMP-dependent protein kinase (PKA) catalytic subunit Tpk2. Furthermore, PKA phosphorylation sites on the anaphase promoting complex specificity factor Cdc20 are required for the recovery defect, indicating a link between the recovery defect and PKA regulation of mitosis. This work identifies a new signaling pathway that can regulate DNA damage checkpoint recovery and implicates the Ras signaling pathway as an important regulator of mitotic events. PMID:20716966

  6. Structures of human Bruton's tyrosine kinase in active and inactive conformations suggest a mechanism of activation for TEC family kinases

    SciTech Connect

    Marcotte, Douglas J.; Liu, Yu-Ting; Arduini, Robert M.; Hession, Catherine A.; Miatkowski, Konrad; Wildes, Craig P.; Cullen, Patrick F.; Hong, Victor; Hopkins, Brian T.; Mertsching, Elisabeth; Jenkins, Tracy J.; Romanowski, Michael J.; Baker, Darren P.; Silvian, Laura F.

    2010-11-15

    Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, plays a crucial role in B-cell maturation and mast cell activation. Although the structures of the unphosphorylated mouse BTK kinase domain and the unphosphorylated and phosphorylated kinase domains of human ITK are known, understanding the kinase selectivity profiles of BTK inhibitors has been hampered by the lack of availability of a high resolution, ligand-bound BTK structure. Here, we report the crystal structures of the human BTK kinase domain bound to either Dasatinib (BMS-354825) at 1.9 {angstrom} resolution or to 4-amino-5-(4-phenoxyphenyl)-7H-pyrrolospyrimidin- 7-yl-cyclopentane at 1.6 {angstrom} resolution. This data provides information relevant to the development of small molecule inhibitors targeting BTK and the TEC family of nonreceptor tyrosine kinases. Analysis of the structural differences between the TEC and Src families of kinases near the Trp-Glu-Ile motif in the N-terminal region of the kinase domain suggests a mechanism of regulation of the TEC family members.

  7. Checkpoint inhibition in myeloma.

    PubMed

    Benson, Don M

    2016-12-02

    Historically, attempts at cancer immunotherapy have emphasized strategies designed to stimulate or augment the immune system into action. In the past decade, a complementary approach has developed, that of releasing immune cells from inhibitory restraint. Discoveries in the fundamental biology of how immunity is regulated, how the immune system interfaces with malignancy, and how cancer cells may exploit these processes to evade detection have all been translated into the rapidly growing field of therapeutic immune checkpoint inhibition for cancer. Myeloma is a malignancy associated with significant immune dysfunction imparted both by the disease itself as well as by many of the immunosuppressive therapies that have been used in the past. The growing body of preclinical data regarding immunoregulatory mechanisms that appear active in myeloma has begun to be translated to clinical trials targeting these signaling axes. This review will attempt to summarize the current understanding of the basic biology of several immune checkpoint pathways that may be important in myeloma and provide an up-to-date overview of recent and ongoing clinical trials of immune checkpoint inhibitors in myeloma. Finally, several current challenges and possible future directions of immune checkpoint blockade in myeloma will be reviewed. © 2016 by The American Society of Hematology. All rights reserved.

  8. Multiple forms of thymidine kinase during human development.

    PubMed

    Bernard, B; Preston, B; Beaupre, P; Elliott, K; Sadava, D

    1977-01-01

    The developmental progression of thymidine kinase from the electrophoretically slow-migrating 'fetal' forms to the fast-migrating 'adult' form was examined in human serum and fetal liver. In fetal liver, only the fetal forms of the enzyme were present at 17 weeks' gestation. A transitional period followed in that both enzyme forms were identified and by 24 weeks only the adult form was detected in fetal liver. This same enzyme changeover pattern--fetal to transitional to adult--occurred at a later time in human serum as it took place between 30 and 40 weeks' gestation.

  9. Mathematical model of the morphogenesis checkpoint in budding yeast

    PubMed Central

    Ciliberto, Andrea; Novak, Bela; Tyson, John J.

    2003-01-01

    The morphogenesis checkpoint in budding yeast delays progression through the cell cycle in response to stimuli that prevent bud formation. Central to the checkpoint mechanism is Swe1 kinase: normally inactive, its activation halts cell cycle progression in G2. We propose a molecular network for Swe1 control, based on published observations of budding yeast and analogous control signals in fission yeast. The proposed Swe1 network is merged with a model of cyclin-dependent kinase regulation, converted into a set of differential equations and studied by numerical simulation. The simulations accurately reproduce the phenotypes of a dozen checkpoint mutants. Among other predictions, the model attributes a new role to Hsl1, a kinase known to play a role in Swe1 degradation: Hsl1 must also be indirectly responsible for potent inhibition of Swe1 activity. The model supports the idea that the morphogenesis checkpoint, like other checkpoints, raises the cell size threshold for progression from one phase of the cell cycle to the next. PMID:14691135

  10. Mathematical model of the morphogenesis checkpoint in budding yeast.

    PubMed

    Ciliberto, Andrea; Novak, Bela; Tyson, John J

    2003-12-22

    The morphogenesis checkpoint in budding yeast delays progression through the cell cycle in response to stimuli that prevent bud formation. Central to the checkpoint mechanism is Swe1 kinase: normally inactive, its activation halts cell cycle progression in G2. We propose a molecular network for Swe1 control, based on published observations of budding yeast and analogous control signals in fission yeast. The proposed Swe1 network is merged with a model of cyclin-dependent kinase regulation, converted into a set of differential equations and studied by numerical simulation. The simulations accurately reproduce the phenotypes of a dozen checkpoint mutants. Among other predictions, the model attributes a new role to Hsl1, a kinase known to play a role in Swe1 degradation: Hsl1 must also be indirectly responsible for potent inhibition of Swe1 activity. The model supports the idea that the morphogenesis checkpoint, like other checkpoints, raises the cell size threshold for progression from one phase of the cell cycle to the next.

  11. Alternative DNA Damage Checkpoint Pathways in Eukaryotes

    DTIC Science & Technology

    2002-04-01

    data suggest that Ches1 functions in an alternative checkpoint pathway. Our goals are to identify genes constituting this pathway, to isolate the...human counterparts, and to compare their structure and activity in normal and cancer tissues. In order to identify the genes involved in the alternative

  12. Structure of Human G Protein-Coupled Receptor Kinase 2 in Complex with the Kinase Inhibitor Balanol

    SciTech Connect

    Tesmer, John J.G.; Tesmer, Valerie M.; Lodowski, David T.; Steinhagen, Henning; Huber, Jochen

    2010-07-19

    G protein-coupled receptor kinase 2 (GRK2) is a pharmaceutical target for the treatment of cardiovascular diseases such as congestive heart failure, myocardial infarction, and hypertension. To better understand how nanomolar inhibition and selectivity for GRK2 might be achieved, we have determined crystal structures of human GRK2 in complex with G{beta}{gamma} in the presence and absence of the AGC kinase inhibitor balanol. The selectivity of balanol among human GRKs is assessed.

  13. Phyllanthus emblica Fruit Extract Activates Spindle Assembly Checkpoint, Prevents Mitotic Aberrations and Genomic Instability in Human Colon Epithelial NCM460 Cells

    PubMed Central

    Guo, Xihan; Wang, Xu

    2016-01-01

    The fruit of Phyllanthus emblica Linn. (PE) has been widely consumed as a functional food and folk medicine in Southeast Asia due to its remarkable nutritional and pharmacological effects. Previous research showed PE delays mitotic progress and increases genomic instability (GIN) in human colorectal cancer cells. This study aimed to investigate the similar effects of PE by the biomarkers related to spindle assembly checkpoint (SAC), mitotic aberrations and GIN in human NCM460 normal colon epithelial cells. Cells were treated with PE and harvested differently according to the biomarkers observed. Frequencies of micronuclei (MN), nucleoplasmic bridge (NPB) and nuclear bud (NB) in cytokinesis-block micronucleus assay were used as indicators of GIN. Mitotic aberrations were assessed by the biomarkers of chromosome misalignment, multipolar division, chromosome lagging and chromatin bridge. SAC activity was determined by anaphase-to- metaphase ratio (AMR) and the expression of core SAC gene budding uninhibited by benzimidazoles related 1 (BubR1). Compared with the control, PE-treated cells showed (1) decreased incidences of MN, NPB and NB (p < 0.01); (2) decreased frequencies of all mitotic aberration biomarkers (p < 0.01); and (3) decreased AMR (p < 0.01) and increased BubR1 expression (p < 0.001). The results revealed PE has the potential to protect human normal colon epithelial cells from mitotic and genomic damages partially by enhancing the function of SAC. PMID:27598149

  14. Phyllanthus emblica Fruit Extract Activates Spindle Assembly Checkpoint, Prevents Mitotic Aberrations and Genomic Instability in Human Colon Epithelial NCM460 Cells.

    PubMed

    Guo, Xihan; Wang, Xu

    2016-09-03

    The fruit of Phyllanthus emblica Linn. (PE) has been widely consumed as a functional food and folk medicine in Southeast Asia due to its remarkable nutritional and pharmacological effects. Previous research showed PE delays mitotic progress and increases genomic instability (GIN) in human colorectal cancer cells. This study aimed to investigate the similar effects of PE by the biomarkers related to spindle assembly checkpoint (SAC), mitotic aberrations and GIN in human NCM460 normal colon epithelial cells. Cells were treated with PE and harvested differently according to the biomarkers observed. Frequencies of micronuclei (MN), nucleoplasmic bridge (NPB) and nuclear bud (NB) in cytokinesis-block micronucleus assay were used as indicators of GIN. Mitotic aberrations were assessed by the biomarkers of chromosome misalignment, multipolar division, chromosome lagging and chromatin bridge. SAC activity was determined by anaphase-to- metaphase ratio (AMR) and the expression of core SAC gene budding uninhibited by benzimidazoles related 1 (BubR1). Compared with the control, PE-treated cells showed (1) decreased incidences of MN, NPB and NB (p < 0.01); (2) decreased frequencies of all mitotic aberration biomarkers (p < 0.01); and (3) decreased AMR (p < 0.01) and increased BubR1 expression (p < 0.001). The results revealed PE has the potential to protect human normal colon epithelial cells from mitotic and genomic damages partially by enhancing the function of SAC.

  15. Identification of "Preferred" Human Kinase Inhibitors for Sleeping Sickness Lead Discovery. Are Some Kinases Better than Others for Inhibitor Repurposing?

    PubMed

    Amata, Emanuele; Xi, Hualin; Colmenarejo, Gonzalo; Gonzalez-Diaz, Rosario; Cordon-Obras, Carlos; Berlanga, Manuela; Manzano, Pilar; Erath, Jessey; Roncal, Norma E; Lee, Patricia J; Leed, Susan E; Rodriguez, Ana; Sciotti, Richard J; Navarro, Miguel; Pollastri, Michael P

    2016-03-11

    A kinase-targeting cell-based high-throughput screen (HTS) against Trypanosoma brucei was recently reported, and this screening set included the Published Kinase Inhibitor Set (PKIS). From the PKIS was identified 53 compounds with pEC50 ≥ 6. Utilizing the published data available for the PKIS, a statistical analysis of these active antiparasitic compounds was performed, allowing identification of a set of human kinases having inhibitors that show a high likelihood for blocking T. brucei cellular proliferation in vitro. This observation was confirmed by testing other established inhibitors of these human kinases and by mining past screening campaigns at GlaxoSmithKline. Overall, although the parasite targets of action are not known, inhibitors of this set of human kinases displayed an enhanced hit rate relative to a random kinase-targeting HTS campaign, suggesting that repurposing efforts should focus primarily on inhibitors of these specific human kinases. We therefore term this statistical analysis-driven approach "preferred lead repurposing".

  16. Protein Kinase A Subunit α Catalytic and A Kinase Anchoring Protein 79 in Human Placental Mitochondria.

    PubMed

    Ma, Maggie Pui Chi; Thomson, Murray

    2012-01-01

    Components of protein phosphorylation signalling systems have been discovered in mitochondria and it has been proposed that these molecules modulate processes including oxidative phosphorylation, apoptosis and steroidogenesis. We used electrophoresis and Western blots probed with specific antibodies to protein kinase A α catalytic subunit (PKAα Cat) and A kinase anchoring protein of approximately 79 kDa molecular weight (AKAP79) to demonstrate the presence of these two proteins in human placental mitochondria. Heavy mitochondria characteristic of cytotrophoblast were separated from light mitochondria characteristic of syncytiotrophoblast by centrifugation. PKAα Cat and AKAP79 were present in both heavy and light mitochondria with no significant difference in concentration. Sucrose density gradient separation of submitochondrial fractions indicated PKAα Cat is located predominantly in the outer membrane whereas AKAP79 is present mainly in the contact site fractions. These data indicate that PKAα Cat is present in the cytoplasm, nucleus and mitochondria of placental cells. AKAP79 is also present in human placental mitochondria but there may be anchoring proteins other than AKAP79 responsible for fixing PKA to the outer membrane. PKA may play roles in mitochondrial protein phosphorylation systems in both cytotrophoblast and syncytiotrophoblast.

  17. ATR kinase is required for global genomic nucleotide excision repair exclusively during S phase in human cells

    PubMed Central

    Auclair, Yannick; Rouget, Raphael; Affar, El Bachir; Drobetsky, Elliot A.

    2008-01-01

    Global-genomic nucleotide excision repair (GG-NER) is the only pathway available to humans for removal, from the genome overall, of highly genotoxic helix-distorting DNA adducts generated by many environmental mutagens and certain chemotherapeutic agents, e.g., UV-induced 6–4 photoproducts (6–4PPs) and cyclobutane pyrimidine dimers (CPDs). The ataxia telangiectasia and rad-3-related kinase (ATR) is rapidly activated in response to UV-induced replication stress and proceeds to phosphorylate a plethora of downstream effectors that modulate primarily cell cycle checkpoints but also apoptosis and DNA repair. To investigate whether this critical kinase might participate in the regulation of GG-NER, we developed a novel flow cytometry-based DNA repair assay that allows precise evaluation of GG-NER kinetics as a function of cell cycle. Remarkably, inhibition of ATR signaling in primary human lung fibroblasts by treatment with caffeine, or with siRNA specifically targeting ATR, resulted in total inhibition of 6–4PP removal during S phase, whereas cells repaired normally during either G0/G1 or G2/M. Similarly striking S-phase-specific defects in GG-NER of both 6–4PPs and CPDs were documented in ATR-deficient Seckel syndrome skin fibroblasts. Finally, among six diverse model human tumor strains investigated, three manifested complete abrogation of 6–4PP repair exclusively in S-phase populations. Our data reveal a highly novel role for ATR in the regulation of GG-NER uniquely during S phase of the cell cycle, and indicate that many human cancers may be characterized by a defect in this regulation. PMID:19004803

  18. Broad specificity of human phosphoglycerate kinase for antiviral nucleoside analogs.

    PubMed

    Gallois-Montbrun, Sarah; Faraj, Abdesslem; Seclaman, Edward; Sommadossi, Jean-Pierre; Deville-Bonne, Dominique; Véron, Michel

    2004-11-01

    Nucleoside analogs used in antiviral therapies need to be phosphorylated to their tri-phospho counterparts in order to be active on their cellular target. Human phosphoglycerate kinase (hPGK) was recently reported to participate in the last step of phosphorylation of cytidine L-nucleotide derivatives [Krishnan PGE, Lam W, Dutschman GE, Grill SP, Cheng YC. Novel role of 3-phosphoglycerate kinase, a glycolytic enzyme, in the activation of L-nucleoside analogs, a new class of anticancer and antiviral agents. J Biol Chem 2003;278:36726-32]. In the present work, we extended the enzymatic study of human PGK specificity to purine and pyrimidine nucleotide derivatives in both D- and L-configuration. Human PGK demonstrated catalytic efficiencies in the 10(4)-10(5)M(-1)s(-1) range for purine ribo-, deoxyribo- and dideoxyribonucleotide derivatives, either in D- or L-configuration. In contrast, it was poorly active with natural pyrimidine D-nucleotides (less than 10(3)M(-1)s(-1)). Pyrimidine L-enantiomers, which are promising therapeutic analogs against B hepatitis, were 2-25 times better substrates than their D-counterparts. The broad specificity of substrate of human PGK suggests that this enzyme may be involved in the cellular activation of several antiviral nucleoside analogs including dideoxyinosine, acyclovir, L-2'-deoxycytosine and L-2'-deoxythymidine.

  19. RAC1 GTPase plays an important role in γ-irradiation induced G2/M checkpoint activation

    PubMed Central

    2012-01-01

    Introduction In response to gamma-irradiation (IR)-induced double-strand DNA breaks, cells undergo cell-cycle arrest, allowing time for DNA repair before reentering the cell cycle. G2/M checkpoint activation involves activation of ataxia telangiectasia mutated (ATM)/ATM- and rad3-related (ATR) kinases and inhibition of Cdc25 phosphatases, resulting in inhibition of Cdc2 kinase and subsequent G2/M cell-cycle arrest. Previous studies from our laboratory showed that the G2/M checkpoint activation after IR exposure of MCF-7 breast cancer cells is dependent on the activation of extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) signaling. In the present studies, we investigated the role of Ras-related C3 botulinum toxin substrate 1 (Rac1) guanosine triphosphatase (GTPase) in IR-induced G2/M checkpoint response and ERK1/2 activation, as well as in cell survival after IR. Methods With Rac1-specific inhibitor, dominant negative mutant Rac1 (N17Rac1) and specific small interfering RNA, the effect of Rac1 on IR-induced G2/M checkpoint response and ERK1/2 activation was examined in human breast cancer cells. In addition, the effect of Rac1 on cell survival after irradiation was assessed by using Rac1-specific inhibitor. Results IR exposure of MCF-7 breast cancer cells was associated with a marked activation of Rac1 GTPase. Furthermore, inhibition of Rac1 by using specific inhibitor, dominant-negative Rac1 mutant, or specific siRNA resulted in attenuation of IR-induced G2/M arrest and concomitant diminution of IR-induced activation of ATM, ATR, Chk1, and Chk2 kinases, as well as phosphorylation of Cdc2-Tyr15. Moreover, Rac1 inhibition or decreased Rac1 expression also abrogated IR-induced phosphorylation of mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) and ERK1/2. Ultimately, inhibition of Rac1 markedly increased cellular sensitivity to IR exposure, which involves induction of apoptosis. Conclusion Studies in this report suggest that Rac1 GTPase plays an

  20. Extending Thymidine Kinase Activity to the Catalytic Repertoire of Human Deoxycytidine Kinase

    SciTech Connect

    Hazra, Saugata; Sabini, Eliszbetta; Ort, Stephan; Konrad, Manfred; Lavie, Arnon

    2009-03-04

    Salvage of nucleosides in the cytosol of human cells is carried out by deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1). Whereas TK1 is only responsible for thymidine phosphorylation, dCK is capable of converting dC, dA, and dG into their monophosphate forms. Using structural data on dCK, we predicted that select mutations at the active site would, in addition to making the enzyme faster, expand the catalytic repertoire of dCK to include thymidine. Specifically, we hypothesized that steric repulsion between the methyl group of the thymine base and Arg104 is the main factor preventing the phosphorylation of thymidine by wild-type dCK. Here we present kinetic data on several dCK variants where Arg104 has been replaced by select residues, all performed in combination with the mutation of Asp133 to an alanine. We show that several hydrophobic residues at position 104 endow dCK with thymidine kinase activity. Depending on the exact nature of the mutations, the enzyme's substrate preference is modified. The R104M-D133A double mutant is a pyrimidine-specific enzyme due to large K{sub m} values with purines. The crystal structure of the double mutant R104M-D133A in complex with the L-form of thymidine supplies a structural explanation for the ability of this variant to phosphorylate thymidine and thymidine analogs. The replacement of Arg104 by a smaller residue allows L-dT to bind deeper into the active site, making space for the C5-methyl group of the thymine base. The unique catalytic properties of several of the mutants make them good candidates for suicide-gene/protein-therapy applications.

  1. Human acylphosphatase cannot replace phosphoglycerate kinase in Saccharomyces cerevisiae.

    PubMed

    Van Hoek, P; Modesti, A; Ramponi, G; Kötter, P; van Dijken, J P; Pron, J T

    2001-10-01

    Human acylphosphatase (h-AP, EC 3.6.1.7) has been reported to catalyse the hydrolysis of the 1-phosphate group of 1,3-diphosphoglycerate. In vivo operation of this reaction in the yeast Saccharomyces cerevisiae would bypass phosphoglycerate kinase and thus reduce the ATP yield from glycolysis. To investigate whether h-AP can indeed replace the S. cerevisiae phosphoglycerate kinase, a multi-copy plasmid carrying the h-AP gene under control of the yeast TDH3 promoter was introduced into a pgk1 delta mutant of S. cerevisiae. A strain carrying the expression vector without the h-AP cassette was used as a reference. For both strains, steady-state carbon- and energy-limited chemostat cultures were obtained at a dilution rate of 0.10 h(-1) on a medium containing a mixture of glucose and ethanol (15% and 85% on a carbon basis, respectively). Although the h-AP strain exhibited a high acylphosphatase activity in cell extracts, switching to glucose as sole carbon and energy source resulted in a complete arrest of glucose consumption and growth. The lack of a functional glycolytic pathway was further evident from the absence of ethanol formation in the presence of excess glucose in the culture. As h-AP cannot replace yeast phosphoglycerate kinase in vivo, the enzyme is not a useful tool to modify the ATP yield of glycolysis in S. cerevisiae.

  2. Biomarkers for Checkpoint Inhibition.

    PubMed

    Weber, Jeffrey S

    2017-01-01

    The identification of predictive biomarkers for the benefit of cancer immunotherapy is the holy grail of the burgeoning immunotherapy field. Recent work has shown that there are a core of concepts that establish the presence of an immune cell-infiltrate, an inflammatory signature of the tumor microenvironment, and the availability of target antigens defined by mutated neoantigens, as critical for the success of the checkpoint blockade. Genetic analyses have shown that resistance to PD-1 blockade, either innate or adaptive, may be due to existing or de novo mutations in signaling pathways critical for T-cell function in a modest proportion of cases. Major hurdles in the field that remain to be overcome are the difficulty of obtaining tumor biopsies for biomarker assessment, the heterogeneity of biomarker expression within tumors and within different tumors from the same patient, and the inducibility of some biomarkers by disease-related processes. Although assessment of peripheral blood or serum biomarkers would be ideal, few data suggest that they would reliably predict outcome with checkpoint blockade. Ultimately, some amalgamated biomarker that includes tumor and host factors will be required to predict which patients are likely to benefit from, or be resistant to, the effects of checkpoint inhibition.

  3. Negative Checkpoint Regulatory Molecule 2B4 (CD244) Upregulation Is Associated with Invariant Natural Killer T Cell Alterations and Human Immunodeficiency Virus Disease Progression

    PubMed Central

    Ahmad, Fareed; Shankar, Esaki M.; Yong, Yean K.; Tan, Hong Y.; Ahrenstorf, Gerrit; Jacobs, Roland; Larsson, Marie; Schmidt, Reinhold E.; Kamarulzaman, Adeeba; Ansari, Abdul W.

    2017-01-01

    The CD1d-restricted invariant natural killer T (iNKT) cells are implicated in innate immune responses against human immunodeficiency virus (HIV). However, the determinants of cellular dysfunction across the iNKT cells subsets are seldom defined in HIV disease. Herein, we provide evidence for the involvement of the negative checkpoint regulator (NCR) 2B4 in iNKT cell alteration in a well-defined cohort of HIV-seropositive anti-retroviral therapy (ART) naïve, ART-treated, and elite controllers (ECs). We report on exaggerated 2B4 expression on iNKT cells of HIV-infected treatment-naïve individuals. In sharp contrast to CD4−iNKT cells, 2B4 expression was significantly higher on CD4+ iNKT cell subset. Notably, an increased level of 2B4 on iNKT cells was strongly correlated with parameters associated with HIV disease progression. Further, iNKT cells from ART-naïve individuals were defective in their ability to produce intracellular IFN-γ. Together, our results suggest that the levels of 2B4 expression and the downstream co-inhibitory signaling events may contribute to impaired iNKT cell responses. PMID:28396665

  4. FOXM1 allows human keratinocytes to bypass the oncogene-induced differentiation checkpoint in response to gain of MYC or loss of p53

    PubMed Central

    Molinuevo, R; Freije, A; de Pedro, I; Stoll, S W; Elder, J T; Gandarillas, A

    2017-01-01

    Tumour suppressor p53 or proto-oncogene MYC is frequently altered in squamous carcinomas, but this is insufficient to drive carcinogenesis. We have shown that overactivation of MYC or loss of p53 via DNA damage triggers an anti-oncogenic differentiation-mitosis checkpoint in human epidermal keratinocytes, resulting in impaired cell division and squamous differentiation. Forkhead box M1 (FOXM1) is a transcription factor recently proposed to govern the expression of a set of mitotic genes. Deregulation of FOXM1 occurs in a wide variety of epithelial malignancies. We have ectopically expressed FOXM1 in keratinocytes of the skin after overexpression of MYC or inactivation of endogenous p53. Ectopic FOXM1 rescues the proliferative capacity of MYC- or p53-mutant cells in spite of higher genetic damage and a larger cell size typical of differentiation. As a consequence, differentiation induced by loss of p53 or MYC is converted into increased proliferation and keratinocytes displaying genomic instability are maintained within the proliferative compartment. The results demonstrate that keratinocyte oncogene-induced differentiation is caused by mitosis control and provide new insight into the mechanisms driving malignant progression in squamous cancer. PMID:27452522

  5. Comparative analysis of human and bovine protein kinases reveals unique relationship and functional diversity.

    PubMed

    Kabir, Nuzhat N; Kazi, Julhash U

    2011-10-01

    Reversible protein phosphorylation by protein kinases and phosphatases is a common event in various cellular processes. The eukaryotic protein kinase superfamily, which is one of the largest superfamilies of eukaryotic proteins, plays several roles in cell signaling and diseases. We identified 482 eukaryotic protein kinases and 39 atypical protein kinases in the bovine genome, by searching publicly accessible genetic-sequence databases. Bovines have 512 putative protein kinases, each orthologous to a human kinase. Whereas orthologous kinase pairs are, on an average, 90.6% identical, orthologous kinase catalytic domain pairs are, on an average, 95.9% identical at the amino acid level. This bioinformatic study of bovine protein kinases provides a suitable framework for further characterization of their functional and structural properties.

  6. Protein kinase C and the antiviral effect of human interferon.

    PubMed

    Cernescu, C; Constantinescu, S N; Baltă, F; Popescu, L M; Cajal, N

    1989-01-01

    Protein kinase C (PKC) inhibitors: Hidaka's compounds H-7 (10 microM) and H-8 (20 microM), palmitoyl-carnitine (10 microM) and phloretin (50 microM), did not modify the antiviral effect of human natural or recombinant interferon alpha and of natural interferon beta. The tumor promoter 12-o-tetradecanoyl-phorbol-13-acetate (TPA) (200 nM), known as activator of PKC induced an antiviral state when tested on human embryo fibroblasts challenged with the vesicular stomatitis virus. The battery of PKC inhibitors used inhibited the antiviral effect induced by TPA. Palmitoyl-carnitine (10 microM) exerted a toxic effect that was reversed by interferon treatment (2,000 IU/ml interferon alpha). These results suggest that PKC, possibly activated by interferon-receptor interaction, is not essential for inducing the antiviral effect of interferon, but, probably, mediates the antiviral effect of TPA.

  7. Cyclin-dependent kinase 5 regulates degranulation in human eosinophils.

    PubMed

    Odemuyiwa, Solomon O; Ilarraza, Ramses; Davoine, Francis; Logan, Michael R; Shayeganpour, Anooshirvan; Wu, Yingqi; Majaesic, Carina; Adamko, Darryl J; Moqbel, Redwan; Lacy, Paige

    2015-04-01

    Degranulation from eosinophils in response to secretagogue stimulation is a regulated process that involves exocytosis of granule proteins through specific signalling pathways. One potential pathway is dependent on cyclin-dependent kinase 5 (Cdk5) and its effector molecules, p35 and p39, which play a central role in neuronal cell exocytosis by phosphorylating Munc18, a regulator of SNARE binding. Emerging evidence suggests a role for Cdk5 in exocytosis in immune cells, although its role in eosinophils is not known. We sought to examine the expression of Cdk5 and its activators in human eosinophils, and to assess the role of Cdk5 in eosinophil degranulation. We used freshly isolated human eosinophils and analysed the expression of Cdk5, p35, p39 and Munc18c by Western blot, RT-PCR, flow cytometry and immunoprecipitation. Cdk5 kinase activity was determined following eosinophil activation. Cdk5 inhibitors were used (roscovitine, AT7519 and small interfering RNA) to determine its role in eosinophil peroxidase (EPX) secretion. Cdk5 was expressed in association with Munc18c, p35 and p39, and phosphorylated following human eosinophil activation with eotaxin/CCL11, platelet-activating factor, and secretory IgA-Sepharose. Cdk5 inhibitors (roscovitine, AT7519) reduced EPX release when cells were stimulated by PMA or secretory IgA. In assays using small interfering RNA knock-down of Cdk5 expression in human eosinophils, we observed inhibition of EPX release. Our findings suggest that in activated eosinophils, Cdk5 is phosphorylated and binds to Munc18c, resulting in Munc18c release from syntaxin-4, allowing SNARE binding and vesicle fusion, with subsequent eosinophil degranulation. Our work identifies a novel role for Cdk5 in eosinophil mediator release by agonist-induced degranulation.

  8. Role for casein kinase 1 in the phosphorylation of Claspin on critical residues necessary for the activation of Chk1.

    PubMed

    Meng, Zheng; Capalbo, Luisa; Glover, David M; Dunphy, William G

    2011-08-15

    The mediator protein Claspin is critical for the activation of the checkpoint kinase Chk1 during checkpoint responses to stalled replication forks. This function involves the Chk1-activating domain (CKAD) of Claspin, which undergoes phosphorylation on multiple conserved sites. These phosphorylations promote binding of Chk1 to Claspin and ensuing activation of Chk1 by ATR. However, despite the importance of this regulatory process, the kinase responsible for these phosphorylations has remained unknown. By using a multifaceted approach, we have found that casein kinase 1 gamma 1 (CK1γ1) carries out this function. CK1γ1 phosphorylates the CKAD of Claspin efficiently in vitro, and depletion of CK1γ1 from human cells by small interfering RNA (siRNA) results in dramatically diminished phosphorylation of Claspin. Consequently, the siRNA-treated cells display impaired activation of Chk1 and resultant checkpoint defects. These results indicate that CK1γ1 is a novel component of checkpoint responses that controls the interaction of a key checkpoint effector kinase with its cognate mediator protein.

  9. Crystal Structures of Human Pyridoxal Kinase in Complex with the Neurotoxins, Ginkgotoxin and Theophylline: Insights into Pyridoxal Kinase Inhibition

    PubMed Central

    Ghatge, Mohini S.; di Salvo, Martino L.; Di Biase, Stefano; Danso-Danquah, Richmond; Musayev, Faik N.; Contestabile, Roberto; Schirch, Verne; Safo, Martin K.

    2012-01-01

    Several drugs and natural compounds are known to be highly neurotoxic, triggering epileptic convulsions or seizures, and causing headaches, agitations, as well as other neuronal symptoms. The neurotoxic effects of some of these compounds, including theophylline and ginkgotoxin, have been traced to their inhibitory activity against human pyridoxal kinase (hPL kinase), resulting in deficiency of the active cofactor form of vitamin B6, pyridoxal 5′-phosphate (PLP). Pyridoxal (PL), an inactive form of vitamin B6 is converted to PLP by PL kinase. PLP is the B6 vitamer required as a cofactor for over 160 enzymatic activities essential in primary and secondary metabolism. We have performed structural and kinetic studies on hPL kinase with several potential inhibitors, including ginkgotoxin and theophylline. The structural studies show ginkgotoxin and theophylline bound at the substrate site, and are involved in similar protein interactions as the natural substrate, PL. Interestingly, the phosphorylated product of ginkgotoxin is also observed bound at the active site. This work provides insights into the molecular basis of hPL kinase inhibition and may provide a working hypothesis to quickly screen or identify neurotoxic drugs as potential hPL kinase inhibitors. Such adverse effects may be prevented by administration of an appropriate form of vitamin B6, or provide clues of how to modify these drugs to help reduce their hPL kinase inhibitory effects. PMID:22879864

  10. Attenuation of G{sub 2} cell cycle checkpoint control in human tumor cells is associated with increased frequencies of unrejoined chromosome breaks but not increased cytotoxicity following radiation exposure

    SciTech Connect

    Schwartz, J.L.; Cowan, J.; Grdina, D.J.

    1997-08-01

    The contribution of G{sub 2} cell cycle checkpoint control to ionizing radiation responses was examined in ten human tumor cell lines. Most of the delay in cell cycle progression seen in the first cell cycle following radiation exposure was due to blocks in G{sub 2} and there were large cell line-to-cell line variations in the length of the G{sub 2} block. Longer delays were seen in cell lines that had mutations in p53. There was a highly significant inverse correlation between the length of G{sub 2} delay and the frequency of unrejoined chromosome breaks seen as chromosome terminal deletions in mitosis, and observation that supports the hypothesis that the signal for G{sub 2} delay in mammalian cells is an unrejoined chromosome break. There were also an inverse correlation between the length of G{sub 2} delay and the level of chromosome aneuploidy in each cell line, suggesting that the G{sub 2} and mitotic spindel checkpoints may be linked to each other. Attenuation in G{sub 2} checkpoint control was not associated with alterations in either the frequency of induced chromosome rearrangements or cell survival following radiation exposure suggesting that chromosome rearrangements, the major radiation-induced lethal lesion in tumor cells, form before cells enters G{sub 2}. Thus, agents that act solely to override G{sub 2} arrest should produce little radiosensitization in human tumor cells.

  11. Chemoimmunotherapy by combining oxaliplatin with immune checkpoint blockades reduced tumor burden in colorectal cancer animal model.

    PubMed

    Wang, Weiwei; Wu, Ling; Zhang, Jiansheng; Wu, Huiguo; Han, Enkun; Guo, Qiang

    2017-05-20

    Colorectal cancer (CRC) is among one of the top common cancers worldwide. Developing novel comprehensive treatment strategies is critical for improving survival of late stage CRC patients. Recent advances in immune checkpoint blockades provided a novel strategy for treating cancers via stimulating the antitumor immune response. However, the effects of immune checkpoint blockades were limited in CRC due to intrinsic resistance. Oxaliplatin (OXA) based chemotherapy was the foundation of CRC adjuvant chemotherapy. Here, we investigated the potential roles of OXA in inducing immunogenicity and synergizing with immune checkpoints in CRC. Immunogenicity of OXA was tested in CRC cell lines. Immune checkpoint blockades sensitive and resistant CRC models were used to study the potential synergistic roles of OXA with immune checkpoint blockades. We found CT26 mouse model was sensitive to immune checkpoint blockades, while MC38 mouse model was resistant. OXA could induce immunogenic cell death in several human and mouse CRC cell lines. Short term OXA treatment increased immune cell infiltration in MC38 mouse model and therefore enhanced the efficacy of immune checkpoint in MC38 mouse model. As a response to the OXA and immune checkpoint blockades combination, inhibitory immune checkpoints were down-regulated in MC38 tumors, while immune enhancing cytokines were up-regulated. Short term OXA treatment induced antitumor immune response in an immune checkpoint blockades resistant mouse model, therefore synergized with immune checkpoint blockades. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Building and exploring an integrated human kinase network: global organization and medical entry points.

    PubMed

    Colinge, Jacques; César-Razquin, Adrián; Huber, Kilian; Breitwieser, Florian P; Májek, Peter; Superti-Furga, Giulio

    2014-07-31

    Biological matter is organized in functional networks of different natures among which kinase-substrate and protein-protein interactions play an important role. Large public data collections allowed us to compile an important corpus of interaction data around human protein kinases. One of the most interesting observations analyzing this network is that coherence in kinase functional activity relies on kinase substrate interactions primarily and not on which protein complexes are formed around them. Further dissecting the two types of interactions at the level of kinase groups (CMGCs, Tyrosine kinases, etc.) we show a prevalence of intra-group interconnectivity, which we can naturally relate to current scenarios of evolution of biological networks. Tracking publication dates we observe high correlation of kinase interaction research focus with general kinase research. We find a similar bias in the targets of kinase inhibitors that feature high redundancy. Finally, intersecting kinase inhibitor specificity with sets of kinases located at specific positions in the kinase network, we propose alternative options for future therapeutic strategies using these compounds. Despite its importance for cellular regulation and the fact that protein kinases feature prominent targets of modern therapeutic approaches, the structure and logic of the global, integrated protein phosphorylation network have not been investigated intensively. To focus on the regulatory skeleton of the phosphorylation network, we contemplated a network consisting of kinases, their substrates, and publicly available physical protein interactions. Analysis of this network at multiple levels allowed establishing a series of interesting properties such as prevalence of kinase substrate interactions as opposed to general protein-protein interactions for establishing a holistic control over kinases activities. Kinases controlling many or a few only other kinases, in addition to non-kinases, were distributed in

  13. Multiple phosphorylation of Rad9 by CDK is required for DNA damage checkpoint activation.

    PubMed

    Wang, Guoliang; Tong, Xiangyan; Weng, Stephanie; Zhou, Huilin

    2012-10-15

    The DNA damage checkpoint controls cell cycle arrest in response to DNA damage, and activation of this checkpoint is in turn cell cycle-regulated. Rad9, the ortholog of mammalian 53BP1, is essential for this checkpoint response and is phosphorylated by the cyclin-dependent kinase (CDK) in the yeast Saccharomyces cerevisiae. Previous studies suggested that the CDK consensus sites of Rad9 are important for its checkpoint activity. However, the precise CDK sites of Rad9 involved have not been determined. Here we show that CDK consensus sites of Rad9 function in parallel to its BRCT domain toward checkpoint activation, analogous to its fission yeast ortholog Crb2. Unlike Crb2, however, mutation of multiple rather than any individual CDK site of Rad9 is required to completely eliminate its checkpoint activity in vivo. Although Dpb11 interacts with CDK-phosphorylated Rad9, we provide evidence showing that elimination of this interaction does not affect DNA damage checkpoint activation in vivo, suggesting that additional pathway(s) exist. Taken together, these findings suggest that the regulation of Rad9 by CDK and the role of Dpb11 in DNA damage checkpoint activation are more complex than previously suggested. We propose that multiple phosphorylation of Rad9 by CDK may provide a more robust system to allow Rad9 to control cell cycle-dependent DNA damage checkpoint activation.

  14. The binding mode of human nucleoside diphosphate kinase B to single-strand DNA.

    PubMed

    Agou, F; Raveh, S; Véron, M

    2000-06-01

    In this paper, we studied the interaction of the human isoform B of nucleoside diphosphate kinase (NDP kinase B) with the nuclease hypersensitive element (NHE) present in the promoter element of the c-myc oncogene. The DNA-binding properties of NDP kinase B and other NDP kinases are compared and the nucleotide requirement for binding are discussed. Using quantitative methods, we identified the DNA-binding sites on the protein and we proposed a structural model for a complex of one hexameric NDP kinase B with an oligonucleotide.

  15. The Abscission Checkpoint: Making It to the Final Cut.

    PubMed

    Nähse, Viola; Christ, Liliane; Stenmark, Harald; Campsteijn, Coen

    2017-01-01

    Cytokinesis is the final stage of cell division and is concluded by abscission of the intercellular bridge to physically separate the daughter cells. Timing of cytokinetic abscission is monitored by a molecular machinery termed the abscission checkpoint. This machinery delays abscission in cells with persistent chromatin in the intercellular bridge. Recent work has also uncovered its response to high membrane tension, nuclear pore defects, and DNA replication stress. Although it is known that the abscission checkpoint depends on persistent activity of the Aurora B protein kinase, we have only recently begun to understand its molecular basis. We propose here a molecular framework for abscission checkpoint signaling and we discuss outstanding questions relating to its function and physiological relevance.

  16. Irreversible Nek2 kinase inhibitors with cellular activity

    PubMed Central

    Henise, Jeffrey C.; Taunton, Jack

    2013-01-01

    A structure-based approach was used to design irreversible, cysteine-targeted inhibitors of the human centrosomal kinase, Nek2. Potent inhibition of Nek2 kinase activity in biochemical and cell-based assays required a noncatalytic cysteine residue (Cys22), located near the glycine-rich loop in a subset of human kinases. Elaboration of an oxindole scaffold led to our most selective compound, oxindole propynamide 16 (JH295). Propynamide 16 irreversibly inhibited cellular Nek2 without affecting the mitotic kinases, Cdk1, Aurora B, or Plk1. Moreover, 16 did not perturb bipolar spindle assembly or the spindle assembly checkpoint. To our knowledge, 16 is the first small molecule shown to inactivate Nek2 kinase activity in cells. PMID:21627121

  17. Rejuvenation of human cardiac progenitor cells with Pim-1 kinase.

    PubMed

    Mohsin, Sadia; Khan, Mohsin; Nguyen, Jonathan; Alkatib, Monique; Siddiqi, Sailay; Hariharan, Nirmala; Wallach, Kathleen; Monsanto, Megan; Gude, Natalie; Dembitsky, Walter; Sussman, Mark A

    2013-10-25

    Myocardial function is enhanced by adoptive transfer of human cardiac progenitor cells (hCPCs) into a pathologically challenged heart. However, advanced age, comorbidities, and myocardial injury in patients with heart failure constrain the proliferation, survival, and regenerative capacity of hCPCs. Rejuvenation of senescent hCPCs will improve the outcome of regenerative therapy for a substantial patient population possessing functionally impaired stem cells. Reverse phenotypic and functional senescence of hCPCs by ex vivo modification with Pim-1. C-kit-positive hCPCs were isolated from heart biopsy samples of patients undergoing left ventricular assist device implantation. Growth kinetics, telomere lengths, and expression of cell cycle regulators showed significant variation between hCPC isolated from multiple patients. Telomere length was significantly decreased in hCPC with slow-growth kinetics concomitant with decreased proliferation and upregulation of senescent markers compared with hCPC with fast-growth kinetics. Desirable youthful characteristics were conferred on hCPCs by genetic modification using Pim-1 kinase, including increases in proliferation, telomere length, survival, and decreased expression of senescence markers. Senescence characteristics of hCPCs are ameliorated by Pim-1 kinase resulting in rejuvenation of phenotypic and functional properties. hCPCs show improved cellular properties resulting from Pim-1 modification, but benefits were more pronounced in hCPC with slow-growth kinetics relative to hCPC with fast-growth kinetics. With the majority of patients with heart failure presenting advanced age, infirmity, and impaired regenerative capacity, the use of Pim-1 modification should be incorporated into cell-based therapeutic approaches to broaden inclusion criteria and address limitations associated with the senescent phenotype of aged hCPC.

  18. Rejuvenation of Human Cardiac Progenitor Cells With Pim-1 Kinase

    PubMed Central

    Mohsin, Sadia; Khan, Mohsin; Nguyen, Jonathan; Alkatib, Monique; Siddiqi, Sailay; Hariharan, Nirmala; Wallach, Kathleen; Monsanto, Megan; Gude, Natalie; Dembitsky, Walter; Sussman, Mark A.

    2014-01-01

    Rationale Myocardial function is enhanced by adoptive transfer of human cardiac progenitor cells (hCPCs) into a pathologically challenged heart. However, advanced age, comorbidities, and myocardial injury in patients with heart failure constrain the proliferation, survival, and regenerative capacity of hCPCs. Rejuvenation of senescent hCPCs will improve the outcome of regenerative therapy for a substantial patient population possessing functionally impaired stem cells. Objective Reverse phenotypic and functional senescence of hCPCs by ex vivo modification with Pim-1. Methods and Results C-kit–positive hCPCs were isolated from heart biopsy samples of patients undergoing left ventricular assist device implantation. Growth kinetics, telomere lengths, and expression of cell cycle regulators showed significant variation between hCPC isolated from multiple patients. Telomere length was significantly decreased in hCPC with slow-growth kinetics concomitant with decreased proliferation and upregulation of senescent markers compared with hCPC with fast-growth kinetics. Desirable youthful characteristics were conferred on hCPCs by genetic modification using Pim-1 kinase, including increases in proliferation, telomere length, survival, and decreased expression of senescence markers. Conclusions Senescence characteristics of hCPCs are ameliorated by Pim-1 kinase resulting in rejuvenation of phenotypic and functional properties. hCPCs show improved cellular properties resulting from Pim-1 modification, but benefits were more pronounced in hCPC with slow-growth kinetics relative to hCPC with fast-growth kinetics. With the majority of patients with heart failure presenting advanced age, infirmity, and impaired regenerative capacity, the use of Pim-1 modification should be incorporated into cell-based therapeutic approaches to broaden inclusion criteria and address limitations associated with the senescent phenotype of aged hCPC. PMID:24044948

  19. ARHGEF17 is an essential spindle assembly checkpoint factor that targets Mps1 to kinetochores

    PubMed Central

    Isokane, Mayumi; Walter, Thomas; Mahen, Robert; Nijmeijer, Bianca; Hériché, Jean-Karim; Miura, Kota; Maffini, Stefano; Ivanov, Miroslav Penchev; Kitajima, Tomoya S.; Peters, Jan-Michael

    2016-01-01

    To prevent genome instability, mitotic exit is delayed until all chromosomes are properly attached to the mitotic spindle by the spindle assembly checkpoint (SAC). In this study, we characterized the function of ARHGEF17, identified in a genome-wide RNA interference screen for human mitosis genes. Through a series of quantitative imaging, biochemical, and biophysical experiments, we showed that ARHGEF17 is essential for SAC activity, because it is the major targeting factor that controls localization of the checkpoint kinase Mps1 to the kinetochore. This mitotic function is mediated by direct interaction of the central domain of ARHGEF17 with Mps1, which is autoregulated by the activity of Mps1 kinase, for which ARHGEF17 is a substrate. This mitosis-specific role is independent of ARHGEF17’s RhoGEF activity in interphase. Our study thus assigns a new mitotic function to ARHGEF17 and reveals the molecular mechanism for a key step in SAC establishment. PMID:26953350

  20. Histone deacetylase inhibitors promote glioma cell death by G2 checkpoint abrogation leading to mitotic catastrophe.

    PubMed

    Cornago, M; Garcia-Alberich, C; Blasco-Angulo, N; Vall-Llaura, N; Nager, M; Herreros, J; Comella, J X; Sanchis, D; Llovera, M

    2014-10-02

    Glioblastoma multiforme is resistant to conventional anti-tumoral treatments due to its infiltrative nature and capability of relapse; therefore, research efforts focus on characterizing gliomagenesis and identifying molecular targets useful on therapy. New therapeutic strategies are being tested in patients, such as Histone deacetylase inhibitors (HDACi) either alone or in combination with other therapies. Here two HDACi included in clinical trials have been tested, suberanilohydroxamic acid (SAHA) and valproic acid (VPA), to characterize their effects on glioma cell growth in vitro and to determine the molecular changes that promote cancer cell death. We found that both HDACi reduce glioma cell viability, proliferation and clonogenicity. They have multiple effects, such as inducing the production of reactive oxygen species (ROS) and activating the mitochondrial apoptotic pathway, nevertheless cell death is not prevented by the pan-caspase inhibitor Q-VD-OPh. Importantly, we found that HDACi alter cell cycle progression by decreasing the expression of G2 checkpoint kinases Wee1 and checkpoint kinase 1 (Chk1). In addition, HDACi reduce the expression of proteins involved in DNA repair (Rad51), mitotic spindle formation (TPX2) and chromosome segregation (Survivin) in glioma cells and in human glioblastoma multiforme primary cultures. Therefore, HDACi treatment causes glioma cell entry into mitosis before DNA damage could be repaired and to the formation of an aberrant mitotic spindle that results in glioma cell death through mitotic catastrophe-induced apoptosis.

  1. WRN Is Required for ATM Activation and the S-Phase Checkpoint in Response to Interstrand Cross-Link–Induced DNA Double-Strand Breaks

    PubMed Central

    Cheng, Wen-Hsing; Muftic, Diana; Muftuoglu, Meltem; Dawut, Lale; Morris, Christa; Helleday, Thomas; Shiloh, Yosef

    2008-01-01

    Werner syndrome (WS) is a human genetic disorder characterized by extensive clinical features of premature aging. Ataxia-telengiectasia (A-T) is a multisystem human genomic instability syndrome that includes premature aging in some of the patients. WRN and ATM, the proteins defective in WS and A-T, respectively, play significant roles in the maintenance of genomic stability and are involved in several DNA metabolic pathways. A role for WRN in DNA repair has been proposed; however, this study provides evidence that WRN is also involved in ATM pathway activation and in a S-phase checkpoint in cells exposed to DNA interstrand cross-link–induced double-strand breaks. Depletion of WRN in such cells by RNA interference results in an intra-S checkpoint defect, and interferes with activation of ATM as well as downstream phosphorylation of ATM target proteins. Treatment of cells under replication stress with the ATM kinase inhibitor KU 55933 results in a S-phase checkpoint defect similar to that observed in WRN shRNA cells. Moreover, γH2AX levels are higher in WRN shRNA cells than in control cells 6 and 16 h after exposure to psoralen DNA cross-links. These results suggest that WRN and ATM participate in a replication checkpoint response, in which WRN facilitates ATM activation in cells with psoralen DNA cross-link–induced collapsed replication forks. PMID:18596239

  2. NAD kinase levels control the NADPH concentration in human cells.

    PubMed

    Pollak, Nadine; Niere, Marc; Ziegler, Mathias

    2007-11-16

    NAD kinases (NADKs) are vital, as they generate the cellular NADP pool. As opposed to three compartment-specific isoforms in plants and yeast, only a single NADK has been identified in mammals whose cytoplasmic localization we established by immunocytochemistry. To understand the physiological roles of the human enzyme, we generated and analyzed cell lines stably deficient in or overexpressing NADK. Short hairpin RNA-mediated down-regulation led to similar (about 70%) decrease of both NADK expression, activity, and the NADPH concentration and was accompanied by increased sensitivity toward H(2)O(2). Overexpression of NADK resulted in a 4-5-fold increase in the NADPH, but not NADP(+), concentration, although the recombinant enzyme phosphorylated preferentially NAD(+). Surprisingly, NADK overexpression and the ensuing increase of the NADPH level only moderately enhanced protection against oxidant treatment. Apparently, to maintain the NADPH level for the regeneration of oxidative defense systems human cells depend primarily on NADP-dependent dehydrogenases (which re-reduce NADP(+)), rather than on a net increase of NADP. The stable shifts of the NADPH level in the generated cell lines were also accompanied by alterations in the expression of peroxiredoxin 5 and Nrf2. Because the basal oxygen radical level in the cell lines was only slightly changed, the redox state of NADP may be a major transmitter of oxidative stress.

  3. Targeting immune checkpoints in malignant glioma

    PubMed Central

    Li, Tete; Liu, Yong-Jun; Chen, Wei; Chen, Jingtao

    2017-01-01

    Malignant glioma is the most common and a highly aggressive cancer in the central nervous system (CNS). Cancer immunotherapy, strategies to boost the bodys anti-cancer immune responses instead of directly targeting tumor cells, recently achieved great success in treating several human solid tumors. Although once considered immune privileged and devoid of normal immunological functions, CNS is now considered a promising target for cancer immunotherapy, featuring the recent progresses in neurobiology and neuroimmunology and a highly immunosuppressive state in malignant glioma. In this review, we focus on immune checkpoint inhibitors, specifically, antagonizing monoclonal antibodies for programmed cell death protein-1 (PD-1), cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4), and indoleamine 2,3-dioxygenase (IDO). We discuss advances in the working mechanisms of these immune checkpoint molecules, their status in malignant glioma, and current preclinical and clinical trials targeting these molecules in malignant glioma. PMID:27756892

  4. Requirements for Linux Checkpoint/Restart

    SciTech Connect

    Duell, Jason; Hargrove, Paul H.; Roman, Eric S.

    2002-02-26

    This document has 4 main objectives: (1) Describe data to be saved and restored during checkpoint/restart; (2) Describe how checkpoint/restart is used within the context of the Scalable Systems environment, and MPI applications; (3) Identify issues for a checkpoint/restart implementation; and (4) Sketch the architecture of a checkpoint/restart implementation.

  5. Focal adhesion kinase overexpression and its impact on human osteosarcoma

    PubMed Central

    Chen, Yong; Yang, Aizhen; Chen, Hui; Zhang, Jian; Wu, Sujia; Shi, Xin; Wang, Chen; Sun, Xiaoliang

    2015-01-01

    Focal adhesion kinase (FAK) has been implicated in tumorigenesis in various malignancies. We sought to examine the expression patterns of FAK and the activated form, phosphorylated FAK (pFAK), in human osteosarcoma and to investigate the correlation of FAK expression with clinicopathologic parameters and prognosis. In addition, the functional consequence of manipulating the FAK protein level was investigated in human osteosarcoma cell lines. Immunohistochemical staining was used to detect FAK and pFAK in pathologic archived materials from 113 patients with primary osteosarcoma. Kaplan-Meier survival and Cox regression analyses were performed to evaluate the prognoses. The role of FAK in the cytological behavior of MG63 and 143B human osteosarcoma cell lines was studied via FAK protein knock down with siRNA. Cell proliferation, migration, invasiveness and apoptosis were assessed using the CCK8, Transwell and Annexin V/PI staining methods. Both FAK and pFAK were overexpressed in osteosarcoma. There were significant differences in overall survival between the FAK-/pFAK- and FAK+/pFAK- groups (P = 0.016), the FAK+/pFAK- and FAK+/pFAK+ groups (P = 0.012) and the FAK-/pFAK- and FAK+/pFAK+ groups (P < 0.001). There were similar differences in metastasis-free survival between groups. The Cox proportional hazards analysis showed that the FAK expression profile was an independent indicator of both overall and metastasis-free survival. siRNA-based knockdown of FAK not only dramatically reduced the migration and invasion of MG63 and 143B cells, but also had a distinct effect on osteosarcoma cell proliferation and apoptosis. These results collectively suggest that FAK overexpression and phosphorylation might predict more aggressive biologic behavior in osteosarcoma and may be an independent predictor of poor prognosis. PMID:26393679

  6. Enantioselectivity of human AMP, dTMP and UMP-CMP kinases

    PubMed Central

    Alexandre, Julie A.C.; Roy, Béatrice; Topalis, Dimitri; Pochet, Sylvie; Périgaud, Christian; Deville-Bonne, Dominique

    2007-01-01

    l-Nucleoside analogues such as lamivudine are active for treating viral infections. Like d-nucleosides, the biological activity of the l-enantiomers requires their stepwise phosphorylation by cellular or viral kinases to give the triphosphate. The enantioselectivity of NMP kinases has not been thoroughly studied, unlike that of deoxyribonucleoside kinases. We have therefore investigated the capacity of l-enantiomers of some natural (d)NMP to act as substrates for the recombinant forms of human uridylate-cytidylate kinase, thymidylate kinase and adenylate kinases 1 and 2. Both cytosolic and mitochondrial adenylate kinases were strictly enantioselective, as they phosphorylated only d-(d)AMP. l-dTMP was a substrate for thymidylate kinase, but with an efficiency 150-fold less than d-dTMP. Both l-dUMP and l-(d)CMP were phosphorylated by UMP-CMP kinase although much less efficiently than their natural counterparts. The stereopreference was conserved with the 2′-azido derivatives of dUMP and dUMP while, unexpectedly, the 2′-azido-d-dCMP was a 4-fold better substrate for UMP-CMP kinase than was CMP. Docking simulations showed that the small differences in the binding of d-(d)NMP to their respective kinases could account for the differences in interactions of the l-isomers with the enzymes. This in vitro information was then used to develop the in vivo activation pathway for l-dT. PMID:17626051

  7. Defining Human Tyrosine Kinase Phosphorylation Networks Using Yeast as an In Vivo Model Substrate.

    PubMed

    Corwin, Thomas; Woodsmith, Jonathan; Apelt, Federico; Fontaine, Jean-Fred; Meierhofer, David; Helmuth, Johannes; Grossmann, Arndt; Andrade-Navarro, Miguel A; Ballif, Bryan A; Stelzl, Ulrich

    2017-08-23

    Systematic assessment of tyrosine kinase-substrate relationships is fundamental to a better understanding of cellular signaling and its profound alterations in human diseases such as cancer. In human cells, such assessments are confounded by complex signaling networks, feedback loops, conditional activity, and intra-kinase redundancy. Here we address this challenge by exploiting the yeast proteome as an in vivo model substrate. We individually expressed 16 human non-receptor tyrosine kinases (NRTKs) in Saccharomyces cerevisiae and identified 3,279 kinase-substrate relationships involving 1,351 yeast phosphotyrosine (pY) sites. Based on the yeast data without prior information, we generated a set of linear kinase motifs and assigned ∼1,300 known human pY sites to specific NRTKs. Furthermore, experimentally defined pY sites for each individual kinase were shown to cluster within the yeast interactome network irrespective of linear motif information. We therefore applied a network inference approach to predict kinase-substrate relationships for more than 3,500 human proteins, providing a resource to advance our understanding of kinase biology. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Identification of kinases, phosphatases, and phosphorylation sites in human and porcine spermatozoa.

    PubMed

    Lackey, Brett R; Gray, Sandra L

    2015-01-01

    Multiple inter-connected signaling pathways, involving kinases and phosphatases, form a framework that controls sperm motility, function, and fertilizing ability. Methods that give a broad view of the proteomic landscape may prove valuable in uncovering new crosstalk connections, as well as in discovering new proteins within this regulatory framework. A multi-immunoblotting strategy was utilized to evaluate this concept on human and porcine spermatozoa samples. In human and porcine spermatozoa, a diversity of kinases were identified including protein kinase A (PKA), protein kinase B (PKB), isoforms of protein kinase C (PKC), calmodulin-dependent kinases (CAMK), casein kinase (CK), and isoforms of glycogen synthase kinase (GSK3). Several phosphatases, such as protein phosphatase (PP)-1, PP2A, PP2C, and mitogen activated protein kinase (MAPK) phosphatase (MKP-1), were identified in human spermatozoa. The phosphorylation epitopes recognized belonged to members of the MAPK family, in addition to α and β isoforms of GSK3 and cAMP response element binding protein (CREB). Proteomic approaches that allow a broad view may aid in understanding the crosstalk between signaling systems in spermatozoal physiology.

  9. Checkpointing in speculative versioning caches

    DOEpatents

    Eichenberger, Alexandre E; Gara, Alan; Gschwind, Michael K; Ohmacht, Martin

    2013-08-27

    Mechanisms for generating checkpoints in a speculative versioning cache of a data processing system are provided. The mechanisms execute code within the data processing system, wherein the code accesses cache lines in the speculative versioning cache. The mechanisms further determine whether a first condition occurs indicating a need to generate a checkpoint in the speculative versioning cache. The checkpoint is a speculative cache line which is made non-speculative in response to a second condition occurring that requires a roll-back of changes to a cache line corresponding to the speculative cache line. The mechanisms also generate the checkpoint in the speculative versioning cache in response to a determination that the first condition has occurred.

  10. Cheminfomatic-based Drug Discovery of Human Tyrosine Kinase Inhibitors

    PubMed Central

    Reid, Terry-Elinor; Fortunak, Joseph M.; Wutoh, Anthony; Wang, Xiang Simon

    2016-01-01

    Receptor Tyrosine Kinases (RTKs) are essential components for regulating cell-cell signaling and communication events in cell growth, proliferation, differentiation, survival and metabolism. Deregulation of RTKs and their associated signaling pathways can lead to a wide variety of human diseases such as immunodeficiency, diabetes, arterosclerosis, psoriasis and cancer. Thus RTKs have become one of the most important drug targets families in recent decade. Pharmaceutical companies have dedicated their research efforts towards the discovery of small-molecule inhibitors of RTKs, many of which had been approved by the U.S. Food and Drug Administration (US FDA) or are currently in clinical trials. The great successes in the development of small-molecule inhibitors of RTKs are largely attributed to the use of modern cheminformatic approaches to identifying lead scaffolds. Those include the quantitative structure-activity relationship (QSAR) modeling, as well as the structure-, and ligand-based pharmacophore modeling techniques in this case. Herein we inspected the literature thoroughly in an effort to conduct a comparative analysis of major findings regarding the essential structure-activity relationships (SARs)/pharmacophore features of known active RTK inhibitors, most of which were collected from cheminformatic modeling approaches. PMID:26369823

  11. Activation of human mitochondrial pantothenate kinase 2 by palmitoylcarnitine.

    PubMed

    Leonardi, Roberta; Rock, Charles O; Jackowski, Suzanne; Zhang, Yong-Mei

    2007-01-30

    The human isoform 2 of pantothenate kinase (PanK2) is localized to the mitochondria, and mutations in this protein are associated with a progressive neurodegenerative disorder. PanK2 inhibition by acetyl-CoA is so stringent (IC50 < 1 microM) that it is unclear how the enzyme functions in the presence of intracellular CoA concentrations. Palmitoylcarnitine was discovered to be a potent activator of PanK2 that functions to competitively antagonize acetyl-CoA inhibition. Acetyl-CoA was a competitive inhibitor of purified PanK2 with respect to ATP. The interaction between PanK2 and acetyl-CoA was stable enough that a significant proportion of the purified protein was isolated as the PanK2.acetyl-CoA complex. The long-chain acylcarnitine activation of PanK2 explains how PanK2 functions in vivo, by providing a positive regulatory mechanism to counteract the negative regulation of PanK2 activity by acetyl-CoA. Our results suggest that PanK2 is located in the mitochondria to sense the levels of palmitoylcarnitine and up-regulate CoA biosynthesis in response to an increased mitochondrial demand for the cofactor to support beta-oxidation.

  12. Expression of immune checkpoint molecules of T cell immunoglobulin and mucin protein 3/galectin-9 for NK cell suppression in human gastrointestinal stromal tumors.

    PubMed

    Komita, Hideo; Koido, Shigeo; Hayashi, Kazumi; Kan, Shin; Ito, Masaki; Kamata, Yuko; Suzuki, Masafumi; Homma, Sadamu

    2015-10-01

    Monoclonal antibody therapy for immune checkpoint blockade has achieved promising results for several types of malignant tumors. For the future treatment of gastrointestinal stromal tumors (GISTs) by immune checkpoint blockade, expression of immune checkpoint-related molecules that suppress antitumor immunity in GISTs was examined. Infiltration of immune cell types into 19 GIST tissues was analyzed by immunohistochemistry, and expression of T cell immunoglobulin and mucin protein 3 (Tim-3) and programmed cell death-1 (PD-1) in the infiltrated immune cells was examined by immunofluorescence microscopy. The expression status of galectin-9 in the GIST tumor cells was also determined by immunohistochemistry. All the GIST tissues showed CD8+ T cell infiltration and 8 showed CD56+ natural killer (NK) cell infiltration, and the numbers of infiltrated CD8+ T and NK cells were strongly correlated. However, these CD8+ T and NK cells were CD69-negative inactivated cells. Tim-3 was expressed in the infiltrated NK cells in 6/8 (75%) of the GIST tissues. Expression of galectin-9, a ligand of Tim-3, was observed in 13/19 (68.4%) GIST tissues and all of the GIST tissues with Tim-3+ NK cell infiltration showed positive galectin-9 expression. No PD-1 expression in the infiltrated NK cells and neither Tim-3 nor PD-1 expression was observed in the infiltrated CD8+ T cells. Interaction between Tim-3 in infiltrated NK cells and galectin-9 in tumor cells may be involved in an immune checkpoint mechanism for suppression of antitumor immunity in GISTs. Blockade of the Tim-3/galectin-9 pathway may become a new strategy for GIST treatment.

  13. Studies on the Orientation of Cyclic AMP-Dependent Protein Kinase in Human Erythrocyte Membranes

    PubMed Central

    Rubin, Charles S.; Rosenfeld, Robert D.; Rosen, Ora M.

    1973-01-01

    The topographic location of cyclic AMP-dependent protein kinase has been studied in preparations of permeable and sealed membranes derived from human erythrocytes. Both the catalytic and cyclic AMP-binding components of protein kinase are localized on the inner, cytoplasmic, surface of the plasma membrane. PMID:4359486

  14. Partial characterization of a highly conserved aspartyl kinase (AK) in normal human liver.

    PubMed

    Arenas-Díaz, G; Marshall, S H

    1990-01-01

    Subcellular fractions from human liver were assayed for aspartyl kinase (AK) activity measured by standard spectrophotometric methods. Along the purification procedure three different fractions displayed the expected enzyme activity. Interestingly, two of these fractions were specifically recognized by antibodies raised against E. coli aspartate kinases, suggesting a high degree of evolutionary conservation for these ubiquitous enzymes for prokaryotes. Since their known function in bacteria is not strictly required in eukaryotes, these observation imply that the presence and activity of aspartyl kinase(s) in mammals might represent putative regulatory roles for these enzymes in eukaryotic cell metabolism.

  15. Abscission checkpoint control: stuck in the middle with Aurora B.

    PubMed

    Carmena, Mar

    2012-07-01

    At the end of cell division, the cytoplasmic bridge joining the daughter cells is severed through a process that involves scission of the plasma membrane. The presence of chromatin bridges 'stuck' in the division plane is sensed by the chromosomal passenger complex (CPC) component Aurora B kinase, triggering a checkpoint that delays abscission until the chromatin bridges have been resolved. Recent work has started to shed some light on the molecular mechanism by which the CPC controls the timing of abscission.

  16. Purification, crystallization and preliminary X-ray diffraction analysis of the kinase domain of human tousled-like kinase 2

    SciTech Connect

    Garrote, Ana M.; Redondo, Pilar; Montoya, Guillermo; Muñoz, Inés G.

    2014-02-19

    The C-terminal kinase domain of TLK2 (a human tousled-like kinase) has been cloned and overexpressed in Escherichia coli followed by purification to homogeneity. Crystallization experiments in the presence of ATP-γ-S yielded crystals suitable for X-ray diffraction analysis belonging to two different space groups: tetragonal I4{sub 1}22 and cubic P2{sub 1}3. Tousled-like kinases (TLKs) are an evolutionarily conserved family of serine/threonine protein kinases involved in chromatin dynamics, including DNA replication and repair, transcription and chromosome segregation. The two members of the family reported in humans, namely TLK1 and TLK2, localize to the cell nucleus and are capable of forming homo- or hetero-oligomers by themselves. To characterize the role of TLK2, its C-terminal kinase domain was cloned and overexpressed in Escherichia coli followed by purification to homogeneity. Crystallization experiments in the presence of ATP-γ-S yielded crystals suitable for X-ray diffraction analysis belonging to two different space groups: tetragonal I4{sub 1}22 and cubic P2{sub 1}3. The latter produced the best diffracting crystal (3.4 Å resolution using synchrotron radiation), with unit-cell parameters a = b = c = 126.05 Å, α = β = γ = 90°. The asymmetric unit contained one protein molecule, with a Matthews coefficient of 4.59 Å{sup 3} Da{sup −1} and a solvent content of 73.23%.

  17. Checkpoint genes and Exo1 regulate nearby inverted repeat fusions that form dicentric chromosomes in Saccharomyces cerevisiae.

    PubMed

    Kaochar, Salma; Shanks, Lisa; Weinert, Ted

    2010-12-14

    Genomic rearrangements are common, occur by largely unknown mechanisms, and can lead to human diseases. We previously demonstrated that some genome rearrangements occur in budding yeast through the fusion of two DNA sequences that contain limited sequence homology, lie in inverted orientation, and are within 5 kb of one another. This inverted repeat fusion reaction forms dicentric chromosomes, which are well-known intermediates to additional rearrangements. We have previously provided evidence indicating that an error of stalled or disrupted DNA replication forks can cause inverted repeat fusion. Here we analyze how checkpoint protein regulatory pathways known to stabilize stalled forks affect this form of instability. We find that two checkpoint pathways suppress inverted repeat fusion, and that their activities are distinguishable by their interactions with exonuclease 1 (Exo1). The checkpoint kinase Rad53 (Chk2) and recombination protein complex MRX(MRN) inhibit Exo1 in one pathway, whereas in a second pathway the ATR-like kinases Mec1 and Tel1, adaptor protein Rad9, and effector kinases Chk1 and Dun1 act independently of Exo1 to prevent inverted repeat fusion. We provide a model that indicates how in Rad53 or MRX mutants, an inappropriately active Exo1 may facilitate faulty template switching between nearby inverted repeats to form dicentric chromosomes. We further investigate the role of Rad53, using hypomorphic alleles of Rad53 and null mutations in Rad9 and Mrc1, and provide evidence that only local, as opposed to global, activity of Rad53 is sufficient to prevent inverted repeat fusion.

  18. Induction of a G1-S checkpoint in fission yeast.

    PubMed

    Bøe, Cathrine A; Krohn, Marit; Rødland, Gro Elise; Capiaghi, Christoph; Maillard, Olivier; Thoma, Fritz; Boye, Erik; Grallert, Beáta

    2012-06-19

    Entry into S phase is carefully regulated and, in most organisms, under the control of a G(1)-S checkpoint. We have previously described a G(1)-S checkpoint in fission yeast that delays formation of the prereplicative complex at chromosomal replication origins after exposure to UV light (UVC). This checkpoint absolutely depends on the Gcn2 kinase. Here, we explore the signal for activation of the Gcn2-dependent G(1)-S checkpoint in fission yeast. If some form of DNA damage can activate the checkpoint, deficient DNA repair should affect the length of the checkpoint-induced delay. We find that the cell-cycle delay differs in repair-deficient mutants from that in wild-type cells. However, the duration of the delay depends not only on the repair capacity of the cells, but also on the nature of the repair deficiency. First, the delay is abolished in cells that are deficient in the early steps of repair. Second, the delay is prolonged in repair mutants that fail to complete repair after the incision stage. We conclude that the G(1)-S delay depends on damage to the DNA and that the activating signal derives not from the initial DNA damage, but from a repair intermediate(s). Surprisingly, we find that activation of Gcn2 does not depend on the processing of DNA damage and that activated Gcn2 alone is not sufficient to delay entry into S phase in UVC-irradiated cells. Thus, the G(1)-S delay depends on at least two different inputs.

  19. [Function of aurora kinase C (AURKC) in human reproduction].

    PubMed

    Harbuz, R; Zouari, R; Dieterich, K; Nikas, Y; Lunardi, J; Hennebicq, S; Ray, P-F

    2009-06-01

    Infertility concerns at least 70 million couples worldwide. An important proportion of cases is believed to have a genetic component, yet few causal genes have been identified so far. Hundreds of genes are probably involved in spermatogenesis and oogenesis and this genetic heterogeneity has so far hindered the identification of genes causing infertility in the human. Careful morphological examination of spermatozoa can provide cues to identify homogeneous cohorts of patients likely to have the same genetic defect. We studied a cohort of North-Africans patients with a rare phenotype of large-headed spermatozoa. Using a homozygosity mapping strategy, we could map the morbid gene and we identified the same homozygous mutation (c.144delC) in the aurora kinase C gene (AURKC) of all patients studied initially. We then genotyped a total of 62 patients. All who had a typical phenotype with close to 100% large-headed spermatozoa were homozygously mutated (n=34), whereas no AURKC mutations were detected in the others. A carrier frequency of 1/50 was established from individuals from the Maghrebian population, indicating that 1 in 10,000 men from North-African can be expected to present this form of infertility, a frequency comparable to that of Y-microdeletions, thus far the only known recurrent genetic event altering spermatogenesis. Then we demonstrated by flow cytometry that all spermatozoa have in fact a homogeneous 4C. We recommend the realisation of a molecular diagnosis to all patients with large-headed spermatozoa. ICSI is formally contraindicated for all homozygous patients who can have recourse to donor sperm or adoption. One cannot be as categorical for the patients not harbouring an AURKC mutation.

  20. Construction of protein phosphorylation networks by data mining, text mining and ontology integration: analysis of the spindle checkpoint

    PubMed Central

    Ross, Karen E.; Arighi, Cecilia N.; Ren, Jia; Huang, Hongzhan; Wu, Cathy H.

    2013-01-01

    Knowledge representation of the role of phosphorylation is essential for the meaningful understanding of many biological processes. However, such a representation is challenging because proteins can exist in numerous phosphorylated forms with each one having its own characteristic protein–protein interactions (PPIs), functions and subcellular localization. In this article, we evaluate the current state of phosphorylation event curation and then present a bioinformatics framework for the annotation and representation of phosphorylated proteins and construction of phosphorylation networks that addresses some of the gaps in current curation efforts. The integrated approach involves (i) text mining guided by RLIMS-P, a tool that identifies phosphorylation-related information in scientific literature; (ii) data mining from curated PPI databases; (iii) protein form and complex representation using the Protein Ontology (PRO); (iv) functional annotation using the Gene Ontology (GO); and (v) network visualization and analysis with Cytoscape. We use this framework to study the spindle checkpoint, the process that monitors the assembly of the mitotic spindle and blocks cell cycle progression at metaphase until all chromosomes have made bipolar spindle attachments. The phosphorylation networks we construct, centered on the human checkpoint kinase BUB1B (BubR1) and its yeast counterpart MAD3, offer a unique view of the spindle checkpoint that emphasizes biologically relevant phosphorylated forms, phosphorylation-state–specific PPIs and kinase–substrate relationships. Our approach for constructing protein phosphorylation networks can be applied to any biological process that is affected by phosphorylation. Database URL: http://www.yeastgenome.org/ PMID:23749465

  1. LYN- and AIRE-mediated tolerance checkpoint defects synergize to trigger organ-specific autoimmunity.

    PubMed

    Proekt, Irina; Miller, Corey N; Jeanne, Marion; Fasano, Kayla J; Moon, James J; Lowell, Clifford A; Gould, Douglas B; Anderson, Mark S; DeFranco, Anthony L

    2016-10-03

    Studies of the genetic factors associated with human autoimmune disease suggest a multigenic origin of susceptibility; however, how these factors interact and through which tolerance pathways they operate generally remain to be defined. One key checkpoint occurs through the activity of the autoimmune regulator AIRE, which promotes central T cell tolerance. Recent reports have described a variety of dominant-negative AIRE mutations that likely contribute to human autoimmunity to a greater extent than previously thought. In families with these mutations, the penetrance of autoimmunity is incomplete, suggesting that other checkpoints play a role in preventing autoimmunity. Here, we tested whether a defect in LYN, an inhibitory protein tyrosine kinase that is implicated in systemic autoimmunity, could combine with an Aire mutation to provoke organ-specific autoimmunity. Indeed, mice with a dominant-negative allele of Aire and deficiency in LYN spontaneously developed organ-specific autoimmunity in the eye. We further determined that a small pool of retinal protein-specific T cells escaped thymic deletion as a result of the hypomorphic Aire function and that these cells also escaped peripheral tolerance in the presence of LYN-deficient dendritic cells, leading to highly destructive autoimmune attack. These findings demonstrate how 2 distinct tolerance pathways can synergize to unleash autoimmunity and have implications for the genetic susceptibility of autoimmune disease.

  2. Lazy checkpoint coordination for bounding rollback propagation

    NASA Technical Reports Server (NTRS)

    Wang, Yi-Min; Fuchs, W. Kent

    1992-01-01

    Independent checkpointing allows maximum process autonomy but suffers from potential domino effects. Coordinated checkpointing eliminates the domino effect by sacrificing a certain degree of process autonomy. In this paper, we propose the technique of lazy checkpoint coordination which preserves process autonomy while employing communication-induced checkpoint coordination for bounding rollback propagation. The introduction of the notion of laziness allows a flexible trade-off between the cost for checkpoint coordination and the average rollback distance. Worst-case overhead analysis provides a means for estimating the extra checkpoint overhead. Communication trace-driven simulation for several parallel programs is used to evaluate the benefits of the proposed scheme for real applications.

  3. Compiler-assisted static checkpoint insertion

    NASA Technical Reports Server (NTRS)

    Long, Junsheng; Fuchs, W. K.; Abraham, Jacob A.

    1992-01-01

    This paper describes a compiler-assisted approach for static checkpoint insertion. Instead of fixing the checkpoint location before program execution, a compiler enhanced polling mechanism is utilized to maintain both the desired checkpoint intervals and reproducible checkpoint 1ocations. The technique has been implemented in a GNU CC compiler for Sun 3 and Sun 4 (Sparc) processors. Experiments demonstrate that the approach provides for stable checkpoint intervals and reproducible checkpoint placements with performance overhead comparable to a previously presented compiler assisted dynamic scheme (CATCH) utilizing the system clock.

  4. Lazy checkpoint coordination for bounding rollback propagation

    NASA Technical Reports Server (NTRS)

    Wang, Yi-Min; Fuchs, W. K.

    1993-01-01

    Independent checkpointing allows maximum process autonomy but suffers from potential domino effects. Coordinated checkpointing eliminates the domino effect by sacrificing a certain degree of process autonomy. In this paper, we propose the technique of lazy checkpoint coordination which preserves process autonomy while employing communication-induced checkpoint coordination for bounding rollback propagation. The introduction of the notion of laziness allows a flexible trade-off between the cost for checkpoint coordination and the average rollback distance. Worst-case overhead analysis provides a means for estimating the extra checkpoint overhead. Communication trace-driven simulation for several parallel programs is used to evaluate the benefits of the proposed scheme for real applications.

  5. Role of the promoter in the sensitivity of human thymidine kinase to lack of Zn2+.

    PubMed Central

    Chesters, J K; Boyne, R; Petrie, L; Lipson, K E

    1995-01-01

    Previous studies had indicated that lack of Zn2+ inhibits the expression of thymidine kinase activity and produces a corresponding reduction in the concentration of its mRNA. The present investigations have shown that with human thymidine kinase this is associated with increased binding of a specific protein to the gene's promoter in the region between -55 and -83 bp 5' to the transcription initiation site. A second binding site for the protein is present within the sixth exon of the human thymidine kinase gene. Images Figure 1 Figure 4 Figure 5 Figure 6 PMID:7772056

  6. A Pleiotropic RNA-Binding Protein Controls Distinct Cell Cycle Checkpoints to Drive Resistance of p53-Defective Tumors to Chemotherapy.

    PubMed

    Cannell, Ian G; Merrick, Karl A; Morandell, Sandra; Zhu, Chang-Qi; Braun, Christian J; Grant, Robert A; Cameron, Eleanor R; Tsao, Ming-Sound; Hemann, Michael T; Yaffe, Michael B

    2015-11-09

    In normal cells, p53 is activated by DNA damage checkpoint kinases to simultaneously control the G1/S and G2/M cell cycle checkpoints through transcriptional induction of p21(cip1) and Gadd45α. In p53-mutant tumors, cell cycle checkpoints are rewired, leading to dependency on the p38/MK2 pathway to survive DNA-damaging chemotherapy. Here we show that the RNA binding protein hnRNPA0 is the "successor" to p53 for checkpoint control. Like p53, hnRNPA0 is activated by a checkpoint kinase (MK2) and simultaneously controls both cell cycle checkpoints through distinct target mRNAs, but unlike p53, this is through the post-transcriptional stabilization of p27(Kip1) and Gadd45α mRNAs. This pathway drives cisplatin resistance in lung cancer, demonstrating the importance of post-transcriptional RNA control to chemotherapy response.

  7. Activity and regulation by growth factors of calmodulin-dependent protein kinase III (elongation factor 2-kinase) in human breast cancer

    PubMed Central

    Parmer, T G; Ward, M D; Yurkow, E J; Vyas, V H; Kearney, T J; Hait, W N

    1999-01-01

    Calmodulin-dependent protein kinase III (CaM kinase III, elongation factor-2 kinase) is a unique member of the Ca2+/CaM-dependent protein kinase family. Activation of CaM kinase III leads to the selective phosphorylation of elongation factor 2 (eEF-2) and transient inhibition of protein synthesis. Recent cloning and sequencing of CaM kinase III revealed that this enzyme represents a new superfamily of protein kinases. The activity of CaM kinase III is selectively activated in proliferating cells; inhibition of the kinase blocked cells in G0/G1-S and decreased viability. To determine the significance of CaM kinase III in breast cancer, we measured the activity of the kinase in human breast cancer cell lines as well as in fresh surgical specimens. The specific activity of CaM kinase III in human breast cancer cell lines was equal to or greater than that seen in a variety of cell lines with similar rates of proliferation. The specific activity of CaM kinase III was markedly increased in human breast tumour specimens compared with that of normal adjacent breast tissue. The activity of this enzyme was regulated by breast cancer mitogens. In serum-deprived MDA-MB-231 cells, the combination of insulin-like growth factor I (IGF-I) and epidermal growth factor (EGF) stimulated cell proliferation and activated CaM kinase III to activities observed in the presence of 10% serum. Inhibition of enzyme activity blocked cell proliferation induced by growth factors. In MCF-7 cells separated by fluorescence-activated cell sorting, CaM kinase III was increased in S-phase over that of other phases of the cell cycle. In summary, the activity of Ca2+/CaM-dependent protein kinase III is controlled by breast cancer mitogens and appears to be constitutively activated in human breast cancer. These results suggest that CaM kinase III may contribute an important link between growth factor/receptor interactions, protein synthesis and the induction of cellular proliferation in human breast

  8. HUMAN KINASES DISPLAY MUTATIONAL HOTSPOTS AT COGNATE POSITIONS WITHIN CANCER.

    PubMed

    Gallion, Jonathan; Wilkins, Angela D; Lichtarge, Olivier

    2016-01-01

    The discovery of driver genes is a major pursuit of cancer genomics, usually based on observing the same mutation in different patients. But the heterogeneity of cancer pathways plus the high background mutational frequency of tumor cells often cloud the distinction between less frequent drivers and innocent passenger mutations. Here, to overcome these disadvantages, we grouped together mutations from close kinase paralogs under the hypothesis that cognate mutations may functionally favor cancer cells in similar ways. Indeed, we find that kinase paralogs often bear mutations to the same substituted amino acid at the same aligned positions and with a large predicted Evolutionary Action. Functionally, these high Evolutionary Action, non-random mutations affect known kinase motifs, but strikingly, they do so differently among different kinase types and cancers, consistent with differences in selective pressures. Taken together, these results suggest that cancer pathways may flexibly distribute a dependence on a given functional mutation among multiple close kinase paralogs. The recognition of this "mutational delocalization" of cancer drivers among groups of paralogs is a new phenomena that may help better identify relevant mechanisms and therefore eventually guide personalized therapy.

  9. Apoptosis and melanogenesis in human melanoma cells induced by anthrax lethal factor inactivation of mitogen-activated protein kinase kinase

    NASA Astrophysics Data System (ADS)

    Koo, Han-Mo; Vanbrocklin, Matt; McWilliams, Mary Jane; Leppla, Stephan H.; Duesbery, Nicholas S.; Vande Woude, George F.

    2002-03-01

    Lethal factor, the principal virulence factor of Bacillus anthracis, inhibits mitogen-activated protein kinase (MAPK) signaling by proteolytically cleaving MAPK kinases. Edema factor, another component of anthrax toxin, is an adenylate cyclase, which increases intracellular cAMP. Inhibition of MAPK signaling with either anthrax lethal toxin (LeTx) or small molecule MAPK kinase inhibitors triggers apoptosis in human melanoma cells. Normal melanocytes do not undergo apoptosis in response to MAPK inhibition but arrest in the G1 phase of the cell cycle. Importantly, in vivo treatment of human melanoma xenograft tumors in athymic nude mice with LeTx results in significant or complete tumor regression without apparent side effects, suggesting that inhibiting the MAPK signaling pathway may be a useful strategy for treating melanoma. Additionally, interrupting MAPK signaling with LeTx and elevating cAMP with anthrax edema toxin in both melanoma cells and melanocytes lead to dramatic melanin production, perhaps explaining the formation of blackened eschars in cutaneous anthrax.

  10. Simultaneous inhibition assay for human and microbial kinases via MALDI-MS/MS.

    PubMed

    Smith, Anne Marie E; Brennan, John D

    2014-03-03

    Selective inhibition of one kinase over another is a critical issue in drug development. For antimicrobial development, it is particularly important to selectively inhibit bacterial kinases, which can phosphorylate antimicrobial compounds such as aminoglycosides, without affecting human kinases. Previous work from our group showed the development of a MALDI-MS/MS assay for the detection of small molecule modulators of the bacterial aminoglycoside kinase APH3'IIIa. Herein, we demonstrate the development of an enhanced kinase MALDI-MS/MS assay involving simultaneous assaying of two kinase reactions, one for APH3'IIIa, and the other for human protein kinase A (PKA), which leads to an output that provides direct information on selectivity and mechanism of action. Specificity of the respective enzyme substrates were verified, and the assay was validated through generation of Z'-factors of 0.55 for APH3'IIIa with kanamycin and 0.60 for PKA with kemptide. The assay was used to simultaneously screen a kinase-directed library of mixtures of ten compounds each against both enzymes, leading to the identification of selective inhibitors for each enzyme as well as one non-selective inhibitor following mixture deconvolution.

  11. Identification of a novel EGF-sensitive cell cycle checkpoint

    SciTech Connect

    Walker, Francesca . E-mail: francesca.walker@ludwig.edu.au; Zhang Huihua; Burgess, Antony W.

    2007-02-01

    The site of action of growth factors on mammalian cell cycle has been assigned to the boundary between the G1 and S phases. We show here that Epidermal Growth Factor (EGF) is also required for mitosis. BaF/3 cells expressing the EGFR (BaF/wtEGFR) synthesize DNA in response to EGF, but arrest in S-phase. We have generated a cell line (BaF/ERX) with defective downregulation of the EGFR and sustained activation of EGFR signalling pathways: these cells undergo mitosis in an EGF-dependent manner. The transit of BaF/ERX cells through G2/M strictly requires activation of EGFR and is abolished by AG1478. This phenotype is mimicked by co-expression of ErbB2 in BaF/wtEGFR cells, and abolished by inhibition of the EGFR kinase, suggesting that sustained signalling of the EGFR, through impaired downregulation of the EGFR or heterodimerization, is required for completion of the cycle. We have confirmed the role of EGFR signalling in the G2/M phase of the cell cycle using a human tumor cell line which overexpresses the EGFR and is dependent on EGFR signalling for growth. These findings unmask an EGF-sensitive checkpoint, helping to understand the link between sustained EGFR signalling, proliferation and the acquisition of a radioresistant phenotype in cancer cells.

  12. Universal quantitative kinase assay based on diagonal SCX chromatography and stable isotope dimethyl labeling provides high-definition kinase consensus motifs for PKA and human Mps1.

    PubMed

    Hennrich, Marco L; Marino, Fabio; Groenewold, Vincent; Kops, Geert J P L; Mohammed, Shabaz; Heck, Albert J R

    2013-05-03

    In order to understand cellular signaling, a clear understanding of kinase-substrate relationships is essential. Some of these relationships are defined by consensus recognition motifs present in substrates making them amendable for phosphorylation by designated kinases. Here, we explore a method that is based on two sequential steps of strong cation exchange chromatography combined with differential stable isotope labeling, to define kinase consensus motifs with high accuracy. We demonstrate the value of our method by evaluating the motifs of two very distinct kinases: cAMP regulated protein kinase A (PKA) and human monopolar spindle 1 (Mps1) kinase, also known as TTK. PKA is a well-studied basophilic kinase with a relatively well-defined motif and numerous known substrates in vitro and in vivo. Mps1, a kinase involved in chromosome segregation, has been less well characterized. Its substrate specificity is unclear and here we show that Mps1 is an acidophilic kinase with a striking tendency for phosphorylation of threonines. The final outcomes of our work are high-definition kinase consensus motifs for PKA and Mps1. Our generic method, which makes use of proteolytic cell lysates as a source for peptide-substrate libraries, can be implemented for any kinase present in the kinome.

  13. Recovery from the DNA Replication Checkpoint.

    PubMed

    Chaudhury, Indrajit; Koepp, Deanna M

    2016-10-28

    Checkpoint recovery is integral to a successful checkpoint response. Checkpoint pathways monitor progress during cell division so that in the event of an error, the checkpoint is activated to block the cell cycle and activate repair pathways. Intrinsic to this process is that once repair has been achieved, the checkpoint signaling pathway is inactivated and cell cycle progression resumes. We use the term "checkpoint recovery" to describe the pathways responsible for the inactivation of checkpoint signaling and cell cycle re-entry after the initial stress has been alleviated. The DNA replication or S-phase checkpoint monitors the integrity of DNA synthesis. When replication stress is encountered, replication forks are stalled, and the checkpoint signaling pathway is activated. Central to recovery from the S-phase checkpoint is the restart of stalled replication forks. If checkpoint recovery fails, stalled forks may become unstable and lead to DNA breaks or unusual DNA structures that are difficult to resolve, causing genomic instability. Alternatively, if cell cycle resumption mechanisms become uncoupled from checkpoint inactivation, cells with under-replicated DNA might proceed through the cell cycle, also diminishing genomic stability. In this review, we discuss the molecular mechanisms that contribute to inactivation of the S-phase checkpoint signaling pathway and the restart of replication forks during recovery from replication stress.

  14. Recovery from the DNA Replication Checkpoint

    PubMed Central

    Chaudhury, Indrajit; Koepp, Deanna M.

    2016-01-01

    Checkpoint recovery is integral to a successful checkpoint response. Checkpoint pathways monitor progress during cell division so that in the event of an error, the checkpoint is activated to block the cell cycle and activate repair pathways. Intrinsic to this process is that once repair has been achieved, the checkpoint signaling pathway is inactivated and cell cycle progression resumes. We use the term “checkpoint recovery” to describe the pathways responsible for the inactivation of checkpoint signaling and cell cycle re-entry after the initial stress has been alleviated. The DNA replication or S-phase checkpoint monitors the integrity of DNA synthesis. When replication stress is encountered, replication forks are stalled, and the checkpoint signaling pathway is activated. Central to recovery from the S-phase checkpoint is the restart of stalled replication forks. If checkpoint recovery fails, stalled forks may become unstable and lead to DNA breaks or unusual DNA structures that are difficult to resolve, causing genomic instability. Alternatively, if cell cycle resumption mechanisms become uncoupled from checkpoint inactivation, cells with under-replicated DNA might proceed through the cell cycle, also diminishing genomic stability. In this review, we discuss the molecular mechanisms that contribute to inactivation of the S-phase checkpoint signaling pathway and the restart of replication forks during recovery from replication stress. PMID:27801838

  15. Characterization and chromosomal localization of the gene for human rhodopsin kinase

    SciTech Connect

    Khani, S.C.; Yamamoto, S.; Dryja, T.P.

    1996-08-01

    G-protein-dependent receptor kinases (GRKs) play a key role in the adapatation of receptors to persistent stimuli. In rod photoreceptors rhodopsin kinase (RK) mediates rapid densensitization of rod photoreceptors to light by catalyzing phosphorylation of the visual pigment rhodopsin. To study the structure and mechanism of FRKs in human photoreceptors, we have isolated and characterized cDNA and genomic clones derived from the human RK locus using a bovine rhodopsin kinase cDNA fragment as a probe. The RK locus, assigned to chromosome 13 band q34, is composed of seven exons that encode a protein 92% identical in amino acid sequence to bovine rhodopsin kinase. The marked difference between the structure of this gene and that of another recently clone human GRK gene suggests the existence of a wide evolutionary gap between members of the GRK gene family. 39 refs., 3 figs.

  16. New checkpoints in cancer immunotherapy.

    PubMed

    Ni, Ling; Dong, Chen

    2017-03-01

    Immune responses must be fine-tuned to allow effective clearance of invading pathogens, while maintain tolerance to self-antigens. T cells are the major effector cells for fighting and killing tumor cells. Immune checkpoints play a pivotal role in T cell activation, and determine the functional outcome of T cell receptor (TCR) signaling. The blockade of immune checkpoints CTLA-4 and PD-1 has already been one of the most successful cancer immunotherapies. In this review, we will focus on three novel inhibitory B7 family checkpoint molecules, B7-H3, B7S1 and VISTA. The aim of this article is to summarize their expressions in tumors as well as their roles in controlling and suppressing T cell immune responses and anti-tumor immunity. These pathways may be explored in future cancer immunotherapy.

  17. Disease severity in a mouse model of ataxia telangiectasia is modulated by the DNA damage checkpoint gene Hus1

    PubMed Central

    Balmus, Gabriel; Zhu, Min; Mukherjee, Sucheta; Lyndaker, Amy M.; Hume, Kelly R.; Lee, Jaesung; Riccio, Mark L.; Reeves, Anthony P.; Sutter, Nathan B.; Noden, Drew M.; Peters, Rachel M.; Weiss, Robert S.

    2012-01-01

    The human genomic instability syndrome ataxia telangiectasia (A-T), caused by mutations in the gene encoding the DNA damage checkpoint kinase ATM, is characterized by multisystem defects including neurodegeneration, immunodeficiency and increased cancer predisposition. ATM is central to a pathway that responds to double-strand DNA breaks, whereas the related kinase ATR leads a parallel signaling cascade that is activated by replication stress. To dissect the physiological relationship between the ATM and ATR pathways, we generated mice defective for both. Because complete ATR pathway inactivation causes embryonic lethality, we weakened the ATR mechanism to different degrees by impairing HUS1, a member of the 911 complex that is required for efficient ATR signaling. Notably, simultaneous ATM and HUS1 defects caused synthetic lethality. Atm/Hus1 double-mutant embryos showed widespread apoptosis and died mid-gestationally. Despite the underlying DNA damage checkpoint defects, increased DNA damage signaling was observed, as evidenced by H2AX phosphorylation and p53 accumulation. A less severe Hus1 defect together with Atm loss resulted in partial embryonic lethality, with the surviving double-mutant mice showing synergistic increases in genomic instability and specific developmental defects, including dwarfism, craniofacial abnormalities and brachymesophalangy, phenotypes that are observed in several human genomic instability disorders. In addition to identifying tissue-specific consequences of checkpoint dysfunction, these data highlight a robust, cooperative configuration for the mammalian DNA damage response network and further suggest HUS1 and related genes in the ATR pathway as candidate modifiers of disease severity in A-T patients. PMID:22575700

  18. Tyrosine phosphorylation on spleen tyrosine kinase (Syk) is differentially regulated in human and murine platelets by protein kinase C isoforms.

    PubMed

    Buitrago, Lorena; Bhavanasi, Dheeraj; Dangelmaier, Carol; Manne, Bhanu Kanth; Badolia, Rachit; Borgognone, Alessandra; Tsygankov, Alexander Y; McKenzie, Steven E; Kunapuli, Satya P

    2013-10-04

    Protein kinase C (PKC) isoforms differentially regulate platelet functional responses downstream of glycoprotein VI (GPVI) signaling, but the role of PKCs regulating upstream effectors such as Syk is not known. We investigated the role of PKC on Syk tyrosine phosphorylation using the pan-PKC inhibitor GF109203X (GFX). GPVI-mediated phosphorylation on Syk Tyr-323, Tyr-352, and Tyr-525/526 was rapidly dephosphorylated, but GFX treatment inhibited this dephosphorylation on Tyr-525/526 in human platelets but not in wild type murine platelets. GFX treatment did not affect tyrosine phosphorylation on FcRγ chain or Src family kinases. Phosphorylation of Lat Tyr-191 and PLCγ2 Tyr-759 was also increased upon treatment with GFX. We evaluated whether secreted ADP is required for such dephosphorylation. Exogenous addition of ADP to GFX-treated platelets did not affect tyrosine phosphorylation on Syk. FcγRIIA- or CLEC-2-mediated Syk tyrosine phosphorylation was also potentiated with GFX in human platelets. Because potentiation of Syk phosphorylation is not observed in murine platelets, PKC-deficient mice cannot be used to identify the PKC isoform regulating Syk phosphorylation. We therefore used selective inhibitors of PKC isoforms. Only PKCβ inhibition resulted in Syk hyperphosphorylation similar to that in platelets treated with GFX. This result indicates that PKCβ is the isoform responsible for Syk negative regulation in human platelets. In conclusion, we have elucidated a novel pathway of Syk regulation by PKCβ in human platelets.

  19. Cell Cycle Regulation by Checkpoints

    PubMed Central

    Barnum, Kevin J.; O’Connell, Matthew J.

    2016-01-01

    Cell cycle checkpoints are surveillance mechanisms that monitor the order, integrity, and fidelity of the major events of the cell cycle. These include growth to the appropriate cell size, the replication and integrity of the chromosomes, and their accurate segregation at mitosis. Many of these mechanisms are ancient in origin and highly conserved, and hence have been heavily informed by studies in simple organisms such as the yeasts. Others have evolved in higher organisms, and control alternative cell fates with significant impact on tumor suppression. Here, we consider these different checkpoint pathways and the consequences of their dysfunction on cell fate. PMID:24906307

  20. Cell cycle regulation by checkpoints.

    PubMed

    Barnum, Kevin J; O'Connell, Matthew J

    2014-01-01

    Cell cycle checkpoints are surveillance mechanisms that monitor the order, integrity, and fidelity of the major events of the cell cycle. These include growth to the appropriate cell size, the replication and integrity of the chromosomes, and their accurate segregation at mitosis. Many of these mechanisms are ancient in origin and highly conserved, and hence have been heavily informed by studies in simple organisms such as the yeasts. Others have evolved in higher organisms, and control alternative cell fates with significant impact on tumor suppression. Here, we consider these different checkpoint pathways and the consequences of their dysfunction on cell fate.

  1. Tumor-initiating cells contribute to radiation resistance in primary human renal clear cell carcinomas by activating the DNA damage checkpoint response.

    PubMed

    Li, Bo; Gao, Yong-Ju; Wu, Xin-Yu; Cui, Jing; Long, Ye; Xu, Jun-Ling; Ding, De-Gang

    2017-09-01

    The use of radiotherapy in patients with clear cell renal carcinoma (ccRCC) is predominantly limited to palliation of metastases or control of local growth, because ccRCC cells readily develop radioresistance. The mechanisms underlying ccRCC resistance remain elusive. The present study demonstrated that ccRCC cells that survive fractionated radiation treatment display tumor-initiating cell (TIC) characteristics, such as high self-renewal and tumorigenic capacities, and overexpress stemness genes. ccRCC cells that survived fractionated radiation exhibited increased activation of the DNA damage checkpoint response and G2/M phase arrest compared with sham-irradiated cells. The results of the present study suggest that ionizing radiation destroys the bulk of tumor cells within ccRCC, but spares TICs; this subpopulation confers ccRCC radioresistance and may cause tumor recurrence or relapse following radiotherapy. Furthermore, these findings indicate that the DNA damage checkpoint response may serve as a potential therapeutic target for overcoming resistance of TICs in patients with ccRCC.

  2. Network support for system initiated checkpoints

    DOEpatents

    Chen, Dong; Heidelberger, Philip

    2013-01-29

    A system, method and computer program product for supporting system initiated checkpoints in parallel computing systems. The system and method generates selective control signals to perform checkpointing of system related data in presence of messaging activity associated with a user application running at the node. The checkpointing is initiated by the system such that checkpoint data of a plurality of network nodes may be obtained even in the presence of user applications running on highly parallel computers that include ongoing user messaging activity.

  3. Interleukin 1 stimulates phosphatidylinositol kinase activity in human fibroblasts.

    PubMed Central

    Ballou, L R; Barker, S C; Postlethwaite, A E; Kang, A H

    1991-01-01

    IL-1 mediates multiple cellular immune and inflammatory responses, but little is known of the intracellular biochemical mechanisms involved in IL-1 actions. We studied the effects of IL-1 on phosphatidylinositol (PtdIns) metabolism and confirmed reports indicating that IL-1 does not stimulate increased PtdIns turnover; however, we observed the accumulation of PtdIns-4-phosphate (PtdInsP) in response to IL-1. Using a fibroblast membrane preparation, we were able to detect stimulated PtdInsP accumulation within 10 s of IL-1 addition. Increased PtdInsP accumulation was due to stimulated PtdIns kinase activity, not the inhibition of PtdInsP hydrolysis by phospholipase(s). PtdIns kinase activity was magnesium dependent, increased as a function of IL-1 concentration, and specifically phosphorylated the D4 position of inositol. Stimulated PtdIns kinase activity could be detected at 10(-12) M IL-1 in fibroblast membranes, a concentration within the physiological range for IL-1 action; half-maximal activity was reached at approximately 10(-10) M IL-1. Heat denaturation of IL-1 or treatment of IL-1 with anti-IL-1 antibody abrogated the IL-1 effect. These findings demonstrate the direct, IL-1-mediated, stimulation of PtdIns kinase. IL-1-stimulated PtdIns kinase activity represents an important physiological regulatory effect by IL-1 as it could control the synthesis and/or maintenance of phosphorylated derivatives of PtdIns which comprise only a very small pool of substrates for the generation of the second messengers inositol 1,4,5-triphosphate and diacylglycerol. PMID:1845871

  4. A first generation inhibitor of human Greatwall kinase, enabled by structural and functional characterisation of a minimal kinase domain construct

    PubMed Central

    Ocasio, Cory A.; Rajasekaran, Mohan B.; Walker, Sarah; Le Grand, Darren; Spencer, John; Pearl, Frances M.G.; Ward, Simon E.; Savic, Velibor; Pearl, Laurence H.; Hochegger, Helfrid; Oliver, Antony W.

    2016-01-01

    MASTL (microtubule-associated serine/threonine kinase-like), more commonly known as Greatwall (GWL), has been proposed as a novel cancer therapy target. GWL plays a crucial role in mitotic progression, via its known substrates ENSA/ARPP19, which when phosphorylated inactivate PP2A/B55 phosphatase. When over-expressed in breast cancer, GWL induces oncogenic properties such as transformation and invasiveness. Conversely, down-regulation of GWL selectively sensitises tumour cells to chemotherapy. Here we describe the first structure of the GWL minimal kinase domain and development of a small-molecule inhibitor GKI-1 (Greatwall Kinase Inhibitor-1). In vitro, GKI-1 inhibits full-length human GWL, and shows cellular efficacy. Treatment of HeLa cells with GKI-1 reduces ENSA/ARPP19 phosphorylation levels, such that they are comparable to those obtained by siRNA depletion of GWL; resulting in a decrease in mitotic events, mitotic arrest/cell death and cytokinesis failure. Furthermore, GKI-1 will be a useful starting point for the development of more potent and selective GWL inhibitors. PMID:27563826

  5. Purification, crystallization and preliminary X-ray diffraction analysis of the kinase domain of human tousled-like kinase 2

    PubMed Central

    Garrote, Ana M.; Redondo, Pilar; Montoya, Guillermo; Muñoz, Inés G.

    2014-01-01

    Tousled-like kinases (TLKs) are an evolutionarily conserved family of serine/threonine protein kinases involved in chromatin dynamics, including DNA replication and repair, transcription and chromosome segregation. The two members of the family reported in humans, namely TLK1 and TLK2, localize to the cell nucleus and are capable of forming homo- or hetero-oligomers by themselves. To characterize the role of TLK2, its C-terminal kinase domain was cloned and overexpressed in Escherichia coli followed by purification to homogeneity. Crystallization experiments in the presence of ATP-γ-S yielded crystals suitable for X-ray diffraction analysis belonging to two different space groups: tetragonal I4122 and cubic P213. The latter produced the best diffracting crystal (3.4 Å resolution using synchrotron radiation), with unit-cell parameters a = b = c = 126.05 Å, α = β = γ = 90°. The asymmetric unit contained one protein molecule, with a Matthews coefficient of 4.59 Å3 Da−1 and a solvent content of 73.23%. PMID:24598926

  6. Evidence that Aurora B is implicated in spindle checkpoint signalling independently of error correction.

    PubMed

    Santaguida, Stefano; Vernieri, Claudio; Villa, Fabrizio; Ciliberto, Andrea; Musacchio, Andrea

    2011-04-20

    Fidelity of chromosome segregation is ensured by a tension-dependent error correction system that prevents stabilization of incorrect chromosome-microtubule attachments. Unattached or incorrectly attached chromosomes also activate the spindle assembly checkpoint, thus delaying mitotic exit until all chromosomes are bioriented. The Aurora B kinase is widely recognized as a component of error correction. Conversely, its role in the checkpoint is controversial. Here, we report an analysis of the role of Aurora B in the spindle checkpoint under conditions believed to uncouple the effects of Aurora B inhibition on the checkpoint from those on error correction. Partial inhibition of several checkpoint and kinetochore components, including Mps1 and Ndc80, strongly synergizes with inhibition of Aurora B activity and dramatically affects the ability of cells to arrest in mitosis in the presence of spindle poisons. Thus, Aurora B might contribute to spindle checkpoint signalling independently of error correction. Our results support a model in which Aurora B is at the apex of a signalling pyramid whose sensory apparatus promotes the concomitant activation of error correction and checkpoint signalling pathways.

  7. DNA replication and damage checkpoints and meiotic cell cycle controls in the fission and budding yeasts.

    PubMed Central

    Murakami, H; Nurse, P

    2000-01-01

    The cell cycle checkpoint mechanisms ensure the order of cell cycle events to preserve genomic integrity. Among these, the DNA-replication and DNA-damage checkpoints prevent chromosome segregation when DNA replication is inhibited or DNA is damaged. Recent studies have identified an outline of the regulatory networks for both of these controls, which apparently operate in all eukaryotes. In addition, it appears that these checkpoints have two arrest points, one is just before entry into mitosis and the other is prior to chromosome separation. The former point requires the central cell-cycle regulator Cdc2 kinase, whereas the latter involves several key regulators and substrates of the ubiquitin ligase called the anaphase promoting complex. Linkages between these cell-cycle regulators and several key checkpoint proteins are beginning to emerge. Recent findings on post-translational modifications and protein-protein interactions of the checkpoint proteins provide new insights into the checkpoint responses, although the functional significance of these biochemical properties often remains unclear. We have reviewed the molecular mechanisms acting at the DNA-replication and DNA-damage checkpoints in the fission yeast Schizosaccharomyces pombe, and the modifications of these controls during the meiotic cell cycle. We have made comparisons with the controls in fission yeast and other organisms, mainly the distantly related budding yeast. PMID:10861204

  8. Restricted distribution of the butyrate kinase pathway among butyrate-producing bacteria from the human colon.

    PubMed

    Louis, Petra; Duncan, Sylvia H; McCrae, Sheila I; Millar, Jacqueline; Jackson, Michelle S; Flint, Harry J

    2004-04-01

    The final steps in butyrate synthesis by anaerobic bacteria can occur via butyrate kinase and phosphotransbutyrylase or via butyryl-coenzyme A (CoA):acetate CoA-transferase. Degenerate PCR and enzymatic assays were used to assess the presence of butyrate kinase among 38 anaerobic butyrate-producing bacterial isolates from human feces that represent three different clostridial clusters (IV, XIVa, and XVI). Only four strains were found to possess detectable butyrate kinase activity. These were also the only strains to give PCR products (verifiable by sequencing) with degenerate primer pairs designed within the butyrate kinase gene or between the linked butyrate kinase/phosphotransbutyrylase genes. Further analysis of the butyrate kinase/phosphotransbutyrylase genes of one isolate, L2-50, revealed similar organization to that described previously from different groups of clostridia, along with differences in flanking sequences and phylogenetic relationships. Butyryl-CoA:acetate CoA-transferase activity was detected in all 38 strains examined, suggesting that it, rather than butyrate kinase, provides the dominant route for butyrate formation in the human colonic ecosystem that contains a constantly high concentration of acetate.

  9. Latex of Euphorbia antiquorum-induced S-phase arrest via active ATM kinase and MAPK pathways in human cervical cancer HeLa cells.

    PubMed

    Hsieh, Wen-Tsong; Lin, Hui-Yi; Chen, Jou-Hsuan; Lin, Wen-Chung; Kuo, Yueh-Hsiung; Wood, W Gibson; Lu, Hsu-Feng; Chung, Jing-Gung

    2015-09-01

    Latex of Euphorbia antiquorum (EA) has demonstrated great chemotherapeutic potential for cancer. However, the mechanisms of anti-proliferation of EA on cancer cell remain to be further investigated. The purpose of this study was to explore the influence of EA in human cervical cancer cells. Here, the cell cycle distribution by flow cytometry was examined and the protein expression by the western blotting methods was analyzed. From the cytometric results it was shown that EA-induced S-phase arrest in a concentration manner both in human cervical cancer HeLa and CaSki cells. According the western blot results it was illustrated that EA could downregulate early cyclin E1-Cdk2; and cyclin A-Cdc2 provides a significant additional quantity of S-phase promotion, that in turn promoted the expression of p21(waf1/cip1) and p27(kip1) which were the inhibitors in the complex of cyclin A and Cdc2 that led to cell cycle arrest. Moreover, EA promoted the activation of ataxia telangiectasia mutated (ATM) and check-point kinase-2 (Chk2); however, it negatively regulated the expression of Topoisomerases I and II, Cdc25A, and Cdc25C signaling. Caffeine, an ATM/ATR inhibitor significantly reversed EA downregulation in the levels of Cdc25A. Furthermore, JNK inhibitor SP600125 and p38 MAPK inhibitor SB203580 both could reverse the EA upregulation of the protein of Chk2 level, significantly. This study, therefore, revealed that EA could downregulate topoisomerase, and activate ATM kinase, which then induce parallel Chk 1/2 and MAPK signaling pathways to promote the degradation of Cdc25A to induced S-phase arrest in human cervical cancer HeLa cells.

  10. Replication Checkpoint: Tuning and Coordination of Replication Forks in S Phase

    PubMed Central

    Hustedt, Nicole; Gasser, Susan M.; Shimada, Kenji

    2013-01-01

    Checkpoints monitor critical cell cycle events such as chromosome duplication and segregation. They are highly conserved mechanisms that prevent progression into the next phase of the cell cycle when cells are unable to accomplish the previous event properly. During S phase, cells also provide a surveillance mechanism called the DNA replication checkpoint, which consists of a conserved kinase cascade that is provoked by insults that block or slow down replication forks. The DNA replication checkpoint is crucial for maintaining genome stability, because replication forks become vulnerable to collapse when they encounter obstacles such as nucleotide adducts, nicks, RNA-DNA hybrids, or stable protein-DNA complexes. These can be exogenously induced or can arise from endogenous cellular activity. Here, we summarize the initiation and transduction of the replication checkpoint as well as its targets, which coordinate cell cycle events and DNA replication fork stability. PMID:24705211

  11. An ATM-independent S-phase checkpoint response involves CHK1 pathway

    NASA Technical Reports Server (NTRS)

    Zhou, Xiang-Yang; Wang, Xiang; Hu, Baocheng; Guan, Jun; Iliakis, George; Wang, Ya

    2002-01-01

    After exposure to genotoxic stress, proliferating cells actively slow down the DNA replication through a S-phase checkpoint to provide time for repair. We report that in addition to the ataxia-telangiectasia mutated (ATM)-dependent pathway that controls the fast response, there is an ATM-independent pathway that controls the slow response to regulate the S-phase checkpoint after ionizing radiation in mammalian cells. The slow response of S-phase checkpoint, which is resistant to wortmannin, sensitive to caffeine and UCN-01, and related to cyclin-dependent kinase phosphorylation, is much stronger in CHK1 overexpressed cells, and it could be abolished by Chk1 antisense oligonucleotides. These results provide evidence that the ATM-independent slow response of S-phase checkpoint involves CHK1 pathway.

  12. An ATM-independent S-phase checkpoint response involves CHK1 pathway

    NASA Technical Reports Server (NTRS)

    Zhou, Xiang-Yang; Wang, Xiang; Hu, Baocheng; Guan, Jun; Iliakis, George; Wang, Ya

    2002-01-01

    After exposure to genotoxic stress, proliferating cells actively slow down the DNA replication through a S-phase checkpoint to provide time for repair. We report that in addition to the ataxia-telangiectasia mutated (ATM)-dependent pathway that controls the fast response, there is an ATM-independent pathway that controls the slow response to regulate the S-phase checkpoint after ionizing radiation in mammalian cells. The slow response of S-phase checkpoint, which is resistant to wortmannin, sensitive to caffeine and UCN-01, and related to cyclin-dependent kinase phosphorylation, is much stronger in CHK1 overexpressed cells, and it could be abolished by Chk1 antisense oligonucleotides. These results provide evidence that the ATM-independent slow response of S-phase checkpoint involves CHK1 pathway.

  13. Analysis of the Functionality of the Mitotic Checkpoints.

    PubMed

    Fraschini, Roberta

    2017-01-01

    During cell division the main goal of the cell is to produce two daughter cells with the same genome as the mother, i.e., maintain its genetic stability. Since this issue is essential to preserve the cell ability to proliferate properly, all eukaryotic cells have developed several pathways, called mitotic checkpoints, that regulate mitotic entry, progression, and exit in response to different cellular signals. Given the evolutive conservation of mechanisms and proteins involved in the cell cycle control from yeast to humans, the budding yeast S. cerevisiae has been very helpful to gain insight in these complex regulations. Here, we describe how the checkpoint can be activated and which cellular phenotypes can be used as markers of checkpoint activation.

  14. Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

    PubMed

    Bretaña, Neil Arvin; Lu, Cheng-Tsung; Chiang, Chiu-Yun; Su, Min-Gang; Huang, Kai-Yao; Lee, Tzong-Yi; Weng, Shun-Long

    2012-01-01

    Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD) is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase-specific phosphorylation site

  15. NcoI and TaqI RFLPs for human M creatine kinase (CKM)

    SciTech Connect

    Perryman, M.B.; Hejtmancik, J.F.; Ashizawa, Tetsuo; Armstrong, R.; Lin, Sunchiang; Roberts, R.; Epstein, H.F. )

    1988-09-12

    Probe pHMCKUT contains a 135 bp cDNA fragment inserted into pGEM 3. The probe corresponds to nucleotides 1,201 to 1,336 located in the 3{prime} untranslated region of human M creatine kinase. The probe is specific for human M creatine kinase and does not hybridize to human B cretine kinase sequences. NcoI identifies a two allele polymorphism of a band at either 2.5 kb or 3.6 kb. TaqI identifies a two allele polymorphism at either 3.8 kb or 4.5 kb. Human M creatine has been localized to chromosome 19q. Autosomal co-dominant inheritance was shown in six informative Caucasian families.

  16. Mutational analysis of the kinase domain of MYLK2 gene in common human cancers.

    PubMed

    Soung, Young Hwa; Lee, Jong Woo; Kim, Su Young; Nam, Suk Woo; Park, Won Sang; Lee, Jung Young; Yoo, Nam Jin; Lee, Sug Hyung

    2006-01-01

    Genetic alterations of the genes encoding protein kinases have been implicated in the development of human cancers. Myosin light chain kinase 2, skeletal muscle (MYLK2) encodes a calcium/calmodulin-dependent serine/threonine kinase. In a recent study, MYLK2 gene was somatically mutated in colorectal carcinomas. The aim of this study was to explore the possibility that other common human carcinomas besides colorectal carcinomas harbored MYLK2 mutations in the kinase domain. We analyzed exons 6 and 7 eccoding the kinase domain of MYLK2 for somatic mutations in 60 gastric, 104 colorectal, 79 non-small cell lung, and 54 breast cancers using a polymerase chain reaction (PCR)-based single-strand conformation polymorphism (SSCP). We found one MYLK2 mutation in lung adenocarcinomas, but not in other cancers. The MYLK2 mutation detected was a missense mutation that would substitute an amino acid (E374D) However, there was no somatic mutation of the MYLK2 gene. These data suggest that the kinase domain of MYLK2 is rarely mutated in common human carcinomas and that it does not play a dominant role in cancer pathogenesis.

  17. Non-volatile memory for checkpoint storage

    SciTech Connect

    Blumrich, Matthias A.; Chen, Dong; Cipolla, Thomas M.; Coteus, Paul W.; Gara, Alan; Heidelberger, Philip; Jeanson, Mark J.; Kopcsay, Gerard V.; Ohmacht, Martin; Takken, Todd E.

    2014-07-22

    A system, method and computer program product for supporting system initiated checkpoints in high performance parallel computing systems and storing of checkpoint data to a non-volatile memory storage device. The system and method generates selective control signals to perform checkpointing of system related data in presence of messaging activity associated with a user application running at the node. The checkpointing is initiated by the system such that checkpoint data of a plurality of network nodes may be obtained even in the presence of user applications running on highly parallel computers that include ongoing user messaging activity. In one embodiment, the non-volatile memory is a pluggable flash memory card.

  18. Checkpointing for a hybrid computing node

    DOEpatents

    Cher, Chen-Yong

    2016-03-08

    According to an aspect, a method for checkpointing in a hybrid computing node includes executing a task in a processing accelerator of the hybrid computing node. A checkpoint is created in a local memory of the processing accelerator. The checkpoint includes state data to restart execution of the task in the processing accelerator upon a restart operation. Execution of the task is resumed in the processing accelerator after creating the checkpoint. The state data of the checkpoint are transferred from the processing accelerator to a main processor of the hybrid computing node while the processing accelerator is executing the task.

  19. Synthetic Physical Interactions Map Kinetochore-Checkpoint Activation Regions

    PubMed Central

    Ólafsson, Guðjón; Thorpe, Peter H.

    2016-01-01

    The spindle assembly checkpoint (SAC) is a key mechanism to regulate the timing of mitosis and ensure that chromosomes are correctly segregated to daughter cells. The recruitment of the Mad1 and Mad2 proteins to the kinetochore is normally necessary for SAC activation. This recruitment is coordinated by the SAC kinase Mps1, which phosphorylates residues at the kinetochore to facilitate binding of Bub1, Bub3, Mad1, and Mad2. There is evidence that the essential function of Mps1 is to direct recruitment of Mad1/2. To test this model, we have systematically recruited Mad1, Mad2, and Mps1 to most proteins in the yeast kinetochore, and find that, while Mps1 is sufficient for checkpoint activation, recruitment of either Mad1 or Mad2 is not. These data indicate an important role for Mps1 phosphorylation in SAC activation, beyond the direct recruitment of Mad1 and Mad2. PMID:27280788

  20. Moderate exercise promotes human RBC-NOS activity, NO production and deformability through Akt kinase pathway.

    PubMed

    Suhr, Frank; Brenig, Julian; Müller, Rebecca; Behrens, Hilke; Bloch, Wilhelm; Grau, Marijke

    2012-01-01

    Nitric oxide (NO) produced by nitric oxide synthase (NOS) in human red blood cells (RBCs) was shown to depend on shear stress and to exhibit important biological functions, such as inhibition of platelet activation. In the present study we hypothesized that exercise-induced shear stress stimulates RBC-NOS activation pathways, NO signaling, and deformability of human RBCs. Fifteen male subjects conducted an exercise test with venous blood sampling before and after running on a treadmill for 1 hour. Immunohistochemical staining as well as western blot analysis were used to determine phosphorylation and thus activation of Akt kinase and RBC-NOS as well as accumulation of cyclic guanylyl monophosphate (cGMP) induced by the intervention. The data revealed that activation of NO upstream located enzyme Akt kinase was significantly increased after the test. Phosphorylation of RBC-NOSSer(1177) was also significantly increased after exercise, indicating activation of RBC-NOS through Akt kinase. Total detectable RBC-NOS content and phosphorylation of RBC-NOSThr(495) were not affected by the intervention. NO production by RBCs, determined by DAF fluorometry, and RBC deformability, measured via laser-assisted-optical-rotational red cell analyzer, were also significantly increased after the exercise test. The content of the NO downstream signaling molecule cGMP increased after the test. Pharmacological inhibition of phosphatidylinositol 3 (PI3)-kinase/Akt kinase pathway led to a decrease in RBC-NOS activation, NO production and RBC deformability. This human in vivo study first-time provides strong evidence that exercise-induced shear stress stimuli activate RBC-NOS via the PI3-kinase/Akt kinase pathway. Actively RBC-NOS-produced NO in human RBCs is critical to maintain RBC deformability. Our data gain insights into human RBC-NOS regulation by exercise and, therefore, will stimulate new therapeutic exercise-based approaches for patients with microvascular disorders.

  1. Identification of “Preferred” Human Kinase Inhibitors for Sleeping Sickness Lead Discovery. Are Some Kinases Better than Others for Inhibitor Repurposing?

    PubMed Central

    2016-01-01

    A kinase-targeting cell-based high-throughput screen (HTS) against Trypanosoma brucei was recently reported, and this screening set included the Published Kinase Inhibitor Set (PKIS). From the PKIS was identified 53 compounds with pEC50 ≥ 6. Utilizing the published data available for the PKIS, a statistical analysis of these active antiparasitic compounds was performed, allowing identification of a set of human kinases having inhibitors that show a high likelihood for blocking T. brucei cellular proliferation in vitro. This observation was confirmed by testing other established inhibitors of these human kinases and by mining past screening campaigns at GlaxoSmithKline. Overall, although the parasite targets of action are not known, inhibitors of this set of human kinases displayed an enhanced hit rate relative to a random kinase-targeting HTS campaign, suggesting that repurposing efforts should focus primarily on inhibitors of these specific human kinases. We therefore term this statistical analysis-driven approach “preferred lead repurposing”. PMID:26998514

  2. The equine herpes virus 4 thymidine kinase is a better suicide gene than the human herpes virus 1 thymidine kinase.

    PubMed

    Loubière, L; Tiraby, M; Cazaux, C; Brisson, E; Grisoni, M; Zhao-Emonet, J; Tiraby, G; Klatzmann, D

    1999-09-01

    The herpes simplex virus type 1 thymidine kinase suicide gene (HSV1tk) together with ganciclovir (GCV) have been successfully used for in vivo treatment of various experimental tumors, and many clinical trials using this system have been launched. With the aim to improve this therapeutic system, we compared the potential efficacy of different herpes virus derived thymidine kinases (HSV1, varicella-zoster virus, equine herpes virus type-4 and Epstein-Barr virus) as suicide genes in association with the nucleoside analogs acyclovir, ganciclovir and bromovinyldeoxyur- idine. Using various murine and human cell lines expressing these viral tk, we show that HSV1- and EHV4tk are the more efficient suicide genes for the different nucleoside analogs tested. Moreover, EHV4tk expressing murine and human cells were three- to 12-fold more sensitive to GCV than HSV1tk expressing cells. This was correlated with the presence of five-fold higher amounts of the toxic triphosphated-GCV in EHV4- versus HSV1tk expressing cells. Altogether, these experiments underline the potential advantages of the EHV4tk as a suicide gene.

  3. Targeting Cyclin-Dependent Kinases in Human Cancers: From Small Molecules to Peptide Inhibitors

    PubMed Central

    Peyressatre, Marion; Prével, Camille; Pellerano, Morgan; Morris, May C.

    2015-01-01

    Cyclin-dependent kinases (CDK/Cyclins) form a family of heterodimeric kinases that play central roles in regulation of cell cycle progression, transcription and other major biological processes including neuronal differentiation and metabolism. Constitutive or deregulated hyperactivity of these kinases due to amplification, overexpression or mutation of cyclins or CDK, contributes to proliferation of cancer cells, and aberrant activity of these kinases has been reported in a wide variety of human cancers. These kinases therefore constitute biomarkers of proliferation and attractive pharmacological targets for development of anticancer therapeutics. The structural features of several of these kinases have been elucidated and their molecular mechanisms of regulation characterized in depth, providing clues for development of drugs and inhibitors to disrupt their function. However, like most other kinases, they constitute a challenging class of therapeutic targets due to their highly conserved structural features and ATP-binding pocket. Notwithstanding, several classes of inhibitors have been discovered from natural sources, and small molecule derivatives have been synthesized through rational, structure-guided approaches or identified in high throughput screens. The larger part of these inhibitors target ATP pockets, but a growing number of peptides targeting protein/protein interfaces are being proposed, and a small number of compounds targeting allosteric sites have been reported. PMID:25625291

  4. Small molecule adenosine 5'-monophosphate activated protein kinase (AMPK) modulators and human diseases.

    PubMed

    Rana, Sandeep; Blowers, Elizabeth C; Natarajan, Amarnath

    2015-01-08

    Adenosine 5'-monophosphate activated protein kinase (AMPK) is a master sensor of cellular energy status that plays a key role in the regulation of whole-body energy homeostasis. AMPK is a serine/threonine kinase that is activated by upstream kinases LKB1, CaMKKβ, and Tak1, among others. AMPK exists as αβγ trimeric complexes that are allosterically regulated by AMP, ADP, and ATP. Dysregulation of AMPK has been implicated in a number of metabolic diseases including type 2 diabetes mellitus and obesity. Recent studies have associated roles of AMPK with the development of cancer and neurological disorders, making it a potential therapeutic target to treat human diseases. This review focuses on the structure and function of AMPK, its role in human diseases, and its direct substrates and provides a brief synopsis of key AMPK modulators and their relevance in human diseases.

  5. Inhibition of Src family kinases with dasatinib blocks migration and invasion of human melanoma cells.

    PubMed

    Buettner, Ralf; Mesa, Tania; Vultur, Adina; Lee, Frank; Jove, Richard

    2008-11-01

    Src family kinases (SFK) are involved in regulating a multitude of biological processes, including cell adhesion, migration, proliferation, and survival, depending on the cellular context. Therefore, although SFKs are currently being investigated as potential targets for treatment strategies in various cancers, the biological responses to inhibition of SFK signaling in any given tumor type are not predictable. Dasatinib (BMS-354825) is a dual Src/Abl kinase inhibitor with potent antiproliferative activity against hematologic malignancies harboring activated BCR-ABL. In this study, we show that dasatinib blocks migration and invasion of human melanoma cells without affecting proliferation and survival. Moreover, dasatinib completely inhibits SFK kinase activity at low nanomolar concentrations in all eight human melanoma cell lines investigated. In addition, two known downstream targets of SFKs, focal adhesion kinase and Crk-associated substrate (p130(CAS)), are inhibited with similar concentrations and kinetics. Consistent with inhibition of these signaling pathways and invasion, dasatinib down-regulates expression of matrix metalloproteinase-9. We also provide evidence that dasatinib directly inhibits kinase activity of the EphA2 receptor tyrosine kinase, which is overexpressed and/or overactive in many solid tumors, including melanoma. Thus, SFKs and downstream signaling are implicated as having key roles in migration and invasion of melanoma cells.

  6. Role of Rho kinase signalling in healthy and varicose human saphenous veins

    PubMed Central

    Cario-Toumaniantz, Chrystelle; Evellin, Sandrine; Maury, Séverine; Baron, Olivier; Pacaud, Pierre; Loirand, Gervaise

    2002-01-01

    The present study was performed to determine the role of Rho-Rho kinase signalling pathway in smooth muscle cells from both healthy and varicose human saphenous vein. The Rho kinase inhibitor Y-27632 inhibited the noradrenaline (NA)-induced contraction in human saphenous veins with IC50 corresponding to 0.5 μM and 10.9 μM in control and varicose veins, respectively. The maximal amplitude of the NA-induced contraction was smaller in varicose vein compared to control (1263±172 mg versus 1974±245 mg, P<0.05). In β-escin permeabilized strips, GTPγS induced a rise in tension that was inhibited by Y-27632. The amplitude of the GTPγS-induced contraction was smaller in varicose compared to control veins (23.1±2.4% versus 41.3±2.2%, P<0.002). In smooth muscle cells, Y-27632 induced disassembly of both actin cytoskeleton and extracellular fibronectin matrix. In comparison to control cells, varicose vein smooth muscle cells show decreased actin cytoskeleton organization and reduction of fibronectin matrix deposition. The Rho proteins Rnd1 and RhoA, and Rho kinase 1 are expressed in human saphenous veins. A 2.6 fold reduction of Rho kinase expression was found in varicose veins. These results indicate that RhoA-Rho kinase mediated Ca2+ sensitization of the contraction and regulated actin cytoskeleton and extracellular fibronectin matrix assembly in human saphenous smooth muscle. The decrease of Rho kinase expression and Rho kinase-dependent functions detected in smooth muscle from varicose veins supports a role of this signalling pathway in the functional alterations of the vein wall occurring in the course of the disease. PMID:12208777

  7. Biochemical properties of human pantothenate kinase 2 isoforms and mutations linked to pantothenate kinase-associated neurodegeneration.

    PubMed

    Zhang, Yong-Mei; Rock, Charles O; Jackowski, Suzanne

    2006-01-06

    The PANK2 gene encodes the human pantothenate kinase 2 protein isoforms, and PANK2 mutations are linked to pantothenate kinase-associated neurodegeneration. Two PanK2 protein forms are proteolytically processed to form a mitochondrially localized, mature PanK2. Another isoform arose from a proposed initiation at a leucine codon and was not processed further. The fifth isoform was postulated to arise from an alternative splicing event and was found to encode an inactive protein. Fourteen mutant PanK2 proteins with single amino acid substitutions, associated with either early or late onset disease, were evaluated for activity. The PanK2(G521R), the most frequent mutation in pantothenate kinase-associated neurodegeneration, was devoid of activity and did not fold properly. However, nine of the mutant proteins associated with disease possessed catalytic activities that were indistinguishable from wild type, including the frequently encountered PanK2(T528M) missense mutation. PanK2 was extremely sensitive to feedback inhibition by CoA thioesters (IC50 values between 250 and 500 nM), and the regulation of the active PanK2 mutants was comparable with that of the wild-type protein. Coexpression of the PanK2(G521R) and wild-type PanK2 did not interfere with wild-type enzyme activity, arguing against a dominant negative effect of the PanK2(G521R) mutation in heterozygous patients. These data described the unique biochemical features of the PanK2 isoforms and suggested that catalytic defects may not be the sole cause for the neurodegenerative phenotype.

  8. The Saccharomyces cerevisiae spindle pole body duplication gene MPS1 is part of a mitotic checkpoint

    PubMed Central

    1996-01-01

    M-phase checkpoints inhibit cell division when mitotic spindle function is perturbed. Here we show that the Saccharomyces cerevisiae MPS1 gene product, an essential protein kinase required for spindle pole body (SPB) duplication (Winey et al., 1991; Lauze et al., 1995), is also required for M-phase check-point function. In cdc31-2 and mps2-1 mutants, conditional failure of SPB duplication results in cell cycle arrest with high p34CDC28 kinase activity that depends on the presence of the wild-type MAD1 checkpoint gene, consistent with checkpoint arrest of mitosis. In contrast, mps1 mutant cells fail to duplicate their SPBs and do not arrest division at 37 degrees C, exhibiting a normal cycle of p34CDC28 kinase activity despite the presence of a monopolar spindle. Double mutant cdc31-2, mps1-1 cells also fail to arrest mitosis at 37 degrees C, despite having SPB structures similar to cdc31-2 single mutants as determined by EM analysis. Arrest of mitosis upon microtubule depolymerization by nocodazole is also conditionally absent in mps1 strains. This is observed in mps1 cells synchronized in S phase with hydroxyurea before exposure to nocodazole, indicating that failure of checkpoint function in mps1 cells is independent of SPB duplication failure. In contrast, hydroxyurea arrest and a number of other cdc mutant arrest phenotypes are unaffected by mps1 alleles. We propose that the essential MPS1 protein kinase functions both in SPB duplication and in a mitotic checkpoint monitoring spindle integrity. PMID:8567717

  9. Reaction of human UMP-CMP kinase with natural and analog substrates.

    PubMed

    Pasti, Claudia; Gallois-Montbrun, Sarah; Munier-Lehmann, Hélène; Veron, Michel; Gilles, Anne-Marie; Deville-Bonne, Dominique

    2003-04-01

    UMP-CMP kinase catalyses an important step in the phosphorylation of UTP, CTP and dCTP. It is also involved in the necessary phosphorylation by cellular kinases of nucleoside analogs used in antiviral therapies. The reactivity of human UMP-CMP kinase towards natural substrates and nucleotide analogs was reexamined. The expression of the recombinant enzyme and conditions for stability of the enzyme were improved. Substrate inhibition was observed for UMP and CMP at concentrations higher than 0.2 mm, but not for dCMP. The antiviral analog l-3TCMP was found to be an efficient substrate phosphorylated into l-3TCDP by human UMP-CMP kinase. However, in the reverse reaction, the enzyme did not catalyse the addition of the third phosphate to l-3TCDP, which was rather an inhibitor. By molecular modelling, l-3TCMP was built in the active site of the enzyme from Dictyostelium. Human UMP-CMP kinase has a relaxed enantiospecificity for the nucleoside monophosphate acceptor site, but it is restricted to d-nucleotides at the donor site.

  10. Completing the structural family portrait of the human EphB tyrosine kinase domains

    PubMed Central

    Overman, Ross C; Debreczeni, Judit E; Truman, Caroline M; McAlister, Mark S; Attwood, Teresa K

    2014-01-01

    The EphB receptors have key roles in cell morphology, adhesion, migration and invasion, and their aberrant action has been linked with the development and progression of many different tumor types. Their conflicting expression patterns in cancer tissues, combined with their high sequence and structural identity, present interesting challenges to those seeking to develop selective therapeutic molecules targeting this large receptor family. Here, we present the first structure of the EphB1 tyrosine kinase domain determined by X-ray crystallography to 2.5Å. Our comparative crystalisation analysis of the human EphB family kinases has also yielded new crystal forms of the human EphB2 and EphB4 catalytic domains. Unable to crystallize the wild-type EphB3 kinase domain, we used rational engineering (based on our new structures of EphB1, EphB2, and EphB4) to identify a single point mutation which facilitated its crystallization and structure determination to 2.2 Å. This mutation also improved the soluble recombinant yield of this kinase within Escherichia coli, and increased both its intrinsic stability and catalytic turnover, without affecting its ligand-binding profile. The partial ordering of the activation loop in the EphB3 structure alludes to a potential cis-phosphorylation mechanism for the EphB kinases. With the kinase domain structures of all four catalytically competent human EphB receptors now determined, a picture begins to emerge of possible opportunities to produce EphB isozyme-selective kinase inhibitors for mechanistic studies and therapeutic applications. PMID:24677421

  11. Phenotypic checkpoints regulate neuronal development.

    PubMed

    Ben-Ari, Yehezkel; Spitzer, Nicholas C

    2010-11-01

    Nervous system development proceeds by sequential gene expression mediated by cascades of transcription factors in parallel with sequences of patterned network activity driven by receptors and ion channels. These sequences are cell type- and developmental stage-dependent and modulated by paracrine actions of substances released by neurons and glia. How and to what extent these sequences interact to enable neuronal network development is not understood. Recent evidence demonstrates that CNS development requires intermediate stages of differentiation providing functional feedback that influences gene expression. We suggest that embryonic neuronal functions constitute a series of phenotypic checkpoint signatures; neurons failing to express these functions are delayed or developmentally arrested. Such checkpoints are likely to be a general feature of neuronal development and constitute presymptomatic signatures of neurological disorders when they go awry.

  12. Transcriptional co-factor Transducin beta-like (TBL) 1 acts as a checkpoint in pancreatic cancer malignancy

    PubMed Central

    Stoy, Christian; Sundaram, Aishwarya; Rios Garcia, Marcos; Wang, Xiaoyue; Seibert, Oksana; Zota, Annika; Wendler, Susann; Männle, David; Hinz, Ulf; Sticht, Carsten; Muciek, Maria; Gretz, Norbert; Rose, Adam J; Greiner, Vera; Hofmann, Thomas G; Bauer, Andrea; Hoheisel, Jörg; Berriel Diaz, Mauricio; Gaida, Matthias M; Werner, Jens; Schafmeier, Tobias; Strobel, Oliver; Herzig, Stephan

    2015-01-01

    Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer fatalities in Western societies, characterized by high metastatic potential and resistance to chemotherapy. Critical molecular mechanisms of these phenotypical features still remain unknown, thus hampering the development of effective prognostic and therapeutic measures in PDAC. Here, we show that transcriptional co-factor Transducin beta-like (TBL) 1 was over-expressed in both human and murine PDAC. Inactivation of TBL1 in human and mouse pancreatic cancer cells reduced cellular proliferation and invasiveness, correlating with diminished glucose uptake, glycolytic flux, and oncogenic PI3 kinase signaling which in turn could rescue TBL1 deficiency-dependent phenotypes. TBL1 deficiency both prevented and reversed pancreatic tumor growth, mediated transcriptional PI3 kinase inhibition, and increased chemosensitivity of PDAC cells in vivo. As TBL1 mRNA levels were also found to correlate with PI3 kinase levels and overall survival in a cohort of human PDAC patients, TBL1 was identified as a checkpoint in the malignant behavior of pancreatic cancer and its expression may serve as a novel molecular target in the treatment of human PDAC. PMID:26070712

  13. Affinity-aware checkpoint restart

    DOE PAGES

    Saini, Ajay; Rezaei, Arash; Mueller, Frank; ...

    2014-12-08

    Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core mapsmore » and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.« less

  14. Affinity-aware checkpoint restart

    SciTech Connect

    Saini, Ajay; Rezaei, Arash; Mueller, Frank; Hargrove, Paul; Roman, Eric

    2014-12-08

    Current checkpointing techniques employed to overcome faults for HPC applications result in inferior application performance after restart from a checkpoint for a number of applications. This is due to a lack of page and core affinity awareness of the checkpoint/restart (C/R) mechanism, i.e., application tasks originally pinned to cores may be restarted on different cores, and in case of non-uniform memory architectures (NUMA), quite common today, memory pages associated with tasks on a NUMA node may be associated with a different NUMA node after restart. Here, this work contributes a novel design technique for C/R mechanisms to preserve task-to-core maps and NUMA node specific page affinities across restarts. Experimental results with BLCR, a C/R mechanism, enhanced with affinity awareness demonstrate significant performance benefits of 37%-73% for the NAS Parallel Benchmark codes and 6-12% for NAMD with negligible overheads instead of up to nearly four times longer an execution times without affinity-aware restarts on 16 cores.

  15. Production of recombinant human apoptosis signal-regulating kinase 1 (ASK1) in Escherichia coli.

    PubMed

    Volynets, Galyna P; Gorbatiuk, Oksana B; Kukharenko, Oleksandr P; Usenko, Mariya O; Yarmoluk, Sergiy M

    2016-10-01

    Apoptosis signal-regulating kinase 1 (ASK1) is a mediator of the MAPK signaling cascade, which regulates different cellular processes including apoptosis, cell survival, and differentiation. The increased activity of ASK1 is associated with a number of human diseases and this protein kinase is considered as promising therapeutic target. In the present study, the kinase domain of human ASK1 was expressed in Escherichia coli (E. coli) in soluble form. The expression level of ASK1 was around 0.3-0.47 g per 1 L after using auto-induction protocol or IPTG induction. A one-step on column method for the efficient purification of recombinant ASK1 was performed. Our approach yields sufficient amount of recombinant ASK1, which can be used for inhibitor screening assays and different crystallographic studies. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Structural changes in human cytomegalovirus cytoplasmic assembly sites in the absence of UL97 kinase activity

    SciTech Connect

    Azzeh, Maysa; Honigman, Alik; Taraboulos, Albert; Rouvinski, Alexander; Wolf, Dana G. . E-mail: wolfd@md.huji.ac.il

    2006-10-10

    Studies of human cytomegalovirus (HCMV) UL97 kinase deletion mutant ({delta}UL97) indicated a multi-step role for this kinase in early and late phases of the viral life cycle, namely, in DNA replication, capsid maturation and nuclear egress. Here, we addressed its possible involvement in cytoplasmic steps of HCMV assembly. Using the {delta}UL97 and the UL97 kinase inhibitor NGIC-I, we demonstrate that the absence of UL97 kinase activity results in a modified subcellular distribution of the viral structural protein assembly sites, from compact structures impacting upon the nucleus to diffuse perinuclear structures punctuated by large vacuoles. Infection by either wild type or {delta}UL97 viruses induced a profound reorganization of wheat germ agglutinin (WGA)-positive Golgi-related structures. Importantly, the viral-induced Golgi remodeling along with the reorganization of the nuclear architecture was substantially altered in the absence of UL97 kinase activity. These findings suggest that UL97 kinase activity might contribute to organization of the viral cytoplasmic assembly sites.

  17. Resistance artery creatine kinase mRNA and blood pressure in humans.

    PubMed

    Karamat, Fares A; Oudman, Inge; Ris-Stalpers, Carrie; Afink, Gijs B; Keijser, Remco; Clark, Joseph F; van Montfrans, Gert A; Brewster, Lizzy M

    2014-01-01

    Hypertension remains the main risk factor for cardiovascular death. Environmental and biological factors are known to contribute to the condition, and circulating creatine kinase was reported to be the main predictor of blood pressure in the general population. This was proposed to be because of high resistance artery creatine kinase-BB rapidly regenerating ATP for vascular contractility. Therefore, we assessed whether creatine kinase isoenzyme mRNA levels in human resistance arteries are associated with blood pressure. We isolated resistance-sized arteries from omental fat donated by consecutive women undergoing uterine fibroid surgery. Blood pressure was measured in the sitting position. Vessels of 13 women were included, 6 normotensive and 7 hypertensive, mean age 42.9 years (SE, 1.6) and mean systolic/diastolic blood pressure, 144.8 (8.0)/86.5 (4.3) mm Hg. Arteriolar creatine kinase isoenzyme mRNA was assessed using quantitative real-time polymerase chain reaction. Normalized creatine kinase B mRNA copy numbers, ranging from 5.2 to 24.4 (mean, 15.0; SE, 1.9), showed a near-perfect correlation with diastolic blood pressure (correlation coefficient, 0.9; 95% confidence interval, 0.6-1.0) and were well correlated with systolic blood pressure, with a 90% relative increase in resistance artery creatine kinase B mRNA in hypertensives compared with normotensives, normalized copy numbers were, respectively, 19.3 (SE, 2.0) versus 10.1 (SE, 2.1), P=0.0045. To our knowledge, this is the first direct evidence suggesting that resistance artery creatine kinase mRNA expression levels concur with blood pressure levels, almost doubling with hypertension. These findings add to the evidence that creatine kinase might be involved in the vasculature's pressor responses.

  18. Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus.

    PubMed

    Sonntag, Eric; Milbradt, Jens; Svrlanska, Adriana; Strojan, Hanife; Häge, Sigrun; Kraut, Alexandra; Hesse, Anne-Marie; Amin, Bushra; Sonnewald, Uwe; Couté, Yohann; Marschall, Manfred

    2017-10-01

    Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

  19. Reducing space overhead for independendent checkpointing

    NASA Technical Reports Server (NTRS)

    Wang, Yi-Min; Chung, Pi-Yu; Lin, In-Jen; Fuchs, W. Kent

    1992-01-01

    The main disadvantages of independent checkpointing are the possible domino effect and the associated storage space overhead for maintaining multiple checkpoints. In most previous work, it has been assumed that only the checkpoints older than the current global recovery line can be discarded. Here, we generalize a notion of recovery line to potential recovery line. Only the checkpoints belonging to at least one of the potential recovery lines cannot be discarded. By using the model of maximum-sized antichains on a partially ordered set, an efficient algorithm is developed for finding all non-discardable checkpoints, and we show that the number of non-discardable checkpoints cannot exceed N(N+1)/2, where N is the number of processors. Communication trace driven simulation for several hypercube programs is performed to show the benefit of the proposed algorithm for real applications.

  20. Differential genetic interactions between Sgs1, DNA-damage checkpoint components and DNA repair factors in the maintenance of chromosome stability.

    PubMed

    Doerfler, Lillian; Harris, Lorena; Viebranz, Emilie; Schmidt, Kristina H

    2011-10-31

    Genome instability is associated with human cancers and chromosome breakage syndromes, including Bloom's syndrome, caused by inactivation of BLM helicase. Numerous mutations that lead to genome instability are known, yet how they interact genetically is poorly understood. We show that spontaneous translocations that arise by nonallelic homologous recombination in DNA-damage-checkpoint-defective yeast lacking the BLM-related Sgs1 helicase (sgs1Δ mec3Δ) are inhibited if cells lack Mec1/ATR kinase. Tel1/ATM, in contrast, acts as a suppressor independently of Mec3 and Sgs1. Translocations are also inhibited in cells lacking Dun1 kinase, but not in cells defective in a parallel checkpoint branch defined by Chk1 kinase. While we had previously shown that RAD51 deletion did not inhibit translocation formation, RAD59 deletion led to inhibition comparable to the rad52Δ mutation. A candidate screen of other DNA metabolic factors identified Exo1 as a strong suppressor of chromosomal rearrangements in the sgs1Δ mutant, becoming even more important for chromosomal stability upon MEC3 deletion. We determined that the C-terminal third of Exo1, harboring mismatch repair protein binding sites and phosphorylation sites, is dispensable for Exo1's roles in chromosomal rearrangement suppression, mutation avoidance and resistance to DNA-damaging agents. Our findings suggest that translocations between related genes can form by Rad59-dependent, Rad51-independent homologous recombination, which is independently suppressed by Sgs1, Tel1, Mec3 and Exo1 but promoted by Dun1 and the telomerase-inhibitor Mec1. We propose a model for the functional interaction between mitotic recombination and the DNA-damage checkpoint in the suppression of chromosomal rearrangements in sgs1Δ cells.

  1. Somatic mutations of the ERBB4 kinase domain in human cancers.

    PubMed

    Soung, Young Hwa; Lee, Jong Woo; Kim, Su Young; Wang, Young Pil; Jo, Keon Hyun; Moon, Seok Whan; Park, Won Sang; Nam, Suk Woo; Lee, Jung Young; Yoo, Nam Jin; Lee, Sug Hyung

    2006-03-15

    The EGFR family consists of 4 receptor tyrosine kinases, EGFR (ERBB1), ERBB2 (HER2), ERBB3 (HER3) and ERBB4 (HER4). Recent reports revealed that the kinase domains of both EGFR (ERBB1) and ERBB2 gene were somatically mutated in human cancers, raising the possibility that the other ERBB members possess somatic mutations in human cancers. Here, we performed mutational analysis of the ERBB4 kinase domain by polymerase chain reaction-single-strand conformation polymorphism assay in 595 cancer tissues from stomach, lung, colon and breast. We detected the ERBB4 somatic mutations in 3 of 180 gastric carcinomas (1.7%), 3 of 104 colorectal carcinomas (2.9%), 5 of 217 nonsmall cell lung cancers (2.3%) and 1 of 94 breast carcinomas (1.1%). The 12 ERBB4 mutations consisted of 1 in-frame duplication mutation and 8 missense mutations in the exons, and 3 mutations in the introns. We simultaneously analyzed the somatic mutations of EGFR, ERBB2, K-RAS, PIK3CA and BRAF genes in the 12 samples with the ERBB4 mutations and found that 1 gastric carcinoma with ERBB4 mutation also harbored K-RAS gene mutation. Our study demonstrated that in addition to EGFR and ERBB2, somatic mutation of the kinase domain of ERBB4 occurs in the common human cancers, and suggested that alterations of ERBB4-mediated signaling pathway by ERBB4 mutations may contribute to the development of human cancers.

  2. Autophosphorylation in the Activation Loop Is Required for Full Kinase Activity In Vivo of Human and Yeast Eukaryotic Initiation Factor 2α Kinases PKR and GCN2

    PubMed Central

    Romano, Patrick R.; Garcia-Barrio, Minerva T.; Zhang, Xiaolong; Wang, Qizhi; Taylor, Deborah R.; Zhang, Fan; Herring, Christopher; Mathews, Michael B.; Qin, Jun; Hinnebusch, Alan G.

    1998-01-01

    The human double-stranded RNA-dependent protein kinase (PKR) is an important component of the interferon response to virus infection. The activation of PKR is accompanied by autophosphorylation at multiple sites, including one in the N-terminal regulatory region (Thr-258) that is required for full kinase activity. Several protein kinases are activated by phosphorylation in the region between kinase subdomains VII and VIII, referred to as the activation loop. We show that Thr-446 and Thr-451 in the PKR activation loop are required in vivo and in vitro for high-level kinase activity. Mutation of either residue to Ala impaired translational control by PKR in yeast cells and COS1 cells and led to tumor formation in mice. These mutations also impaired autophosphorylation and eukaryotic initiation factor 2 subunit α (eIF2α) phosphorylation by PKR in vitro. Whereas the Ala-446 substitution substantially reduced PKR function, the mutant kinase containing Ala-451 was completely inactive. PKR specifically phosphorylated Thr-446 and Thr-451 in synthetic peptides in vitro, and mass spectrometry analysis of PKR phosphopeptides confirmed that Thr-446 is an autophosphorylation site in vivo. Substitution of Glu-490 in subdomain X of PKR partially restored kinase activity when combined with the Ala-451 mutation. This finding suggests that the interaction between subdomain X and the activation loop, described previously for MAP kinase, is a regulatory feature conserved in PKR. We found that the yeast eIF2α kinase GCN2 autophosphorylates at Thr-882 and Thr-887, located in the activation loop at exactly the same positions as Thr-446 and Thr-451 in PKR. Thr-887 was more critically required than was Thr-882 for GCN2 kinase activity, paralleling the relative importance of Thr-446 and Thr-451 in PKR. These results indicate striking similarities between GCN2 and PKR in the importance of autophosphorylation and the conserved Thr residues in the activation loop. PMID:9528799

  3. BRIT1/MCPH1 is a DNA damage responsive protein that regulates the Brca1–Chk1 pathway, implicating checkpoint dysfunction in microcephaly

    PubMed Central

    Lin, Shiaw-Yih; Rai, Rekha; Li, Kaiyi; Xu, Zhi-Xiang; Elledge, Stephen J.

    2005-01-01

    BRIT1 [BRCT-repeat inhibitor of hTERT expression], a repressor of human telomerase function, is implicated in cellular immortalization. Here, we find that BRIT1 acts as a regulator of both the intra-S and G2/M checkpoints. When BRIT1 expression is depleted, cells lose the ionizing radiation (IR)-induced cell cycle arrest and become IR sensitive. BRIT1 is a chromatin-associated protein that forms irradiation-induced nuclear foci that colocalize with γ-H2AX foci. BRIT1 is also required for the expression of both BRCA1 and the checkpoint kinase Chk1 and phosphorylation of Nbs1. Thus, the checkpoint defects in the absence of BRIT1 are likely to result from its regulation of Nbs1, BRCA1, and Chk1. BRIT1 is identical to the recently discovered MCPH1 gene, found mutant in patients with primary microcephaly. The ataxia telangiectasia mutated-Rad3 related (ATR)–Chk1 pathway is defective in Seckel syndrome, another microcephaly disorder. We propose that the microcephaly observed in patients with MCPH1 deficiencies is due to disruption of the ATR–BRCA1–Chk1 signaling pathway that is also disrupted in Seckel syndrome patients. PMID:16217032

  4. The Cdc25A is involved in S-phase checkpoint induced by benzo(a)pyrene.

    PubMed

    Yao, Biyun; Fu, Juanling; Hu, Entan; Qi, Yanmin; Zhou, Zongcan

    2007-07-31

    Environmental carcinogen benzo(a)pyrene (BaP) generates electrophilic products BaP diolepoxide (BPDE) that react covalently with genomic DNA. Cells that acquire BaP/BPDE-induced DNA damage undergo S-phase arrest in a p53-independent manner. However, the role of Cdc25A in the BaP/BPDE-induced checkpoint is not clear. In the present study, we investigated the change of checkpoint kinase 1 (Chk1) and Cdc25A in S-phase arrest elicited by BaP. The results indicated that BaP (10microM, with S9 mixture) treatment induced S-phase arrest in both human lung carcinoma A549 cells and human bronchial epithelial cells line 16HBE cells, increasing the proportions of cells in S-phase 19.0% and 21.1%, respectively, at 12h after treatment, compared with DMSO control (p<0.01). Then, the S-phase arrest was weakened after 24h. The level of phorsphorylated Chk1 obviously increased and Cdc25A protein level decreased in both two cell lines after treatment with BaP. The results of RT-PCR indicate Cdc25A mRNA in both A549 cells and 16HBE cells was not changed after BaP treatment 12h, and 24h. The treatment of the proteasome inhibitor MG132 greatly increased Cdc25A protein in abundance. Over all, our results indicated Chk1-Cdc25A checkpoint pathway is involved in BaP-induced S-phase arrest. Moreover, transcription of Cdc25A did not change in BaP induced S-phase arrest, the decrease of Cdc25A level was due to increased degradation through the ubiqutin-proteasome pathway.

  5. Human thrombopoiesis depends on Protein kinase Cδ/protein kinase Cε functional couple

    PubMed Central

    Carubbi, Cecilia; Masselli, Elena; Martini, Silvia; Galli, Daniela; Aversa, Franco; Mirandola, Prisco; Italiano, Joseph E.; Gobbi, Giuliana; Vitale, Marco

    2016-01-01

    A deeper understanding of the molecular events driving megakaryocytopoiesis and thrombopoiesis is essential to regulate in vitro and in vivo platelet production for clinical applications. We previously documented the crucial role of PKCε in the regulation of human and mouse megakaryocyte maturation and platelet release. However, since several data show that different PKC isoforms fulfill complementary functions, we targeted PKCε and PKCδ, which show functional and phenotypical reciprocity, at the same time as boosting platelet production in vitro. Results show that PKCδ, contrary to PKCε, is persistently expressed during megakaryocytic differentiation, and a forced PKCδ down-modulation impairs megakaryocyte maturation and platelet production. PKCδ and PKCε work as a functional couple with opposite roles on thrombopoiesis, and the modulation of their balance strongly impacts platelet production. Indeed, we show an imbalance of PKCδ/PKCε ratio both in primary myelofibrosis and essential thrombocythemia, featured by impaired megakaryocyte differentiation and increased platelet production, respectively. Finally, we demonstrate that concurrent molecular targeting of both PKCδ and PKCε represents a strategy for in vitro platelet factories. PMID:27081176

  6. Immune Checkpoint Modulators: An Emerging Antiglioma Armamentarium

    PubMed Central

    Kim, Eileen S.; Kim, Jennifer E.; Patel, Mira A.; Mangraviti, Antonella; Ruzevick, Jacob; Lim, Michael

    2016-01-01

    Immune checkpoints have come to the forefront of cancer therapies as a powerful and promising strategy to stimulate antitumor T cell activity. Results from recent preclinical and clinical studies demonstrate how checkpoint inhibition can be utilized to prevent tumor immune evasion and both local and systemic immune suppression. This review encompasses the key immune checkpoints that have been found to play a role in tumorigenesis and, more specifically, gliomagenesis. The review will provide an overview of the existing preclinical and clinical data, antitumor efficacy, and clinical applications for each checkpoint with respect to GBM, as well as a summary of combination therapies with chemotherapy and radiation. PMID:26881264

  7. Salinomycin causes migration and invasion of human fibrosarcoma cells by inducing MMP-2 expression via PI3-kinase, ERK-1/2 and p38 kinase pathways.

    PubMed

    Yu, Seon-Mi; Kim, Song Ja

    2016-06-01

    Salinomycin (SAL) is a polyether ionophore antibiotic that has recently been shown to regulate a variety of cellular responses in various human cancer cells. However, the effects of SAL on metastatic capacity of HT1080 human fibrosarcoma cells have not been elucidated. We investigated the effect of SAL on migration and invasion, with emphasis on the expression and activation of matrix metalloproteinase (MMP)-2 in HT1080 human fibrosarcoma cells. Treatment of SAL promoted the expression and activation of MMP-2 in a dose- and time-dependent manner, as detected by western blot analysis, gelatin zymography, and real-time polymerase chain reaction. SAL also increased metastatic capacities, as determined by an increase in the migration and invasion of cells using the wound healing assay and the invasion assay, respectively. To confirm the detailed molecular mechanisms of these effects, we measured the activation of phosphoinositide 3 kinase (PI3-kinase) and mitogen-activated protein kinase (MAPK)s (ERK-1/2 and p38 kinase), as detected by the phosphorylated proteins through western blot analysis. SAL treatment increased the phosphorylation of Akt and MAPKs. Inhibition of PI3-kinase, ERK-1/2, and p38 kinase with LY294002, PD98059, and SB203580, respectively, in the presence of SAL suppressed the metastatic capacity by reducing MMP-2 expression, as determined by gelatin zymography. Our results indicate that the PI3-kinase and MAPK signaling pathways are involved in migration and invasion of HT1080 through induction of MMP-2 expression and activation. In conclusion, SAL significantly increases the metastatic capacity of HT1080 cells by inducing MMP-2 expression via PI3-kinase and MAPK pathways. Our results suggest that SAL may be a potential agent for the study of cancer metastatic capacities.

  8. ERK kinases modulate the activation of PI3 kinase related kinases (PIKKs) in DNA damage response.

    PubMed

    Lin, Xiaozeng; Yan, Judy; Tang, Damu

    2013-12-01

    DNA damage response (DDR) is the critical surveillance mechanism in maintaining genome integrity. The mechanism activates checkpoints to prevent cell cycle progression in the presence of DNA lesions, and mediates lesion repair. DDR is coordinated by three apical PI3 kinase related kinases (PIKKs), including ataxia-telangiectasia mutated (ATM), ATM- and Rad3-related (ATR), and DNA-PKcs (the catalytic subunit of the DNA dependent protein kinase). These kinases are activated in response to specific DNA damage or lesions, resulting in checkpoint activation and DNA lesion repair. While it is clear that the pathways of ATM, ATR, and DNA-PK are the core components of DDR, there is accumulating evidence revealing the involvement of other cellular pathways in regulating DDR; this is in line with the concept that in addition to being a nuclear event DDR is also a cellular process. One of these pathways is the extracellular signal-regulated kinase (ERK) MAPK (mitogen-activated protein kinase) pathway. ERK is a converging point of multiple signal transduction pathways involved in cell proliferation, differentiation, and apoptosis. Adding to this list of pathways is the recent development of ERK in DDR. The ERK kinases (ERK1 and ERK2) contribute to the proper execution of DDR in terms of checkpoint activation and the repair of DNA lesions. This review summarizes the contributions of ERK to DDR with emphasis on the relationship of ERK kinases with the activation of ATM, ATR, and DNA-PKcs.

  9. Transcriptional upregulation of the human MRP2 gene expression by serine/threonine protein kinase inhibitors.

    PubMed

    Pułaski, L; Szemraj, J; Uchiumi, T; Kuwano, M; Bartosz, G

    2005-01-01

    Transcriptional regulation by cellular signalling pathways of multidrug resistance proteins that pump anticancer drugs out of cells is one of key issues in the development of the multidrug resistance phenotype. In our study, we have used the reporter gene approach as well as determination of mRNA levels in two cancer cell lines of human origin, MCF-7 and A549, to study the regulation of multidrug resistance proteins 2 and 3 (MRP2 AND MRP3) by serine/threonine protein kinases. Since a prototypic PKC inducer, PMA, caused a marked upregulation of transcription from both human MRP2 and MRP3 promoters, a role for PKC isoforms in positive control of expression of these proteins could be postulated. Interestingly, broad-spectrum serine-threonine protein kinase inhibitors which also inhibit PKC, staurosporine and H-7, stimulated expression from the MRP2 promoter instead of inhibiting it. This effect was not seen for MRP3. MRP2 induction by staurosporine and H-7 was shown to have phenotypic consequences in whole cells, rendering them more resistant to etoposide and increasing their ability to export calcein through the plasma membrane. These results point to the involvement of serine/threonine protein kinases in negative regulation of the human MRP2 gene and to the necessity of testing novel anti-cancer drugs acting as protein kinase inhibitors with regard to their potential ability to induce multidrug resistance.

  10. Regular exercise enhances insulin activation of IRS-1-associated PI3-kinase in human skeletal muscle.

    PubMed

    Kirwan, J P; del Aguila, L F; Hernandez, J M; Williamson, D L; O'Gorman, D J; Lewis, R; Krishnan, R K

    2000-02-01

    Insulin action in skeletal muscle is enhanced by regular exercise. Whether insulin signaling in human skeletal muscle is affected by habitual exercise is not well understood. Phosphatidylinositol 3-kinase (PI3-kinase) activation is an important step in the insulin-signaling pathway and appears to regulate glucose metabolism via GLUT-4 translocation in skeletal muscle. To examine the effects of regular exercise on PI3-kinase activation, 2-h hyperinsulinemic (40 mU. m(-2). min(-1))-euglycemic (5.0 mM) clamps were performed on eight healthy exercise-trained [24 +/- 1 yr, 71.8 +/- 2.0 kg, maximal O(2) uptake (VO(2 max)) of 56.1 +/- 2.5 ml. kg(-1). min(-1)] and eight healthy sedentary men and women (24 +/- 1 yr, 64.7 +/- 4.4 kg, VO(2 max) of 44.4 +/- 2.7 ml. kg(-1). min(-1)). A [6, 6-(2)H]glucose tracer was used to measure hepatic glucose output. A muscle biopsy was obtained from the vastus lateralis muscle at basal and at 2 h of hyperinsulinemia to measure insulin receptor substrate-1(IRS-1)-associated PI3-kinase activation. Insulin concentrations during hyperinsulinemia were similar for both groups (293 +/- 22 and 311 +/- 22 pM for trained and sedentary, respectively). Insulin-mediated glucose disposal rates (GDR) were greater (P < 0.05) in the exercise-trained compared with the sedentary control group (9.22 +/- 0.95 vs. 6.36 +/- 0.57 mg. kg fat-free mass(-1). min(-1)). Insulin-stimulated PI3-kinase activation was also greater (P < 0.004) in the trained compared with the sedentary group (3.8 +/- 0.5- vs. 1.8 +/- 0.2-fold increase from basal). Endurance capacity (VO(2 max)) was positively correlated with PI3-kinase activation (r = 0.53, P < 0.04). There was no correlation between PI3-kinase and muscle morphology. However, increases in GDR were positively related to PI3-kinase activation (r = 0.60, P < 0.02). We conclude that regular exercise leads to greater insulin-stimulated IRS-1-associated PI3-kinase activation in human skeletal muscle, thus facilitating enhanced

  11. Genome-wide inhibitory impact of the AMPK activator metformin on [kinesins, tubulins, histones, auroras and polo-like kinases] M-phase cell cycle genes in human breast cancer cells.

    PubMed

    Oliveras-Ferraros, Cristina; Vazquez-Martin, Alejandro; Menendez, Javier A

    2009-05-15

    Prompted by the ever-growing scientific rationale for examining the antidiabetic drug metformin as a potential antitumor agent in breast cancer disease, we recently tested the hypothesis that the assessment of metformin-induced global changes in gene expression-as identified using 44 K (double density) Agilent's whole human genome arrays-could reveal gene-expression signatures that would allow proper selection of breast cancer patients who should be considered for metformin-based clinical trials. Using Database for Annotation, Visualization and Integrated Discovery bioinformatics (DAVID) resources we herein reveal that, at doses that lead to activation of the AMP-activated protein kinase (AMPK), metformin not only downregulates genes coding for ribosomal proteins (i.e., protein and macromolecule biosynthesis) but unexpectedly suppresses numerous mitosis-related gene families including kinesins, tubulins, histones, auroras and polo-like kinases. This is, to our knowledge, the first genome-scale evidence of a mitotic core component in the transcriptional response of human breast cancer cells to metformin. These findings further support a tight relationship between the activation status of AMPK and the chromosomal and cytoskeletal checkpoints of cell mitosis at the transcriptional level.

  12. Preparation and characterization of human ADCK3, a putative atypical kinase.

    PubMed

    Wheeler, Brody; Jia, Zongchao

    2015-04-01

    AarF domain containing kinase 3 (ADCK3) is a mitochondrial protein known to have a role in the electron transport chain. Despite being required for the biosynthesis of coenzyme Q10, a lipid-soluble electron transporter found to be essential for aerobic cellular respiration, the precise biological function of ADCK3 remains unknown. Patients with mutations in ADCK3 experience an onset of neurological disorders from childhood, including cerebellar ataxia and exercise intolerance. After extensive screening for soluble recombinant protein expression, an N-terminal fusion of maltose-binding protein was found to facilitate the overexpression of the human ADCK3 kinase domain in Escherichia coli as a soluble and biologically active entity. For the first time our work reveals Mg(2+)-dependent ATPase activity of ADCK3, providing strong support for the theoretical prediction of this protein being a functional atypical kinase.

  13. Activation of S6 kinase in human neutrophils by calcium pyrophosphate dihydrate crystals: protein kinase C-dependent and phosphatidylinositol-3-kinase-independent pathways.

    PubMed Central

    Tudan, C; Jackson, J K; Charlton, L; Pelech, S L; Sahl, B; Burt, H M

    1998-01-01

    Phosphatidylinositol 3-kinase (PI 3-kinase) has been shown previously to be a central enzyme in crystal-induced neutrophil activation. Since activation of the 70 kDa S6 kinase (p70S6K) has been shown to be dependent on PI 3-kinase activation in mammalian cells, and since the former is a key enzyme in the transmission of signals to the cell nucleus, activation of p70(S6K) was investigated in crystal-stimulated neutrophils. Cytosolic fractions from calcium pyrophosphate dihydrate (CPPD)-crystal-activated neutrophils were separated by Mono Q chromatography and analysed for phosphotransferase activity using a range of substrates and probed by Western analysis using antibodies to p70(S6K) and mitogen-activated protein kinase (MAP kinase). CPPD crystals induced a robust, transient activation (peak activity at 2 min) of p70(S6K) that was fully inhibited by pretreatment with rapamycin. This is the first report of the activation of p70(S6K) in neutrophil signal transduction pathways induced by an agonist. This crystal-induced activation of p70(S6K) could also be inhibited by a protein kinase C (PKC) inhibitor (Compound 3), but not by the PI 3-kinase inhibitor wortmannin. CPPD crystals also activated the ERK1 and ERK2 forms of MAP kinase (wortmannin insensitive), PKC (Compound 3 sensitive) and protein kinase B (wortmannin sensitive) in neutrophils. These data suggest that activation of p70(S6K) may proceed through a PI 3-kinase- and protein kinase B-independent but PKC-dependent pathway in crystal-activated neutrophils. PMID:9531494

  14. [Role of protein kinases of human red cell membrane in deformability and aggregation changes].

    PubMed

    Murav'ev, A V; Maĭmistova, A A; Tikhomirova, I A; Bulaeva, S V; Mikhaĭlov, P V; Murav'ev, A A

    2012-01-01

    The proteomic analysis has showed that red cell membrane contains several kinases and phosphatases. Therefore the aim of this study was to investigate the role of protein kinases of human red cell membrane in deformability and aggregation changes. Exposure of red blood cells (RBCs) to some chemical compounds led to change in the RBC microrheological properties. When forskolin (10 microM), an adenylyl cyclase (AC) and a protein kinase A (PKA) stimulator was added to RBC suspension, the RBC deformability (RBCD) was increased by 20% (p < 0.05). Somewhat more significant deformability rise appeared after RBC incubation with dB-AMP (by 26%; p < 0.01). Red cell aggregation (RBCA) was significantly decreased under these conditions (p < 0.01). Markedly less changes of deformability was found after RBC incubation with protein kinase stimulator C (PKC)--phorbol 12-myristate 13-acetate (PMA). This drug reduced red cell aggregation only slightly. It was inhibited red cell tyrosine phosphotase activity by N-vanadat and was obtained a significant RBCD rise and RBCA lowering. The similar effect was found when cells were incubated with cisplatin as a tyrosine protein kinase (TPK) activator. It is important to note that a selective TPK inhibitor--lavendustin eliminated the above mention effects. On the whole the total data clearly show that the red cell aggregation and deformation changes were connected with an activation of the different intracellular signaling pathways.

  15. Structural insight into nucleotide recognition by human death-associated protein kinase

    SciTech Connect

    McNamara, Laurie K.; Watterson, D.M.; Brunzelle, Joseph S.

    2009-06-01

    Death-associated protein kinase (DAPK) is a member of the Ca{sup 2+}/calmodulin-regulated family of serine/threonine protein kinases. The role of the kinase activity of DAPK in eukaryotic cell apoptosis and the ability of bioavailable DAPK inhibitors to rescue neuronal death after brain injury have made it a drug-discovery target for neurodegenerative disorders. In order to understand the recognition of nucleotides by DAPK and to gain insight into DAPK catalysis, the crystal structure of human DAPK was solved in complex with ADP and Mg{sup 2+} at 1.85 {angstrom} resolution. ADP is a product of the kinase reaction and product release is considered to be the rate-limiting step of protein kinase catalytic cycles. The structure of DAPK-ADP-Mg{sup 2+} was compared with a newly determined DAPK-AMP-PNP-Mg{sup 2+} structure and the previously determined apo DAPK structure (PDB code 1 jks). The comparison shows that nucleotide-induced changes are localized to the glycine-rich loop region of DAPK.

  16. Protein kinase CK2 is necessary for the adipogenic differentiation of human mesenchymal stem cells.

    PubMed

    Schwind, Lisa; Wilhelm, Nadine; Kartarius, Sabine; Montenarh, Mathias; Gorjup, Erwin; Götz, Claudia

    2015-10-01

    CK2 is a serine/threonine protein kinase, which is so important for many aspects of cellular regulation that life without CK2 is impossible. Here, we analysed CK2 during adipogenic differentiation of human mesenchymal stem cells (hMSCs). With progress of the differentiation CK2 protein level and the kinase activity decreased. Whereas CK2α remained in the nucleus during differentiation, the localization of CK2β showed a dynamic shuttling in the course of differentiation. Over the last years a large number of inhibitors of CK2 kinase activity were generated with the idea to use them in cancer therapy. Our results show that two highly specific inhibitors of CK2, CX-4945 and quinalizarin, reduced its kinase activity in proliferating hMSC with a similar efficiency. CK2 inhibition by quinalizarin resulted in nearly complete inhibition of differentiation whereas, in the presence of CX-4945, differentiation proceeded similar to the controls. In this case, differentiation was accompanied by the loss of CX-4945 inhibitory function. By analysing the subcellular localization of PPARγ2, we found a shift from a nuclear localization at the beginning of differentiation to a more cytoplasmic localization in the presence of quinalizarin. Our data further show for the first time that a certain level of CK2 kinase activity is required for adipogenic stem cell differentiation and that inhibition of CK2 resulted in an altered localization of PPARγ2, an early regulator of differentiation.

  17. p38 mitogen-activated protein kinase activation by ultraviolet A radiation in human dermal fibroblasts.

    PubMed

    Le Panse, Rozen; Dubertret, Louis; Coulomb, Bernard

    2003-08-01

    UVA radiation penetrates deeply into the skin reaching both the epidermis and the dermis. We thus investigated the effects of naturally occurring doses of UVA radiation on mitogen-activated protein kinase (MAPK) activities in human dermal fibroblasts. We demonstrated that UVA selectively activates p38 MAPK with no effect on extracellular-regulated kinases (ERK1-ERK2) or JNK-SAPK (cJun NH2-terminal kinase-stress-activated protein kinase) activities. We then investigated the signaling pathway used by UVA to activate p38 MAPK. L-Histidine and sodium azide had an inhibitory effect on UVA activation of p38 MAPK, pointing to a role of singlet oxygen in transduction of the UVA effect. Afterward, using prolonged cell treatments with growth factors to desensitize their signaling pathways or suramin to block growth factor receptors, we demonstrated that UVA signaling pathways shared elements with growth factor signaling pathways. In addition, using emetine (a translation inhibitor altering ribosome functioning) we detected the involvement of ribotoxic stress in p38 MAPK activation by UVA. Our observations suggest that p38 activation by UVA in dermal fibroblasts involves singlet oxygen-dependent activation of ligand-receptor signaling pathways or ribotoxic stress mechanism (or both). Despite the activation of these two distinct signaling mechanisms, the selective activation of p38 MAPK suggests a critical role of this kinase in the effects of UVA radiation.

  18. Calcium pyrophosphate dihydrate crystals activate MAP kinase in human neutrophils: inhibition of MAP kinase, oxidase activation and degranulation responses of neutrophils by taxol.

    PubMed Central

    Jackson, J K; Tudan, C; Sahl, B; Pelech, S L; Burt, H M

    1997-01-01

    The activation of MAP kinase in human neutrophils stimulated by both uncoated and plasma-opsonized crystals of triclinic calcium pyrophosphate dihydrate (CPPD) was investigated. The effect of taxol on MAP kinase activation and on the responses of neutrophils stimulated by plasma-opsonized crystals was determined. MAP kinase activation was identified and quantified in Mono Q chromatography separated fractions of neutrophils that had been incubated with CPPD crystals by measuring [gamma-32P]adenosine triphosphate (ATP) phosphorylation of myelin basic protein and using immunoblotting techniques. Human neutrophils were incubated with taxol (0-50 microM), added to plasma-opsonized CPPD (50 mg/ml) and MAP kinase activation, chemiluminescence, superoxide anion generation, lysozyme and myeloperoxidase release were monitored. Both uncoated and plasma coated CPPD crystals induced a large increase in MAP kinase activity in neutrophils over control levels within 1 min of incubation. Pretreatment of neutrophils with taxol was able to suppress this activation of MAP kinase. Taxol produced a concentration-dependent inhibition of opsonized CPPD-induced neutrophil chemiluminescence, superoxide anion production and myeloperoxide release. Taxol at 28 microM also significantly inhibited chemiluminescence, superoxide anion production and myeloperoxidase release from neutrophils stimulated by opsonized zymosan. This is the first report of crystal-induced activation of MAP kinase in neutrophils. Microtubule-associated processes, such as signal transduction, secretion and phagocytosis are involved in particulate-induced neutrophil responses. We have suggested that the inhibitory effect of taxol observed in this work is due to its stabilizing effect on microtubules and disruption of MAP kinase activation associated with microtubules. Images Figure 1 Figure 3 PMID:9176102

  19. Localization of a human receptor tyrosine kinase (ETK1) to chromosome region 3p11. 2

    SciTech Connect

    Wicks, I.P.; Boyd, A.W. ); Lapsys, N.M.; Baker, E.; Sutherland, G.R. ); Campbell, L.J. )

    1994-01-01

    The authors have recently described a human receptor tyrosine kinase (hek) that is expressed by some pre-B and thymic T cell lines, but is not detectable on normal adult human tissues. Gene cloning studies established that hek is a new member of the EPH family of receptor tyrosine kinases. The expression of hek may normally be developmentally regulated and inappropriate expression may contribute to oncogenesis. In the present study, they have used Southern blot analysis of somatic cell hybrids and fluorescence in situ hybridization to localize the hek gene to human chromosome region 3p11.2. Karyotype analysis of the cell lines that over-express hek showed no cytogenetically visible abnormality involving the hek locus. 29 refs., 1 fig., 2 tabs.

  20. The Drosophila chk2 gene loki is essential for embryonic DNA double-strand-break checkpoints induced in S phase or G2.

    PubMed

    Masrouha, Nisrine; Yang, Long; Hijal, Sirine; Larochelle, Stéphane; Suter, Beat

    2003-03-01

    Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the occurrence of the next cell cycle transition. The Chk2 family of kinases is known to play a central role in mediating the cellular responses to DNA damage or DNA replication blocks in various organisms. Here we show through a phylogenetic study that the Drosophila melanogaster serine/threonine kinase Loki is the homolog of the yeast Mek1p, Rad53p, Dun1p, and Cds1 proteins as well as the human Chk2. Functional analyses allowed us to conclude that, in flies, chk2 is involved in monitoring double-strand breaks (DSBs) caused by irradiation during S and G2 phases. In this process it plays an essential role in inducing a cell cycle arrest in embryonic cells. Our results also show that, in contrast to C. elegans chk2, Drosophila chk2 is not essential for normal meiosis and recombination, and it also appears to be dispensable for the MMS-induced DNA damage checkpoint and the HU-induced DNA replication checkpoint during larval development. In addition, Drosophila chk2 does not act at the same cell cycle phases as its yeast homologs, but seems rather to be involved in a pathway similar to the mammalian one, which involves signaling through the ATM/Chk2 pathway in response to genotoxic insults. As mutations in human chk2 were linked to several cancers, these similarities point to the usefulness of the Drosophila model system.

  1. The Drosophila chk2 gene loki is essential for embryonic DNA double-strand-break checkpoints induced in S phase or G2.

    PubMed Central

    Masrouha, Nisrine; Yang, Long; Hijal, Sirine; Larochelle, Stéphane; Suter, Beat

    2003-01-01

    Cell cycle checkpoints are signal transduction pathways that control the order and timing of cell cycle transitions, ensuring that critical events are completed before the occurrence of the next cell cycle transition. The Chk2 family of kinases is known to play a central role in mediating the cellular responses to DNA damage or DNA replication blocks in various organisms. Here we show through a phylogenetic study that the Drosophila melanogaster serine/threonine kinase Loki is the homolog of the yeast Mek1p, Rad53p, Dun1p, and Cds1 proteins as well as the human Chk2. Functional analyses allowed us to conclude that, in flies, chk2 is involved in monitoring double-strand breaks (DSBs) caused by irradiation during S and G2 phases. In this process it plays an essential role in inducing a cell cycle arrest in embryonic cells. Our results also show that, in contrast to C. elegans chk2, Drosophila chk2 is not essential for normal meiosis and recombination, and it also appears to be dispensable for the MMS-induced DNA damage checkpoint and the HU-induced DNA replication checkpoint during larval development. In addition, Drosophila chk2 does not act at the same cell cycle phases as its yeast homologs, but seems rather to be involved in a pathway similar to the mammalian one, which involves signaling through the ATM/Chk2 pathway in response to genotoxic insults. As mutations in human chk2 were linked to several cancers, these similarities point to the usefulness of the Drosophila model system. PMID:12663536

  2. Intellectual property issues of immune checkpoint inhibitors

    PubMed Central

    Storz, Ulrich

    2016-01-01

    Immune checkpoint inhibitors are drugs that interfere with tumor escape responses. Some members of this class are already approved, and expected to be blockbusters in the future. Many companies have developed patent activities in this field. This article focuses on the patent landscape, and discusses key players and cases related to immune checkpoint inhibitors. PMID:26466763

  3. Alternative DNA Damage Checkpoint Pathways in Eukaryotes

    DTIC Science & Technology

    2000-04-01

    checkpoint pathway in Saccharomyces cerevisiae. Our hypothesis is that CHES1 does so by activating an alternative DNA damage-induced checkpoint pathway. The...difficulties, therefore we also tried the candidate gene and the yeast 2-hybrid approaches but with no success. In this report, we proposed alternative

  4. Zipper-interacting protein kinase interacts with human cell division cycle 14A phosphatase.

    PubMed

    Wu, Wei; Hu, Haiying; Ye, Zi; Leong, Mancheong; He, Min; Li, Qin; Hu, Renming; Zhang, Shuo

    2015-04-01

    Zipper‑interacting protein kinase (ZIPK) is a novel serine/threonine protein kinase and a member of a large family of protein kinases, known as the death‑associated protein kinases. However, the function of ZIPK has yet to be fully elucidated, as few physiological substrates have currently been identified. In the present study, a yeast two‑hybrid screen was used and the human cell division cycle 14A (HsCdc14A) phosphatase was identified as a novel ZIPK binding protein. To the best of our knowledge, this is the first study to report the interaction between these proteins. The interaction between ZIPK and HsCdc14A was confirmed by in vitro experiments. In addition, ZIPK‑mediated phosphorylation was shown to activate the phosphatase activity of HsCdc14A. These findings indicated that ZIPK may also be involved in the regulation of the cell cycle in human cells, by interacting with HsCdc14A.

  5. Dibutyltin activates MAP kinases in human natural killer cells, in vitro.

    PubMed

    Odman-Ghazi, Sabah O; Abraha, Abraham; Isom, Erica Taylor; Whalen, Margaret M

    2010-10-01

    Previous studies have shown that dibutyltin (DBT) interferes with the function of human natural killer (NK) cells, diminishing their capacity to destroy tumor cells, in vitro. DBT is a widespread environmental contaminant and has been found in human blood. As NK cells are our primary immune defense against tumor cells, it is important to understand the mechanism by which DBT interferes with their function. The current study examines the effects of DBT exposures on key enzymes in the signaling pathway that regulates NK responsiveness to tumor cells. These include several protein tyrosine kinases (PTKs), mitogen-activated protein kinases (MAPKs), and mitogen-activated protein kinase kinases (MAP2Ks). The results showed that in vitro exposures of NK cells to DBT had no effect on PTKs. However, exposures to DBT for as little as 10 min were able to increase the phosphorylation (activation) of the MAPKs. The DBT-induced activations of these MAPKs appear to be due to DBT-induced activations of the immediate upstream activators of the MAPKs, MAP2Ks. The results suggest that DBT-interference with the MAPK signaling pathway is a consequence of DBT exposures, which could account for DBT-induced decreases in NK function.

  6. A human NDP-kinase B specifically binds single-stranded poly-pyrimidine sequences.

    PubMed Central

    Hildebrandt, M; Lacombe, M L; Mesnildrey, S; Véron, M

    1995-01-01

    Recently, a DNA binding protein 'PUF' was purified that binds to a poly-pyrimidine rich element in the human c-myc promoter. Cloning of the corresponding gene surprisingly identified this putative transcription factor as isoform B of the enzyme nucleoside diphosphate kinase (NDPK-B) [Postel et al. (1993) Science, 261, 478-480], the product of the potential metastasis suppressor gene nm23-H2. Using different recombinant NDP kinases, we demonstrate by electrophoretic mobility shift analysis (EMSA) that the NDP kinase DNA binding properties are predominantly observed with human isoform B. Unlike typical DNA binding proteins that are involved in transcriptional regulation, binding occurs to single-stranded DNA rather than to a double-stranded oligonucleotide. As a consequence, complexes of single-stranded DNA and NDPK-B are generated from double-stranded oligonucleotide hybrids in an ATP independent manner. In addition to the c-myc element, NDPK-B is binding in vitro to a variety of poly-pyrimidine rich sequences including dC or dT homo-oligomers, (CT)n dinucleotide repeats, the initiator region of the Adenovirus major late promoter and even poly-pyrimidine rich RNAs. The possible consequences of these findings in understanding the multiple roles of NDP kinase are discussed. Images PMID:7479028

  7. Action of the Src family kinase inhibitor, dasatinib (BMS-354825), on human prostate cancer cells.

    PubMed

    Nam, Sangkil; Kim, Donghwa; Cheng, Jin Q; Zhang, Shumin; Lee, Ji-Hyun; Buettner, Ralf; Mirosevich, Janni; Lee, Francis Y; Jove, Richard

    2005-10-15

    Src family kinases (SFK) are currently being investigated as targets for treatment strategies in various cancers. The novel SFK/Abl inhibitor, dasatinib (BMS-354825), is a promising therapeutic agent with oral bioavailability. Dasatinib has been shown to inhibit growth of Bcr-Abl-dependent chronic myeloid leukemia xenografts in nude mice. Dasatinib also has been shown to have activity against cultured human prostate and breast cancer cells. However, the molecular mechanism by which dasatinib acts on epithelial tumor cells remains unknown. In this study, we show that dasatinib blocks the kinase activities of the SFKs, Lyn, and Src, in human prostate cancer cells at low nanomolar concentrations. Moreover, focal adhesion kinase and Crk-associated substrate (p130(CAS)) signaling downstream of SFKs are also inhibited at similar concentrations of dasatinib. Consistent with inhibition of these signaling pathways, dasatinib suppresses cell adhesion, migration, and invasion of prostate cancer cells at low nanomolar concentrations. Therefore, dasatinib has potential as a therapeutic agent for metastatic prostate cancers harboring activated SFK and focal adhesion kinase signaling.

  8. Genetic instability in cancer cells by impaired cell cycle checkpoints.

    PubMed

    Nakanishi, Makoto; Shimada, Midori; Niida, Hiroyuki

    2006-10-01

    Cells continuously encounter DNA damage caused either by damaging agents, including oxygen radicals and DNA replication errors caused by stalled replication forks, or by extracellular environments such as ultraviolet or ionizing irradiation. Such DNA damage poses a great threat to genome stability, potentially leading to loss or amplification of chromosome activity, which may result in cellular senescence, cancer or apoptosis. The DNA damage checkpoints coordinate an arrest in cell cycle progression with the DNA repair process, suppressing either mitotic catastrophe or proliferation of cells with damaged DNA. Numerous key players have been identified in terms of damage sensor proteins, transducer kinases and effectors, but their coordination and interconnectedness in damage control have only recently become evident. In this review, we discuss changes in chromatin structure, recruitment of mediator proteins and activation of transducer kinases in response to DNA damage. These cellular responses are important for determining the potential effects of current cancer therapies in terms of toxicity and efficacy.

  9. Overlapped checkpointing with hardware assist

    SciTech Connect

    Mitchell, Christopher J; Nunez, James A; Wang, Jun

    2009-01-01

    We present a new approach to handling the demanding I/O workload incurred during checkpoint writes encountered in High Performance Computing. Prior efforts to improve performance have been primarily bound by mechanical limitations of the hard drive. Our research surpasses this limitation by providing a method to: (1) write checkpoint data to a high-speed, non-volatile buffer, and (2) asynchronously write this data to permanent storage while resuming computation. This removes the hard drive from the critical data path because our I/O node based buffers isolate the compute nodes from the storage servers. This solution is feasible because of industry declines in cost for high-capacity, non-volatile storage technologies. Testing was conducted on a small-scale cluster to prove the design, and then scaled at Los Alamos National Laboratory. Results show a definitive speedup factor for select workloads over writing directly to a typical global parallel file system; the Panasas ActiveScale File System.

  10. LTB4 stimulates growth of human pancreatic cancer cells via MAPK and PI-3 kinase pathways

    SciTech Connect

    Tong, W.-G.; Ding, X.-Z.; Talamonti, Mark S.; Bell, Richard H.; Adrian, Thomas E. . E-mail: tadrian@northwestern.edu

    2005-09-30

    We have previously shown the importance of LTB4 in human pancreatic cancer. LTB4 receptor antagonists block growth and induce apoptosis in pancreatic cancer cells both in vitro and in vivo. Therefore, we investigated the effect of LTB4 on proliferation of human pancreatic cancer cells and the mechanisms involved. LTB4 stimulated DNA synthesis and proliferation of both PANC-1 and AsPC-1 human pancreatic cancer cells, as measured by thymidine incorporation and cell number. LTB4 stimulated rapid and transient activation of MEK and ERK1/2 kinases. The MEK inhibitors, PD98059 and U0126, blocked LTB4-stimulated ERK1/2 activation and cell proliferation. LTB4 also stimulated phosphorylation of p38 MAPK; however, the p38 MAPK inhibitor, SB203580, failed to block LTB4-stimulated growth. The activity of JNK/SAPK was not affected by LTB4 treatment. Phosphorylation of Akt was also induced by LTB4 and this effect was blocked by the PI-3 kinase inhibitor wortmannin, which also partially blocked LTB4-stimulated cell proliferation. In conclusion, LTB4 stimulates proliferation of human pancreatic cancer cells through MEK/ERK and PI-3 kinase/Akt pathways, while p38 MPAK and JNK/SAPK are not involved.

  11. Role of the Yes and Csk tyrosine kinases in the development of a pathological state in the human retina.

    PubMed

    Baranova, Lyudmila; Emelyanova, Valentina; Volotovski, Igor

    2010-07-01

    Amplification and a cloning of fragments of genes of human retina tyrosine kinases, the nucleotide sequences of which feature a high homology to the gene families of the Yes and Csk tyrosine kinases, and a cloning of the complete coding sequence of the cDNA of the Csk tyrosine kinase gene of the human lymphocytes have been carried out. It has been established that this sequence contains 1,624 bp and encodes a protein that, with a 99% homology, corresponds to the human tyrosine kinase. A comparative analysis of the nucleotide sequences of the full-size cDNA of the Csk tyrosine kinase of the lymphocytes of healthy donors and of patients with an eye choroidal melanoma has shown that a risk of development of an eye choroidal melanoma can be estimated by the frequency of occurrence of a mutant allele in the 10th exon.

  12. Nucleotide binding to nucleoside diphosphate kinases: X-ray structure of human NDPK-A in complex with ADP and comparison to protein kinases.

    PubMed

    Chen, Yuxing; Gallois-Montbrun, Sarah; Schneider, Benoit; Véron, Michel; Moréra, Solange; Deville-Bonne, Dominique; Janin, Joel

    2003-09-26

    NDPK-A, product of the nm23-H1 gene, is one of the two major isoforms of human nucleoside diphosphate kinase. We analyzed the binding of its nucleotide substrates by fluorometric methods. The binding of nucleoside triphosphate (NTP) substrates was detected by following changes of the intrinsic fluorescence of the H118G/F60W variant, a mutant protein engineered for that purpose. Nucleoside diphosphate (NDP) substrate binding was measured by competition with a fluorescent derivative of ADP, following the fluorescence anisotropy of the derivative. We also determined an X-ray structure at 2.0A resolution of the variant NDPK-A in complex with ADP, Ca(2+) and inorganic phosphate, products of ATP hydrolysis. We compared the conformation of the bound nucleotide seen in this complex and the interactions it makes with the protein, with those of the nucleotide substrates, substrate analogues or inhibitors present in other NDP kinase structures. We also compared NDP kinase-bound nucleotides to ATP bound to protein kinases, and showed that the nucleoside monophosphate moieties have nearly identical conformations in spite of the very different protein environments. However, the beta and gamma-phosphate groups are differently positioned and oriented in the two types of kinases, and they bind metal ions with opposite chiralities. Thus, it should be possible to design nucleotide analogues that are good substrates of one type of kinase, and poor substrates or inhibitors of the other kind.

  13. Radiosensitization of Human Leukemic HL-60 Cells by ATR Kinase Inhibitor (VE-821): Phosphoproteomic Analysis

    PubMed Central

    Šalovská, Barbora; Fabrik, Ivo; Ďurišová, Kamila; Link, Marek; Vávrová, Jiřina; Řezáčová, Martina; Tichý, Aleš

    2014-01-01

    DNA damaging agents such as ionizing radiation or chemotherapy are frequently used in oncology. DNA damage response (DDR)—triggered by radiation-induced double strand breaks—is orchestrated mainly by three Phosphatidylinositol 3-kinase-related kinases (PIKKs): Ataxia teleangiectasia mutated (ATM), DNA-dependent protein kinase (DNA-PK) and ATM and Rad3-related kinase (ATR). Their activation promotes cell-cycle arrest and facilitates DNA damage repair, resulting in radioresistance. Recently developed specific ATR inhibitor, VE-821 (3-amino-6-(4-(methylsulfonyl)phenyl)-N-phenylpyrazine-2-carboxamide), has been reported to have a significant radio- and chemo-sensitizing effect delimited to cancer cells (largely p53-deficient) without affecting normal cells. In this study, we employed SILAC-based quantitative phosphoproteomics to describe the mechanism of the radiosensitizing effect of VE-821 in human promyelocytic leukemic cells HL-60 (p53-negative). Hydrophilic interaction liquid chromatography (HILIC)-prefractionation with TiO2-enrichment and nano-liquid chromatography—tandem mass spectrometry (LC-MS/MS) analysis revealed 9834 phosphorylation sites. Proteins with differentially up-/down-regulated phosphorylation were mostly localized in the nucleus and were involved in cellular processes such as DDR, all phases of the cell cycle, and cell division. Moreover, sequence motif analysis revealed significant changes in the activities of kinases involved in these processes. Taken together, our data indicates that ATR kinase has multiple roles in response to DNA damage throughout the cell cycle and that its inhibitor VE-821 is a potent radiosensitizing agent for p53-negative HL-60 cells. PMID:25003641

  14. Re-replication induced by geminin depletion occurs from G2 and is enhanced by checkpoint activation

    PubMed Central

    Klotz-Noack, Kathleen; McIntosh, Debbie; Schurch, Nicholas; Pratt, Norman; Blow, J. Julian

    2012-01-01

    Summary To prevent re-replication of DNA in a single cell cycle, the licensing of replication origins by Mcm2-7 is prevented during S and G2 phases. Animal cells achieve this by cell cycle regulated proteolysis of the essential licensing factor Cdt1 and inhibition of Cdt1 by geminin. Here we investigate the consequences of ablating geminin in synchronised human U2OS cells. Following geminin loss, cells complete an apparently normal S phase, but a proportion arrest at the G2/M boundary. When Cdt1 accumulates in these cells, DNA re-replicates, suggesting that the key role of geminin is to prevent re-licensing in G2. If cell cycle checkpoints are inhibited in cells lacking geminin, cells progress through mitosis and less re-replication occurs. Checkpoint kinases thereby amplify re-replication into an all-or-nothing response by delaying geminin-depleted cells in G2. Deep DNA sequencing revealed no preferential re-replication of specific genomic regions after geminin depletion. This is consistent with the observation that cells in G2 have lost their replication timing information. In contrast, when Cdt1 is overexpressed or is stabilised by the Neddylation inhibitor MLN4924, re-replication can occur throughout S phase. PMID:22366459

  15. Skp2 is required for Aurora B activation in cell mitosis and spindle checkpoint.

    PubMed

    Wu, Juan; Huang, Yu-Fan; Zhou, Xin-Ke; Zhang, Wei; Lian, Yi-Fan; Lv, Xiao-Bin; Gao, Xiu-Rong; Lin, Hui-Kuan; Zeng, Yi-Xin; Huang, Jian-Qing

    2015-01-01

    The Aurora B kinase plays a critical role in cell mitosis and spindle checkpoint. Here, we showed that the ubiquitin E3-ligase protein Skp2, also as a cell-cycle regulatory protein, was required for the activation of Aurora B and its downstream protein. When we restored Skp2 knockdown Hela cells with Skp2 and Skp2-LRR E3 ligase dead mutant we found that Skp2 could rescue the defect in the activation of Aurora B, but the mutant failed to do so. Furthermore, we discovered that Skp2 could interact with Aurora B and trigger Aurora B Lysine (K) 63-linked ubiquitination. Finally, we demonstrated the essential role of Skp2 in cell mitosis progression and spindle checkpoint, which was Aurora B dependent. Our results identified a novel ubiquitinated substrate of Skp2, and also indicated that Aurora B ubiquitination might serve as an important event for Aurora B activation in cell mitosis and spindle checkpoint.

  16. Unconventional Functions of Mitotic Kinases in Kidney Tumorigenesis

    PubMed Central

    Hascoet, Pauline; Chesnel, Franck; Le Goff, Cathy; Le Goff, Xavier; Arlot-Bonnemains, Yannick

    2015-01-01

    Human tumors exhibit a variety of genetic alterations, including point mutations, translocations, gene amplifications and deletions, as well as aneuploid chromosome numbers. For carcinomas, aneuploidy is associated with poor patient outcome for a large variety of tumor types, including breast, colon, and renal cell carcinoma. The Renal cell carcinoma (RCC) is a heterogeneous carcinoma consisting of different histologic types. The clear renal cell carcinoma (ccRCC) is the most common subtype and represents 85% of the RCC. Central to the biology of the ccRCC is the loss of function of the Von Hippel–Lindau gene, but is also associated with genetic instability that could be caused by abrogation of the cell cycle mitotic spindle checkpoint and may involve the Aurora kinases, which regulate centrosome maturation. Aneuploidy can also result from the loss of cell–cell adhesion and apical–basal cell polarity that also may be regulated by the mitotic kinases (polo-like kinase 1, casein kinase 2, doublecortin-like kinase 1, and Aurora kinases). In this review, we describe the “non-mitotic” unconventional functions of these kinases in renal tumorigenesis. PMID:26579493

  17. Single strand DNA specificity analysis of human nucleoside diphosphate kinase B.

    PubMed

    Agou, F; Raveh, S; Mesnildrey, S; Véron, M

    1999-07-09

    Nucleoside diphosphate kinases (NDP kinases) form a family of oligomeric enzymes present in all organisms. Eukaryotic NDP kinases are hexamers composed of identical subunits (approximately 17 kDa). A distinctive property of human NDPK-B encoded by the gene nm23-H2 is its ability to stimulate the gene transcription. This property is independent of its catalytic activity and is possibly related to the role of this protein in cellular events including differentiation and tumor metastasis. In this paper, we report the first characterization of human NDPK-B.DNA complex formation using a filter-binding assay and fluorescence spectroscopy. We analyzed the binding of several oligonucleotides mimicking the promoter region of the c-myc oncogene including variants in sequence, structure, and length of both strands. We show that NDPK-B binds to single-stranded oligonucleotides in a nonsequence specific manner, but that it exhibits a poor binding activity to double-stranded oligonucleotides. This indicates that the specificity of recognition to DNA is a function of the structural conformation of DNA rather than of its specific sequence. Moreover, competition experiments performed with all nucleotides provide evidence for the contribution of the six active sites in the DNA.protein complex formation. We propose a mechanism through which human NDPK-B could stimulate transcription of c-myc or possibly other genes involved in cellular differentiation.

  18. Induction of Central Host Signaling Kinases during Pneumococcal Infection of Human THP-1 Cells

    PubMed Central

    Kohler, Thomas P.; Scholz, Annemarie; Kiachludis, Delia; Hammerschmidt, Sven

    2016-01-01

    Streptococcus pneumoniae is a widespread colonizer of the mucosal epithelia of the upper respiratory tract of human. However, pneumococci are also responsible for numerous local as well as severe systemic infections, especially in children under the age of five and the elderly. Under certain conditions, pneumococci are able to conquer the epithelial barrier, which can lead to a dissemination of the bacteria into underlying tissues and the bloodstream. Here, specialized macrophages represent an essential part of the innate immune system against bacterial intruders. Recognition of the bacteria through different receptors on the surface of macrophages leads thereby to an uptake and elimination of bacteria. Accompanied cytokine release triggers the migration of leukocytes from peripheral blood to the site of infection, where monocytes differentiate into mature macrophages. The rearrangement of the actin cytoskeleton during phagocytosis, resulting in the engulfment of bacteria, is thereby tightly regulated by receptor-mediated phosphorylation cascades of different protein kinases. The molecular cellular processes including the modulation of central protein kinases are only partially solved. In this study, the human monocytic THP-1 cell line was used as a model system to examine the activation of Fcγ and complement receptor-independent signal cascades during infection with S. pneumoniae. Pneumococci cultured either in chemically defined or complex medium showed no significant differences in pneumococcal phagocytosis by phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 cells. Double immuno-fluorescence microscopy and antibiotic protection assays demonstrated a time-dependent uptake and killing of S. pneumoniae 35A inside of macrophages. Infections of THP-1 cells in the presence of specific pharmacological inhibitors revealed a crucial role of actin polymerization and importance of the phosphoinositide 3-kinase (PI3K) and Protein kinase B (Akt) as well during

  19. Expression of immune checkpoints in T cells of esophageal cancer patients

    PubMed Central

    Xie, Jinhua; Wang, Ji; Cheng, Shouliang; Zheng, Liangfeng; Ji, Feiyue; Yang, Lin; Zhang, Yan; Ji, Haoming

    2016-01-01

    Inhibition of immune checkpoint proteins (checkpoints) has become a promising anti-esophageal cancer strategy. We here tested expressions of immune checkpoints in human esophageal cancers. Our results showed the expressions of many immune checkpoints, including CD28, CD27, CD137L, programmed death 1 (PD-1), T cell immunoglobulin mucin-3 (TIM-3), T cell Ig and ITIM domain (TIGIT), CD160, cytotoxic T lymphocyte antigen 4 (CTLA-4), CD200, CD137 and CD158, were dysregulated in peripheral T cells of esophageal cancer patients. Further, the expressions of PD-1, TIM-3 and TIGIT were upregulated in tumor infiltrating lymphocytes (TILs), which might be associated with TILs exhaustion. Meanwhile, the expressions of PD-1 and TIM-3 on CD4+ T cells were closely associated with clinic pathological features of esophageal cancer patients. These results indicate that co-inhibitory receptors PD-1, TIM-3 and TIGIT may be potential therapeutic oncotargets for esophageal cancer. PMID:27577071

  20. A Human Proteome Array Approach to Identifying Key Host Proteins Targeted by Toxoplasma Kinase ROP18.

    PubMed

    Yang, Zhaoshou; Hou, Yongheng; Hao, Taofang; Rho, Hee-Sool; Wan, Jun; Luan, Yizhao; Gao, Xin; Yao, Jianping; Pan, Aihua; Xie, Zhi; Qian, Jiang; Liao, Wanqin; Zhu, Heng; Zhou, Xingwang

    2017-03-01

    Toxoplasma kinase ROP18 is a key molecule responsible for the virulence of Toxoplasma gondii; however, the mechanisms by which ROP18 exerts parasite virulence via interaction with host proteins remain limited to a small number of identified substrates. To identify a broader array of ROP18 substrates, we successfully purified bioactive mature ROP18 and used it to probe a human proteome array. Sixty eight new putative host targets were identified. Functional annotation analysis suggested that these proteins have a variety of functions, including metabolic process, kinase activity and phosphorylation, cell growth, apoptosis and cell death, and immunity, indicating a pleiotropic role of ROP18 kinase. Among these proteins, four candidates, p53, p38, UBE2N, and Smad1, were further validated. We demonstrated that ROP18 targets p53, p38, UBE2N, and Smad1 for degradation. Importantly, we demonstrated that ROP18 phosphorylates Smad1 Ser-187 to trigger its proteasome-dependent degradation. Further functional characterization of the substrates of ROP18 may enhance understanding of the pathogenesis of Toxoplasma infection and provide new therapeutic targets. Similar strategies could be used to identify novel host targets for other microbial kinases functioning at the pathogen-host interface.

  1. ELUCIDATION OF HUMAN CHOLINE KINASE CRYSTAL STRUCTURES IN COMPLEX WITH THE PRODUCTS ADP OR PHOSPHOCHOLINE

    PubMed Central

    Malito, Enrico; Sekulic, Nikolina; Too, Wei Cun See; Konrad, Manfred; Lavie, Arnon

    2006-01-01

    Summary Choline kinase, responsible for the phosphorylation of choline to phosphocholine as the first step of the CDP-choline pathway for the biosynthesis of phosphatidylcholine, has been recognized as a new target for anticancer therapy. Crystal structures of human choline kinase in its apo, ADP- and phosphocholine-bound complexes, respectively, reveal the molecular details of the substrate binding sites. ATP binds in a cavity where residues from both the N- and C-terminal lobes contribute to form a cleft, while the choline-binding site constitutes a deep hydrophobic groove in the C-terminal domain with a rim composed of negatively charged residues. Upon binding of choline, the enzyme undergoes conformational changes independently affecting the N-terminal domain and the ATP-binding loop. From this structural analysis and comparison with other kinases, and from mutagenesis data on the homologous C. elegans choline kinase, a model of the ternary ADP·Phosphocholine complex was built that reveals the molecular basis for the phosphoryl transfer activity of this enzyme. PMID:17007874

  2. Functional Analysis of Protein Kinase CK2 of the Human Malaria Parasite Plasmodium falciparum▿ †

    PubMed Central

    Holland, Zoë; Prudent, Renaud; Reiser, Jean-Baptiste; Cochet, Claude; Doerig, Christian

    2009-01-01

    Protein kinase CK2 (casein kinase 2) is a eukaryotic serine/threonine protein kinase with multiple substrates and roles in diverse cellular processes, including differentiation, proliferation, and translation. The mammalian holoenzyme consists of two catalytic alpha or alpha′ subunits and two regulatory beta subunits. We report the identification and characterization of a Plasmodium falciparum CK2α orthologue, PfCK2α, and two PfCK2β orthologues, PfCK2β1 and PfCK2β2. Recombinant PfCK2α possesses protein kinase activity, exhibits similar substrate and cosubstrate preferences to those of CK2α subunits from other organisms, and interacts with both of the PfCK2β subunits in vitro. Gene disruption experiments show that the presence of PfCK2α is crucial to asexual blood stage parasites and thereby validate the enzyme as a possible drug target. PfCK2α is amenable to inhibitor screening, and we report differential susceptibility between the human and P. falciparum CK2α enzymes to a small molecule inhibitor. Taken together, our data identify PfCK2α as a potential target for antimalarial chemotherapeutic intervention. PMID:19114502

  3. Crystal structure of a human cyclin-dependent kinase 6 complexwith a flavonol inhibitor, Fisetin

    SciTech Connect

    Lu, Heshu; Chang, Debbie J.; Baratte, Blandine; Meijer, Laurent; Schulze-Gahmen, Ursula

    2005-01-10

    Cyclin-dependent kinases (CDKs) play a central role in cell cycle control, apoptosis, transcription and neuronal functions. They are important targets for the design of drugs with anti-mitotic and/or anti-neurodegenerative effects. CDK4 and CDK6 form a subfamily among the CDKs in mammalian cells, as defined by sequence similarities. Compared to CDK2 and CDK5, structural information on CDK4 and CDK6 is sparse. We describe here the crystal structure of human CDK6 in complex with a viral cyclin and a flavonol inhibitor, fisetin. Fisetin binds to the active form of CDK6, forming hydrogen bonds with the side chains of residues in the binding pocket that undergo large conformational changes during CDK activation by cyclin binding. The 4-keto group and the 3-hydroxyl group of fisetin are hydrogen bonded with the backbone in the hinge region between the N-terminal and C-terminal kinase domain, as has been observed for many CDK inhibitors. However, CDK2 and HCK kinase in complex with other flavone inhibitors such as quercetin and flavopiridol showed a different binding mode with the inhibitor rotated by about 180. The structural information of the CDK6-fisetin complex is correlated with the binding affinities of different flavone inhibitors for CDK6. This complex structure is the first description of an inhibitor complex with a kinase from the CDK4/6 subfamily and can provide a basis for selecting and designing inhibitor compounds with higher affinity and specificity.

  4. Inhibition of focal adhesion kinase induces apoptosis in human osteosarcoma SAOS-2 cells.

    PubMed

    Wang, Jialiang; Zu, Jianing; Xu, Gongping; Zhao, Wei; Jinglong, Yan

    2014-02-01

    Focal adhesion kinase (FAK), a non-receptor tyrosine kinase protein, acts as an early modulator of integrin signaling cascade, regulating basic cellular functions. In transformed cells, unopposed FAK signaling has been considered to promote tumor growth, progression, and metastasis. The aim of this study was to assess the role of focal adhesion kinase in human osteosarcoma SAOS-2 cells. SAOS-2 cells were transfected with PGPU6/GFP/shNC, and PGPU6/GFP/FAK-334 (shRNA-334), respectively. Expression of FAK was detected by real-time PCR and western blots. MTT assay was used to examine changes in cell proliferation. Cell apoptosis was analyzed by flow cytometry. The expression of caspase-3,-7,-9 was measured by Western blots. The expression of FAK in SAOS-2 cells significantly decreased in shRNA-334 group contrast to the control group (P < 0.01). Cells proliferation was inhibited by shRNA-334 and shRNA-334 + cisplatin, and the effects were clearly enhanced when cells treated with the anticancer agents. The level of cell apoptosis in shRNA-334 and shRNA-334 + cisplatin group was higher than in the control group (P < 0.01). The current data support evidence that down-regulation of FAK could induce SAOS-2 apoptosis through the caspase-dependent cell death pathway. Inhibition of the kinases may be important for therapies designed to enhance the apoptosis in osteosarcoma.

  5. Kinome-wide RNAi studies in human multiple myeloma identify vulnerable kinase targets, including a lymphoid-restricted kinase, GRK6

    PubMed Central

    Zhu, Yuan Xiao; Schmidt, Jessica; Yin, Hongwei; Shi, Chang-Xin; Que, Qiang; Basu, Gargi; Azorsa, David; Perkins, Louise M.; Braggio, Esteban; Fonseca, Rafael; Bergsagel, P. Leif; Mousses, Spyro; Stewart, A. Keith

    2010-01-01

    A paucity of validated kinase targets in human multiple myeloma has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in myeloma tumor lines bearing common t(4;14), t(14;16), and t(11;14) translocations to identify critically vulnerable kinases in myeloma tumor cells without regard to preconceived mechanistic notions. Fifteen kinases were repeatedly vulnerable in myeloma cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. Whereas several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly G protein–coupled receptor kinase, GRK6, appeared selectively vulnerable in myeloma. GRK6 inhibition was lethal to 6 of 7 myeloma tumor lines but was tolerated in 7 of 7 human cell lines. GRK6 exhibits lymphoid-restricted expression, and from coimmunoprecipitation studies we demonstrate that expression in myeloma cells is regulated via direct association with the heat shock protein 90 (HSP90) chaperone. GRK6 silencing causes suppression of signal transducer and activator of transcription 3 (STAT3) phosphorylation associated with reduction in MCL1 levels and phosphorylation, illustrating a potent mechanism for the cytotoxicity of GRK6 inhibition in multiple myeloma (MM) tumor cells. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma. PMID:19996089

  6. Anti-KIT Monoclonal Antibody Treatment Enhances the Anti-Tumor Activity of Immune Checkpoint Inhibitors by Reversing Tumor-Induced Immunosuppression.

    PubMed

    Garton, Andrew J; Seibel, Scott; Lopresti-Morrow, Lori; Crew, Linda; Janson, Neal; Mandiyan, Sreekala; Trombetta, E Sergio; Pankratz, Shannon; LaVallee, Theresa M; Gedrich, Richard

    2017-01-30

    The receptor tyrosine kinase KIT is an established oncogenic driver of tumor growth in certain tumor types including gastrointestinal stromal tumors (GIST), in which constitutively active mutant forms of KIT represent an actionable target for small molecule tyrosine kinase inhibitors. There is also considerable potential for KIT to influence tumor growth indirectly based on its expression and function in cell types of the innate immune system, most notably mast cells. We have evaluated syngeneic mouse tumor models for anti-tumor effects of an inhibitory KIT monoclonal antibody (mAb), dosed either alone or in combination with immune checkpoint inhibitors. Anti-KIT mAb treatment enhanced the anti-tumor activity of anti-CTLA-4 and anti-PD-1 mAbs, and promoted immune responses by selectively reducing the immunosuppressive monocytic myeloid-derived suppressor cell (M-MDSC) population and restoring CD8+ and CD4+ T-cell populations to levels observed in naïve mice. These data provide a rationale for clinical investigation of the human KIT-specific mAb KTN0158 in novel immuno-oncology combinations with immune checkpoint inhibitors and other immunotherapeutic agents across a range of tumor types.

  7. Checkpoint inhibitors in Hodgkin's lymphoma.

    PubMed

    Jezeršek Novaković, Barbara

    2016-04-01

    Hodgkin's lymphoma is unusual among cancers in that it consists of a small number of malignant Hodgkin/Reed-Sternberg cells in a sea of immune system cells, including T cells. Most of these T cells are reversibly inactivated in different ways and their reactivation may induce a very strong immune response to cancer cells. One way of reactivation of T cells is with antibodies blocking the CTLA-4 and especially with antibodies directed against PD-1 or the PD-L1 ligand thereby reversing the tumor-induced downregulation of T-cell function and augmenting antitumor immune activity at the priming (CTLA-4) or tissue effector (PD-1) phase. Immune checkpoint inhibitors have been evidenced as an additional treatment option with substantial effectiveness and acceptable toxicity in heavily pretreated patients with Hodgkin's lymphoma. Particularly, PD-1 blockade with nivolumab and pembrolizumab has demonstrated significant single-agent activity in this select population.

  8. Inhibition of SRC tyrosine kinase as treatment for human pancreatic cancer growing orthotopically in nude mice.

    PubMed

    Yezhelyev, Maksim V; Koehl, Gudrun; Guba, Markus; Brabletz, Thomas; Jauch, Karl-Walter; Ryan, Anderson; Barge, Alan; Green, Tim; Fennell, Michael; Bruns, Christiane J

    2004-12-01

    The Src family comprises a family of nonreceptor intracellular tyrosine kinases that mediate a variety of cellular pathways. Src kinases are overexpressed in a variety of human tumors, including cancer of the colon, breast, and pancreas, and they are an integral part of tumor cell signaling pathways associated with migration, proliferation, adhesion, and angiogenesis. We investigated whether the blockade of Src kinase by daily oral administration of the novel Src tyrosine kinase inhibitor AZM475271 [kindly provided by AstraZeneca (Macclesfield, United Kingdom)], alone or in combination with intraperitoneal gemcitabine, can inhibit growth and metastasis of orthotopically implanted human pancreatic carcinoma cells in nude mice. Treatment with AZM475271 alone reduced the primary pancreatic tumor volume by approximately 40%, whereas AZM475271 plus gemcitabine reduced tumor volume by 90%. Furthermore, treatment with AZM475271 and gemcitabine significantly reduced metastasis: none of eight animals who received the combination treatment had lymph node or liver metastases, compared with five of five and three of five animals, respectively, in the control group (P = 0.001). Src inhibition by AZM475271 (alone or with gemcitabine) was associated with significantly reduced tumor cell proliferation, decreased tumor microvessel density, and increased apoptosis in vivo. Moreover, these effects were all significantly increased when gemcitabine was combined with AZM475271 compared with gemcitabine alone. Src inhibition by AZM475271, either alone or in combination with gemcitabine, demonstrated significant antitumor and antimetastatic activity in an orthotopic nude mouse model for human pancreatic cancer. The combination of AZM475271 with gemcitabine sensitized tumor cells to the cytotoxic effect of gemcitabine.

  9. Parasite Mitogen-Activated Protein Kinases as Drug Discovery Targets to Treat Human Protozoan Pathogens

    PubMed Central

    Brumlik, Michael J.; Pandeswara, Srilakshmi; Ludwig, Sara M.; Murthy, Kruthi; Curiel, Tyler J.

    2011-01-01

    Protozoan pathogens are a highly diverse group of unicellular organisms, several of which are significant human pathogens. One group of protozoan pathogens includes obligate intracellular parasites such as agents of malaria, leishmaniasis, babesiosis, and toxoplasmosis. The other group includes extracellular pathogens such as agents of giardiasis and amebiasis. An unfortunate unifying theme for most human protozoan pathogens is that highly effective treatments for them are generally lacking. We will review targeting protozoan mitogen-activated protein kinases (MAPKs) as a novel drug discovery approach towards developing better therapies, focusing on Plasmodia, Leishmania, and Toxoplasma, about which the most is known. PMID:21637385

  10. Replication, checkpoint suppression and structure of centromeric DNA.

    PubMed

    Romeo, Francesco; Falbo, Lucia; Costanzo, Vincenzo

    2016-11-01

    Human centromeres contain large amounts of repetitive DNA sequences known as α satellite DNA, which can be difficult to replicate and whose functional role is unclear. Recently, we have characterized protein composition, structural organization and checkpoint response to stalled replication forks of centromeric chromatin reconstituted in Xenopus laevis egg extract. We showed that centromeric DNA has high affinity for SMC2-4 subunits of condensins and for CENP-A, it is enriched for DNA repair factors and suppresses the ATR checkpoint to ensure its efficient replication. We also showed that centromeric chromatin forms condensins enriched and topologically constrained DNA loops, which likely contribute to the overall structure of the centromere. These findings have important implications on how chromosomes are organized and genome stability is maintained in mammalian cells.

  11. Depletion of ATR selectively sensitizes ATM-deficient human mammary epithelial cells to ionizing radiation and DNA-damaging agents.

    PubMed

    Cui, Yuxia; Palii, Stela S; Innes, Cynthia L; Paules, Richard S

    2014-01-01

    DNA damage response (DDR) to double strand breaks is coordinated by 3 phosphatidylinositol 3-kinase-related kinase (PIKK) family members: the ataxia-telangiectasia mutated kinase (ATM), the ATM and Rad3-related (ATR) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs). ATM and ATR are central players in activating cell cycle checkpoints and function as an active barrier against genome instability and tumorigenesis in replicating cells. Loss of ATM function is frequently reported in various types of tumors, thus placing more reliance on ATR for checkpoint arrest and cell survival following DNA damage. To investigate the role of ATR in the G2/M checkpoint regulation in response to ionizing radiation (IR), particularly when ATM is deficient, cell lines deficient of ATM, ATR, or both were generated using a doxycycline-inducible lentiviral system. Our data suggests that while depletion of ATR or ATM alone in wild-type human mammary epithelial cell cultures (HME-CCs) has little effect on radiosensitivity or IR-induced G2/M checkpoint arrest, depletion of ATR in ATM-deficient cells causes synthetic lethality following IR, which correlates with severe G2/M checkpoint attenuation. ATR depletion also inhibits IR-induced autophagy, regardless of the ATM status, and enhances IR-induced apoptosis particularly when ATM is deficient. Collectively, our results clearly demonstrate that ATR function is required for the IR-induced G2/M checkpoint activation and subsequent survival of cells with ATM deficiency. The synthetic lethal interaction between ATM and ATR in response to IR supports ATR as a therapeutic target for improved anti-cancer regimens, especially in tumors with a dysfunctional ATM pathway.

  12. Chloroplast division checkpoint in eukaryotic algae

    PubMed Central

    Sumiya, Nobuko; Fujiwara, Takayuki; Era, Atsuko; Miyagishima, Shin-ya

    2016-01-01

    Chloroplasts evolved from a cyanobacterial endosymbiont. It is believed that the synchronization of endosymbiotic and host cell division, as is commonly seen in existing algae, was a critical step in establishing the permanent organelle. Algal cells typically contain one or only a small number of chloroplasts that divide once per host cell cycle. This division is based partly on the S-phase–specific expression of nucleus-encoded proteins that constitute the chloroplast-division machinery. In this study, using the red alga Cyanidioschyzon merolae, we show that cell-cycle progression is arrested at the prophase when chloroplast division is blocked before the formation of the chloroplast-division machinery by the overexpression of Filamenting temperature-sensitive (Fts) Z2-1 (Fts72-1), but the cell cycle progresses when chloroplast division is blocked during division-site constriction by the overexpression of either FtsZ2-1 or a dominant-negative form of dynamin-related protein 5B (DRP5B). In the cells arrested in the prophase, the increase in the cyclin B level and the migration of cyclin-dependent kinase B (CDKB) were blocked. These results suggest that chloroplast division restricts host cell-cycle progression so that the cell cycle progresses to the metaphase only when chloroplast division has commenced. Thus, chloroplast division and host cell-cycle progression are synchronized by an interactive restriction that takes place between the nucleus and the chloroplast. In addition, we observed a similar pattern of cell-cycle arrest upon the blockage of chloroplast division in the glaucophyte alga Cyanophora paradoxa, raising the possibility that the chloroplast division checkpoint contributed to the establishment of the permanent organelle. PMID:27837024

  13. Structure of the catalytic domain of human polo-like kinase 1.

    PubMed

    Kothe, Michael; Kohls, Darcy; Low, Simon; Coli, Rocco; Cheng, Alan C; Jacques, Suzanne L; Johnson, Theresa L; Lewis, Cristina; Loh, Christine; Nonomiya, Jim; Sheils, Alissa L; Verdries, Kimberly A; Wynn, Thomas A; Kuhn, Cyrille; Ding, Yuan-Hua

    2007-05-22

    Polo-like kinase 1 (Plk1) is an attractive target for the development of anticancer agents due to its importance in regulating cell-cycle progression. Overexpression of Plk1 has been detected in a variety of cancers, and expression levels often correlate with poor prognosis. Despite high interest in Plk1-targeted therapeutics, there is currently no structure publicly available to guide structure-based drug design of specific inhibitors. We determined the crystal structures of the T210V mutant of the kinase domain of human Plk1 complexed with the nonhydrolyzable ATP analogue adenylylimidodiphosphate (AMPPNP) or the pyrrolo-pyrazole inhibitor PHA-680626 at 2.4 and 2.1 A resolution, respectively. Plk1 adopts the typical kinase domain fold and crystallized in a conformation resembling the active state of other kinases. Comparison of the kinetic parameters determined for the (unphosphorylated) wild-type enzyme, as well as the T210V and T210D mutants, shows that the mutations primarily affect the kcat of the reaction, with little change in the apparent Km for the protein or nucleotide substrates (kcat = 0.0094, 0.0376, and 0.0049 s-1 and Km(ATP) = 3.2, 4.0, and 3.0 microM for WT, T210D, and T210V, respectively). The structure highlights features of the active site that can be exploited to obtain Plk1-specific inhibitors with selectivity over other kinases and Plk isoforms. These include the presence of a phenylalanine at the bottom of the ATP pocket, combined with a cysteine (as opposed to the more commonly found leucine) in the roof of the binding site, a pocket created by Leu132 in the hinge region, and a cluster of positively charged residues in the solvent-exposed area outside of the adenine pocket adjacent to the hinge region.

  14. Human Biliverdin Reductase Suppresses Goodpasture Antigen-binding Protein (GPBP) Kinase Activity

    PubMed Central

    Miralem, Tihomir; Gibbs, Peter E. M.; Revert, Fernando; Saus, Juan; Maines, Mahin D.

    2010-01-01

    The Ser/Thr/Tyr kinase activity of human biliverdin reductase (hBVR) and the expression of Goodpasture antigen-binding protein (GPBP), a nonconventional Ser/Thr kinase for the type IV collagen of basement membrane, are regulated by tumor necrosis factor (TNF-α). The pro-inflammatory cytokine stimulates kinase activity of hBVR and activates NF-κB, a transcriptional regulator of GPBP mRNA. Increased GPBP activity is associated with several autoimmune conditions, including Goodpasture syndrome. Here we show that in HEK293A cells hBVR binds to GPBP and down-regulates its TNF-α-stimulated kinase activity; this was not due to a decrease in GPBP expression. Findings with small interfering RNA to hBVR and to the p65 regulatory subunit of NF-κB show the hBVR role in the initial stimulation of GPBP expression by TNF-α-activated NF-κB; hBVR was not a factor in mediating GPBP mRNA stability. The interacting domain was mapped to the 281CX10C motif in the C-terminal 24 residues of hBVR. A 7-residue peptide, KKRILHC281, corresponding to the core of the consensus D(δ)-Box motif in the interacting domain, was as effective as the intact 296-residue hBVR polypeptide in inhibiting GPBP kinase activity. GPBP neither regulated hBVR expression nor TNF-α dependent NF-κB expression. Collectively, our data reveal that hBVR is a regulator of the TNF-α-GPBP-collagen type IV signaling cascade and uncover a novel biological interaction that may be of relevance in autoimmune pathogenesis. PMID:20177069

  15. Structural insight into nucleotide recognition by human death-associated protein kinase

    SciTech Connect

    McNamara, Laurie K.; Watterson, D. Martin; Brunzelle, Joseph S.

    2009-03-01

    The crystal structures of DAPK–ADP–Mg{sup 2+} and DAPK–AMP-PNP–Mg{sup 2+} complexes were determined at 1.85 and 2.00 Å resolution, respectively. Comparison of the two nucleotide-bound states with apo DAPK revealed localized changes in the glycine-rich loop region that were indicative of a transition from a more open state to a more closed state on binding of the nucleotide substrate and to an intermediate state with the bound nucleotide product. Death-associated protein kinase (DAPK) is a member of the Ca{sup 2+}/calmodulin-regulated family of serine/threonine protein kinases. The role of the kinase activity of DAPK in eukaryotic cell apoptosis and the ability of bioavailable DAPK inhibitors to rescue neuronal death after brain injury have made it a drug-discovery target for neurodegenerative disorders. In order to understand the recognition of nucleotides by DAPK and to gain insight into DAPK catalysis, the crystal structure of human DAPK was solved in complex with ADP and Mg{sup 2+} at 1.85 Å resolution. ADP is a product of the kinase reaction and product release is considered to be the rate-limiting step of protein kinase catalytic cycles. The structure of DAPK–ADP–Mg{sup 2+} was compared with a newly determined DAPK–AMP-PNP–Mg{sup 2+} structure and the previously determined apo DAPK structure (PDB code http://scripts.iucr.org/cgi-bin/cr.cgi?rm). The comparison shows that nucleotide-induced changes are localized to the glycine-rich loop region of DAPK.

  16. Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining.

    PubMed

    Jessulat, Matthew; Malty, Ramy H; Nguyen-Tran, Diem-Hang; Deineko, Viktor; Aoki, Hiroyuki; Vlasblom, James; Omidi, Katayoun; Jin, Ke; Minic, Zoran; Hooshyar, Mohsen; Burnside, Daniel; Samanfar, Bahram; Phanse, Sadhna; Freywald, Tanya; Prasad, Bhanu; Zhang, Zhaolei; Vizeacoumar, Franco; Krogan, Nevan J; Freywald, Andrew; Golshani, Ashkan; Babu, Mohan

    2015-07-01

    The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmid-based NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  17. Spindle Checkpoint Factors Bub1 and Bub2 Promote DNA Double-Strand Break Repair by Nonhomologous End Joining

    PubMed Central

    Jessulat, Matthew; Malty, Ramy H.; Nguyen-Tran, Diem-Hang; Deineko, Viktor; Aoki, Hiroyuki; Vlasblom, James; Omidi, Katayoun; Jin, Ke; Minic, Zoran; Hooshyar, Mohsen; Burnside, Daniel; Samanfar, Bahram; Phanse, Sadhna; Freywald, Tanya; Prasad, Bhanu; Zhang, Zhaolei; Vizeacoumar, Franco; Krogan, Nevan J.; Freywald, Andrew

    2015-01-01

    The nonhomologous end-joining (NHEJ) pathway is essential for the preservation of genome integrity, as it efficiently repairs DNA double-strand breaks (DSBs). Previous biochemical and genetic investigations have indicated that, despite the importance of this pathway, the entire complement of genes regulating NHEJ remains unknown. To address this, we employed a plasmid-based NHEJ DNA repair screen in budding yeast (Saccharomyces cerevisiae) using 369 putative nonessential DNA repair-related components as queries. Among the newly identified genes associated with NHEJ deficiency upon disruption are two spindle assembly checkpoint kinases, Bub1 and Bub2. Both observation of resulting phenotypes and chromatin immunoprecipitation demonstrated that Bub1 and -2, either alone or in combination with cell cycle regulators, are recruited near the DSB, where phosphorylated Rad53 or H2A accumulates. Large-scale proteomic analysis of Bub kinases phosphorylated in response to DNA damage identified previously unknown kinase substrates on Tel1 S/T-Q sites. Moreover, Bub1 NHEJ function appears to be conserved in mammalian cells. 53BP1, which influences DSB repair by NHEJ, colocalizes with human BUB1 and is recruited to the break sites. Thus, while Bub is not a core component of NHEJ machinery, our data support its dual role in mitotic exit and promotion of NHEJ repair in yeast and mammals. PMID:25963654

  18. Protein Kinase-A Inhibition Is Sufficient to Support Human Neural Stem Cells Self-Renewal.

    PubMed

    Georges, Pauline; Boissart, Claire; Poulet, Aurélie; Peschanski, Marc; Benchoua, Alexandra

    2015-12-01

    Human pluripotent stem cell-derived neural stem cells offer unprecedented opportunities for producing specific types of neurons for several biomedical applications. However, to achieve it, protocols of production and amplification of human neural stem cells need to be standardized, cost effective, and safe. This means that small molecules should progressively replace the use of media containing cocktails of protein-based growth factors. Here we have conducted a phenotypical screening to identify pathways involved in the regulation of hNSC self-renewal. We analyzed 80 small molecules acting as kinase inhibitors and identified compounds of the 5-isoquinolinesulfonamide family, described as protein kinase A (PKA) and protein kinase G inhibitors, as candidates to support hNSC self-renewal. Investigating the mode of action of these compounds, we found that modulation of PKA activity was central in controlling the choice between self-renewal or terminal neuronal differentiation of hNSC. We finally demonstrated that the pharmacological inhibition of PKA using the small molecule HA1004 was sufficient to support the full derivation, propagation, and long-term maintenance of stable hNSC in absence of any other extrinsic signals. Our results indicated that tuning of PKA activity is a core mechanism regulating hNSC self-renewal and differentiation and delineate the minimal culture media requirement to maintain undifferentiated hNSC in vitro. © 2015 AlphaMed Press.

  19. Structural basis of CX-4945 binding to human protein kinase CK2

    SciTech Connect

    Ferguson, Andrew D.; Sheth, Payal R.; Basso, Andrea D.; Paliwal, Sunil; Gray, Kimberly; Fischmann, Thierry O.; Le, Hung V.

    2012-02-07

    Protein kinase CK2 (CK2), a constitutively active serine/threonine kinase, is involved in a variety of roles essential to the maintenance of cellular homeostasis. Elevated levels of CK2 expression results in the dysregulation of key signaling pathways that regulate transcription, and has been implicated in cancer. The adenosine-5'-triphosphate-competitive inhibitor CX-4945 has been reported to show broad spectrum anti-proliferative activity in multiple cancer cell lines. Although the enzymatic IC{sub 50} of CX-4945 has been reported, the thermodynamics and structural basis of binding to CK2{alpha} remained elusive. Presented here are the crystal structures of human CK2{alpha} in complex with CX-4945 and adenylyl phosphoramidate at 2.7 and 1.3 {angstrom}, respectively. Biophysical analysis of CX-4945 binding is also described. This data provides the structural rationale for the design of more potent inhibitors against this emerging cancer target.

  20. Functional Significance of Aurora Kinases-p53 Protein Family Interactions in Cancer.

    PubMed

    Sasai, Kaori; Treekitkarnmongkol, Warapen; Kai, Kazuharu; Katayama, Hiroshi; Sen, Subrata

    2016-01-01

    Aurora kinases play critical roles in regulating spindle assembly, chromosome segregation, and cytokinesis to ensure faithful segregation of chromosomes during mitotic cell division cycle. Molecular and cell biological studies have revealed that Aurora kinases, at physiological levels, orchestrate complex sequential cellular processes at distinct subcellular locations through functional interactions with its various substrates. Aberrant expression of Aurora kinases, on the other hand, cause defects in mitotic spindle assembly, checkpoint response activation, and chromosome segregation leading to chromosomal instability. Elevated expression of Aurora kinases correlating with chromosomal instability is frequently detected in human cancers. Recent genomic profiling of about 3000 human cancer tissue specimens to identify various oncogenic signatures in The Cancer Genome Atlas project has reported that recurrent amplification and overexpression of Aurora kinase-A characterize distinct subsets of human tumors across multiple cancer types. Besides the well-characterized canonical pathway interactions of Aurora kinases in regulating assembly of the mitotic apparatus and chromosome segregation, growing evidence also supports the notion that deregulated expression of Aurora kinases in non-canonical pathways drive transformation and genomic instability by antagonizing tumor suppressor and exacerbating oncogenic signaling through direct interactions with critical proteins. Aberrant expression of the Aurora kinases-p53 protein family signaling axes appears to be critical in the abrogation of p53 protein family mediated tumor suppressor pathways frequently deregulated during oncogenic transformation process. Recent findings reveal the existence of feedback regulatory loops in mRNA expression and protein stability of these protein families and their consequences on downstream effectors involved in diverse physiological functions, such as mitotic progression, checkpoint response

  1. Extraction of human kinase mutations from literature, databases and genotyping studies.

    PubMed

    Krallinger, Martin; Izarzugaza, Jose M G; Rodriguez-Penagos, Carlos; Valencia, Alfonso

    2009-08-27

    There is a considerable interest in characterizing the biological role of specific protein residue substitutions through mutagenesis experiments. Additionally, recent efforts related to the detection of disease-associated SNPs motivated both the manual annotation, as well as the automatic extraction, of naturally occurring sequence variations from the literature, especially for protein families that play a significant role in signaling processes such as kinases. Systematic integration and comparison of kinase mutation information from multiple sources, covering literature, manual annotation databases and large-scale experiments can result in a more comprehensive view of functional, structural and disease associated aspects of protein sequence variants. Previously published mutation extraction approaches did not sufficiently distinguish between two fundamentally different variation origin categories, namely natural occurring and induced mutations generated through in vitro experiments. We present a literature mining pipeline for the automatic extraction and disambiguation of single-point mutation mentions from both abstracts as well as full text articles, followed by a sequence validation check to link mutations to their corresponding kinase protein sequences. Each mutation is scored according to whether it corresponds to an induced mutation or a natural sequence variant. We were able to provide direct literature links for a considerable fraction of previously annotated kinase mutations, enabling thus more efficient interpretation of their biological characterization and experimental context. In order to test the capabilities of the presented pipeline, the mutations in the protein kinase domain of the kinase family were analyzed. Using our literature extraction system, we were able to recover a total of 643 mutations-protein associations from PubMed abstracts and 6,970 from a large collection of full text articles. When compared to state-of-the-art annotation

  2. Digital cloning: identification of human cDNAs homologous to novel kinases through expressed sequence tag database searching.

    PubMed

    Chen, H C; Kung, H J; Robinson, D

    1998-01-01

    Identification of novel kinases based on their sequence conservation within kinase catalytic domain has relied so far on two major approaches, low-stringency hybridization of cDNA libraries, and PCR method using degenerate primers. Both of these approaches at times are technically difficult and time-consuming. We have developed a procedure that can significantly reduce the time and effort involved in searching for novel kinases and increase the sensitivity of the analysis. This procedure exploits the computer analysis of a vast resource of human cDNA sequences represented in the expressed sequence tag (EST) database. Seventeen novel human cDNA clones showing significant homology to serine/threonine kinases, including STE-20, CDK- and YAK-related family kinases, were identified by searching EST database. Further sequence analysis of these novel kinases obtained either directly from EST clones or from PCR-RACE products confirmed their identity as protein kinases. Given the rapid accumulation of the EST database and the advent of powerful computer analysis software, this approach provides a fast, sensitive, and economical way to identify novel kinases as well as other genes from EST database.

  3. Elucidation of the active conformation of the APS-kinase domain of human PAPS synthetase 1.

    PubMed

    Sekulic, Nikolina; Dietrich, Kristen; Paarmann, Ingo; Ort, Stephan; Konrad, Manfred; Lavie, Arnon

    2007-03-23

    Bifunctional human PAPS synthetase (PAPSS) catalyzes, in a two-step process, the formation of the activated sulfate carrier 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The first reaction involves the formation of the 5'-adenosine phosphosulfate (APS) intermediate from ATP and inorganic sulfate. APS is then further phosphorylated on its 3'-hydroxyl group by an additional ATP molecule to generate PAPS. The former reaction is catalyzed by the ATP-sulfurylase domain and the latter by the APS-kinase domain. Here, we report the structure of the APS-kinase domain of PAPSS isoform 1 (PAPSS1) representing the Michaelis complex with the products ADP-Mg and PAPS. This structure provides a rare glimpse of the active conformation of an enzyme catalyzing phosphoryl transfer without resorting to substrate analogs, inactivating mutations, or catalytically non-competent conditions. Our structure shows the interactions involved in the binding of the magnesium ion and PAPS, thereby revealing residues critical for catalysis. The essential magnesium ion is observed bridging the phosphate groups of the products. This function of the metal ion is made possible by the DGDN-loop changing its conformation from that previously reported, and identifies these loop residues unambiguously as a Walker B motif. Furthermore, the second aspartate residue of this motif is the likely candidate for initiating nucleophilic attack on the ATP gamma-phosphate group by abstracting the proton from the 3'-hydroxyl group of the substrate APS. We report the structure of the APS-kinase domain of human PAPSS1 in complex with two APS molecules, demonstrating the ability of the ATP/ADP-binding site to bind APS. Both structures reveal extended N termini that approach the active site of the neighboring monomer. Together, these results significantly increase our understandings of how catalysis is achieved by APS-kinase.

  4. Human cervical cancer cells use Ca2+ signalling, protein tyrosine phosphorylation and MAP kinase in regulatory volume decrease

    PubMed Central

    Shen, Meng-Ru; Chou, Cheng-Yang; Browning, Joseph A; Wilkins, Robert J; Ellory, J Clive

    2001-01-01

    This study was aimed at identifying the signalling pathways involved in the activation of volume-regulatory mechanisms of human cervical cancer cells. Osmotic swelling of human cervical cancer cells induced a substantial increase in intracellular Ca2+ ([Ca2+]i) by the activation of Ca2+ entry across the cell membrane, as well as Ca2+ release from intracellular stores. This Ca2+ signalling was critical for the normal regulatory volume decrease (RVD) response. The activation of swelling-activated ion and taurine transport was significantly inhibited by tyrosine kinase inhibitors (genistein and tyrphostin AG 1478) and potentiated by the tyrosine phosphatase inhibitor Na3VO4. However, the Src family of tyrosine kinases was not involved in regulation of the swelling-activated Cl− channel. Cell swelling triggered mitogen-activated protein (MAP) kinase cascades leading to the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) and p38 kinase. The volume-responsive ERK1/ERK2 signalling pathway linked with the activation of K+ and Cl− channels, and taurine transport. However, the volume-regulatory mechanism was independent of the activation of p38 MAP kinase. The phosphorylated ERK1/ERK2 expression following a hypotonic shock was up-regulated by protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and down-regulated by PKC inhibitor staurosporine. The response of ERK activation to hypotonicity also required Ca2+ entry and depended on tyrosine kinase and mitogen-activated/ERK-activating kinase (MEK) activity. Considering the results overall, osmotic swelling promotes the activation of tyrosine kinase and ERK1/ERK2 and raises intracellular Ca2+, all of which play a crucial role in the volume-regulatory mechanism of human cervical cancer cells. PMID:11731569

  5. Human cervical cancer cells use Ca2+ signalling, protein tyrosine phosphorylation and MAP kinase in regulatory volume decrease.

    PubMed

    Shen, M R; Chou, C Y; Browning, J A; Wilkins, R J; Ellory, J C

    2001-12-01

    1. This study was aimed at identifying the signalling pathways involved in the activation of volume-regulatory mechanisms of human cervical cancer cells. 2. Osmotic swelling of human cervical cancer cells induced a substantial increase in intracellular Ca2+ ([Ca2+]i) by the activation of Ca2+ entry across the cell membrane, as well as Ca2+ release from intracellular stores. This Ca2+ signalling was critical for the normal regulatory volume decrease (RVD) response. 3. The activation of swelling-activated ion and taurine transport was significantly inhibited by tyrosine kinase inhibitors (genistein and tyrphostin AG 1478) and potentiated by the tyrosine phosphatase inhibitor Na3VO4. However, the Src family of tyrosine kinases was not involved in regulation of the swelling-activated Cl- channel. 4. Cell swelling triggered mitogen-activated protein (MAP) kinase cascades leading to the activation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2) and p38 kinase. The volume-responsive ERK1/ERK2 signalling pathway linked with the activation of K+ and Cl- channels, and taurine transport. However, the volume-regulatory mechanism was independent of the activation of p38 MAP kinase. 5. The phosphorylated ERK1/ERK2 expression following a hypotonic shock was up-regulated by protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) and down-regulated by PKC inhibitor staurosporine. The response of ERK activation to hypotonicity also required Ca2+ entry and depended on tyrosine kinase and mitogen-activated/ERK-activating kinase (MEK) activity. 6. Considering the results overall, osmotic swelling promotes the activation of tyrosine kinase and ERK1/ERK2 and raises intracellular Ca2+, all of which play a crucial role in the volume-regulatory mechanism of human cervical cancer cells.

  6. Immune checkpoint therapy for pancreatic cancer

    PubMed Central

    Johansson, Henrik; Andersson, Roland; Bauden, Monika; Hammes, Sarah; Holdenrieder, Stefan; Ansari, Daniel

    2016-01-01

    Novel treatment modalities are necessary for pancreatic cancer. Immunotherapy with immune checkpoint inhibition has shown effect in other solid tumors, and could have a place in pancreatic cancer treatment. Most available clinical studies on immune checkpoint inhibitors for pancreatic cancer are not yet completed and are still recruiting patients. Among the completed trials, there have been findings of a preliminary nature such as delayed disease progression and enhanced overall survival after treatment with immune checkpoint inhibitors in mono- or combination therapy. However, due to small sample sizes, major results are not yet identifiable. The present article provides a clinical overview of immune checkpoint inhibition in pancreatic cancer. PubMed, ClinicalTrials.gov and American Society of Clinical Oncology’s meeting abstracts were systematically searched for relevant clinical studies. Four articles, five abstracts and 25 clinical trials were identified and analyzed in detail. PMID:27920468

  7. Optimal message log reclamation for uncoordinated checkpointing

    NASA Technical Reports Server (NTRS)

    Wang, Yi-Min; Fuchs, W. K.

    1994-01-01

    Uncoordinated checkpointing for message-passing systems allows maximum process autonomy and general nondeterministic execution, but suffers from potential domino effect and the large space overhead for maintaining checkpoints and message logs. Traditionally, it has been assumed that only obsolete checkpoints and message logs before the global recovery line can be garbage-collected. Recently, an approach to identifying all garbage checkpoints based on recovery line transformation and decomposition has been developed. We show in this paper that the same approach can be applied to the problem of identifying all garbage message logs for systems requiring message logging to record in-transit messages. Communication trace-driven simulation for several parallel programs is used to evaluate the proposed algorithm.

  8. Optimal message log reclamation for uncoordinated checkpointing

    NASA Technical Reports Server (NTRS)

    Wang, Yi-Min; Fuchs, W. K.

    1994-01-01

    Uncoordinated checkpointing for message-passing systems allows maximum process autonomy and general nondeterministic execution, but suffers from potential domino effect and the large space overhead for maintaining checkpoints and message logs. Traditionally, it has been assumed that only obsolete checkpoints and message logs before the global recovery line can be garbage-collected. Recently, an approach to identifying all garbage checkpoints based on recovery line transformation and decomposition has been developed. We show in this paper that the same approach can be applied to the problem of identifying all garbage message logs for systems requiring message logging to record in-transit messages. Communication trace-driven simulation for several parallel programs is used to evaluate the proposed algorithm.

  9. Crystal structure of the kinase domain of human protein tyrosine kinase 6 (PTK6) at 2.33 Å resolution.

    PubMed

    Thakur, Manish Kumar; Kumar, Amit; Birudukota, Swarnakumari; Swaminathan, Srinivasan; Tyagi, Rajiv; Gosu, Ramachandraiah

    2016-09-16

    Human Protein tyrosine kinase 6 (PTK6) (EC:2.7.10.2), also known as the breast tumor kinase (BRK), is an intracellular non-receptor Src-related tyrosine kinase expressed in a majority of human breast tumors and breast cancer cell lines, but its expression is low or completely absent in normal mammary glands. In the recent past, several studies have suggested that PTK6 is a potential therapeutic target in cancer. To understand its structural and functional properties, the PTK6 kinase domain (PTK6-KD) gene was cloned, overexpressed in a baculo-insect cell system, purified and crystallized at room temperature. X-ray diffraction data to 2.33 Å resolution was collected on a single PTK6-KD crystal, which belonged to the triclinic space group P1. The Matthews coefficient calculation suggested the presence of four protein molecules per asymmetric unit, with a solvent content of ∼50%.The structure has been solved by molecular replacement and crystal structure data submitted to the protein data bank under the accession number 5D7V. This is the first report of apo PTK6-KD structure crystallized in DFG-in and αC-helix-out conformation.

  10. ProNormz--an integrated approach for human proteins and protein kinases normalization.

    PubMed

    Subramani, Suresh; Raja, Kalpana; Natarajan, Jeyakumar

    2014-02-01

    The task of recognizing and normalizing protein name mentions in biomedical literature is a challenging task and important for text mining applications such as protein-protein interactions, pathway reconstruction and many more. In this paper, we present ProNormz, an integrated approach for human proteins (HPs) tagging and normalization. In Homo sapiens, a greater number of biological processes are regulated by a large human gene family called protein kinases by post translational phosphorylation. Recognition and normalization of human protein kinases (HPKs) is considered to be important for the extraction of the underlying information on its regulatory mechanism from biomedical literature. ProNormz distinguishes HPKs from other HPs besides tagging and normalization. To our knowledge, ProNormz is the first normalization system available to distinguish HPKs from other HPs in addition to gene normalization task. ProNormz incorporates a specialized synonyms dictionary for human proteins and protein kinases, a set of 15 string matching rules and a disambiguation module to achieve the normalization. Experimental results on benchmark BioCreative II training and test datasets show that our integrated approach achieve a fairly good performance and outperforms more sophisticated semantic similarity and disambiguation systems presented in BioCreative II GN task. As a freely available web tool, ProNormz is useful to developers as extensible gene normalization implementation, to researchers as a standard for comparing their innovative techniques, and to biologists for normalization and categorization of HPs and HPKs mentions in biomedical literature. URL: http://www.biominingbu.org/pronormz. Copyright © 2013 Elsevier Inc. All rights reserved.

  11. The DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) Are stimulated by bulky adduct-containing DNA.

    PubMed

    Kemp, Michael G; Lindsey-Boltz, Laura A; Sancar, Aziz

    2011-06-03

    A variety of environmental, carcinogenic, and chemotherapeutic agents form bulky lesions on DNA that activate DNA damage checkpoint signaling pathways in human cells. To identify the mechanisms by which bulky DNA adducts induce damage signaling, we developed an in vitro assay using mammalian cell nuclear extract and plasmid DNA containing bulky adducts formed by N-acetoxy-2-acetylaminofluorene or benzo(a)pyrene diol epoxide. Using this cell-free system together with a variety of pharmacological, genetic, and biochemical approaches, we identified the DNA damage response kinases DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) as bulky DNA damage-stimulated kinases that phosphorylate physiologically important residues on the checkpoint proteins p53, Chk1, and RPA. Consistent with these results, purified DNA-PK and ATM were directly stimulated by bulky adduct-containing DNA and preferentially associated with damaged DNA in vitro. Because the DNA damage response kinase ATM and Rad3-related (ATR) is also stimulated by bulky DNA adducts, we conclude that a common biochemical mechanism exists for activation of DNA-PK, ATM, and ATR by bulky adduct-containing DNA.

  12. Identification of human and rat FAD-AMP lyase (cyclic FMN forming) as ATP-dependent dihydroxyacetone kinases.

    PubMed

    Cabezas, Alicia; Costas, María Jesús; Pinto, Rosa María; Couto, Ana; Cameselle, José Carlos

    2005-12-30

    Rat liver FAD-AMP lyase or FMN cyclase is the only known enzymatic source of the unusual flavin nucleotide riboflavin 4',5'-cyclic phosphate. To determine its molecular identity, a peptide-mass fingerprint of the purified rat enzyme was obtained. It pointed to highly related, mammalian hypothetical proteins putatively classified as dihydroxyacetone (Dha) kinases due to weaker homologies to biochemically proven Dha kinases of plants, yeasts, and bacteria. The human protein LOC26007 cDNA was used to design PCR primers. The product amplified from human brain cDNA was cloned, sequenced (GenBank Accession No. ), and found to differ from protein LOC26007 cDNA by three SNPs. Its heterologous expression yielded a protein active both as FMN cyclase and ATP-dependent Dha kinase, each activity being inhibited by the substrate(s) of the other. Cyclase and kinase activities copurified from rat liver extracts. Evidence supports that a single protein sustains both activities, probably in a single active center. Putative Dha kinases from other mammals are likely to be FMN cyclases too. Future work will profit from the availability of the structure of Citrobacter freundii Dha kinase, which contains substrate-interacting residues conserved in human Dha kinase/FMN cyclase.

  13. Analysis of kinase gene expression patterns across 5681 human tissue samples reveals functional genomic taxonomy of the kinome.

    PubMed

    Kilpinen, Sami; Ojala, Kalle; Kallioniemi, Olli

    2010-12-03

    Kinases play key roles in cell signaling and represent major targets for drug development, but the regulation of their activation and their associations with health and disease have not been systematically analyzed. Here, we carried out a bioinformatic analysis of the expression levels of 459 human kinase genes in 5681 samples consisting of 44 healthy and 55 malignant human tissues. Defining the tissues where the kinase genes were transcriptionally active led to a functional genomic taxonomy of the kinome and a classification of human tissues and disease types based on the similarity of their kinome gene expression. The co-expression network around each of the kinase genes was defined in order to determine the functional context, i.e. the biological processes that were active in the cells and tissues where the kinase gene was expressed. Strong associations for individual kinases were found for mitosis (69 genes, including AURKA and BUB1), cell cycle control (73 genes, including PLK1 and AURKB), DNA repair (49 genes, including CHEK1 and ATR), immune response (72 genes, including MATK), neuronal (131 genes, including PRKCE) and muscular (72 genes, including MYLK2) functions. We then analyzed which kinase genes gain or lose transcriptional activity in the development of prostate and lung cancers and elucidated the functional associations of individual cancer associated kinase genes. In summary, we report here a systematic classification of kinases based on the bioinformatic analysis of their expression in human tissues and diseases, as well as grouping of tissues and tumor types according to the similarity of their kinome transcription.

  14. Analysis of Kinase Gene Expression Patterns across 5681 Human Tissue Samples Reveals Functional Genomic Taxonomy of the Kinome

    PubMed Central

    Kilpinen, Sami; Ojala, Kalle; Kallioniemi, Olli

    2010-01-01

    Kinases play key roles in cell signaling and represent major targets for drug development, but the regulation of their activation and their associations with health and disease have not been systematically analyzed. Here, we carried out a bioinformatic analysis of the expression levels of 459 human kinase genes in 5681 samples consisting of 44 healthy and 55 malignant human tissues. Defining the tissues where the kinase genes were transcriptionally active led to a functional genomic taxonomy of the kinome and a classification of human tissues and disease types based on the similarity of their kinome gene expression. The co-expression network around each of the kinase genes was defined in order to determine the functional context, i.e. the biological processes that were active in the cells and tissues where the kinase gene was expressed. Strong associations for individual kinases were found for mitosis (69 genes, including AURKA and BUB1), cell cycle control (73 genes, including PLK1 and AURKB), DNA repair (49 genes, including CHEK1 and ATR), immune response (72 genes, including MATK), neuronal (131 genes, including PRKCE) and muscular (72 genes, including MYLK2) functions. We then analyzed which kinase genes gain or lose transcriptional activity in the development of prostate and lung cancers and elucidated the functional associations of individual cancer associated kinase genes. In summary, we report here a systematic classification of kinases based on the bioinformatic analysis of their expression in human tissues and diseases, as well as grouping of tissues and tumor types according to the similarity of their kinome transcription. PMID:21151926

  15. Verotoxin activates mitogen-activated protein kinase in human peripheral blood monocytes: role in apoptosis and proinflammatory cytokine release

    PubMed Central

    Cameron, Pamela; Smith, Susan J; Giembycz, Mark A; Rotondo, Dino; Plevin, Robin

    2003-01-01

    In this study, we examined the role of mitogen-activated protein (MAP) kinases in the effects of verotoxins (VTs), from Escherichia coli O157:H7, upon both apoptosis and the release of tumour necrosis factor alpha (TNF-α) and granulocyte–macrophage colony-stimulated factor (GM-CSF) from human monocytes. Both VT1 and VT2 stimulated a weak, transient increase in c-Jun-N-terminal kinase (JNK) activity and a strong activation of both p38 mitogen-activated protein kinase (MAP kinase) and extracellular-regulated kinase (ERK) activity in human monocytes, which was sustained in the case of p38 MAP kinase. Stimulation of human monocytes with VT2 (100 ng ml−1) did not result in an increase in apoptosis; however, the toxin stimulated the release of both TNF-α and GM-CSF. Pretreatment of human monocytes with the p38 MAP kinase inhibitor SB203580, at concentrations from 100 nM to 10 μM, significantly decreased the VT1- and VT2-induced TNF-α and GM-CSF release from monocytes. In contrast, inhibition of MEK1 with PD98059 only significantly decreased GM-CSF release. Pretreatment of monocytes with SP600125 inhibited both GM-CSF and TNF-α production; however, significant effects upon p38 MAP kinase and ERK activation were observed. Taken together, these results suggest a role for p38 MAP kinase and ERK in cytokine generation in response to the verotoxins. A role for JNK remains undetermined. PMID:14597601

  16. Dietary flavonoid fisetin induces a forced exit from mitosis by targeting the mitotic spindle checkpoint

    PubMed Central

    Salmela, Anna-Leena; Pouwels, Jeroen; Varis, Asta; Kukkonen, Anu M.; Toivonen, Pauliina; Halonen, Pasi K.; Perälä, Merja; Kallioniemi, Olli; Gorbsky, Gary J.; Kallio, Marko J.

    2009-01-01

    Fisetin is a natural flavonol present in edible vegetables, fruits and wine at 2–160 μg/g concentrations and an ingredient in nutritional supplements with much higher concentrations. The compound has been reported to exert anticarcinogenic effects as well as antioxidant and anti-inflammatory activity via its ability to act as an inhibitor of cell proliferation and free radical scavenger, respectively. Our cell-based high-throughput screen for small molecules that override chemically induced mitotic arrest identified fisetin as an antimitotic compound. Fisetin rapidly compromised microtubule drug-induced mitotic block in a proteasome-dependent manner in several human cell lines. Moreover, in unperturbed human cancer cells fisetin caused premature initiation of chromosome segregation and exit from mitosis without normal cytokinesis. To understand the molecular mechanism behind these mitotic errors, we analyzed the consequences of fisetin treatment on the localization and phoshorylation of several mitotic proteins. Aurora B, Bub1, BubR1 and Cenp-F rapidly lost their kinetochore/centromere localization and others became dephosphorylated upon addition of fisetin to the culture medium. Finally, we identified Aurora B kinase as a novel direct target of fisetin. The activity of Aurora B was significantly reduced by fisetin in vitro and in cells, an effect that can explain the observed forced mitotic exit, failure of cytokinesis and decreased cell viability. In conclusion, our data propose that fisetin perturbs spindle checkpoint signaling, which may contribute to the antiproliferative effects of the compound. PMID:19395653

  17. A mechanism for tunable autoinhibition in the structure of a human Ca2+/calmodulin-dependent kinase II holoenzyme

    PubMed Central

    Chao, Luke H.; Stratton, Margaret M.; Lee, Il-Hyung; Rosenberg, Oren S.; Levitz, Joshua; Mandell, Daniel J.; Kortemme, Tanja; Groves, Jay T.; Schulman, Howard; Kuriyan, John

    2011-01-01

    Summary Calcium/calmodulin-dependent kinase II (CaMKII) forms a highly conserved dodecameric assembly that is sensitive to the frequency of calcium pulse trains. Neither the structure of the dodecameric assembly nor how it regulates CaMKII are known. We present the crystal structure of an autoinhibited full-length human CaMKII holoenzyme, revealing an unexpected compact arrangement of kinase domains docked against a central hub, with the calmodulin binding sites completely inaccessible. We show that this compact docking is important for the autoinhibition of the kinase domains and for setting the calcium response of the holoenzyme. Comparison of CaMKII isoforms, which differ in the length of the linker between the kinase domain and the hub, demonstrates that these interactions can be strengthened or weakened by changes in linker length. This equilibrium between autoinhibited states provides a simple mechanism for tuning the calcium response without changes in either the hub or the kinase domains. PMID:21884935

  18. Knockdown of Minichromosome Maintenance Proteins Inhibits Foci Forming of Mediator of DNA-Damage Checkpoint 1 in Response to DNA Damage in Human Esophageal Squamous Cell Carcinoma TE-1 Cells.

    PubMed

    Yu, Jinzhong; Wang, Ruijie; Wu, Jinfeng; Dang, Zhongqin; Zhang, Qinsheng; Li, Bo

    2016-10-01

    Esophageal squamous cell carcinoma (ESCC) has a high morbidity in China and its treatment depends greatly on adjuvant chemotherapy. However, DNA damage repair in cancer cells severely affects the outcome of treatment. This study investigated the potential mechanism regarding mediator of DNA-damage checkpoint 1 (MDC1) and minichromosome maintenance proteins (MCMs) during DNA damage in ESCC. Recombinant vectors of MDC1 and MCMs with tags were constructed and transfected into human ESCC cell line TE-1. Immunoprecipitation and mass spectrometry were performed to screen the MCMs interacting with MDC1, and direct interaction was confirmed by glutathione S-transferase (GST) pull-down assay in vitro. MCM2 and MCM6 were knocked down by shRNAs, after which chromatin fraction and foci forming of MDC1 upon bleomycin-induced DNA damage were examined. The results showed that MCM2/3/5/6 were immunoprecipitated by antibodies against the tag of MDC1 in TE-1 nuclei, and the GST pull-down assay indicated the direct interaction. Knockdown of MCM2 or MCM6 reduced the chromatin fraction of MDC1 according to Western blot results. Moreover, knockdown of MCM2 or MCM6 could significantly inhibit foci forming of MDC1 in TE-1 nuclei in response to bleomycin-induced DNA damage (p < 0.001). This study indicates the direct interaction between MDC1 and MCMs in TE-1 nuclei. Downregulation of MCMs can inhibit chromatin fraction and foci forming of MDC1 in TE-1 cells upon DNA damage, which suggests MCMs and MDC1 as potential targets to improve the outcome of chemotherapy in ESCC.

  19. Toward an optimal online checkpoint solution under a two-level HPC checkpoint model

    DOE PAGES

    Di, Sheng; Robert, Yves; Vivien, Frederic; ...

    2016-03-29

    The traditional single-level checkpointing method suffers from significant overhead on large-scale platforms. Hence, multilevel checkpointing protocols have been studied extensively in recent years. The multilevel checkpoint approach allows different levels of checkpoints to be set (each with different checkpoint overheads and recovery abilities), in order to further improve the fault tolerance performance of extreme-scale HPC applications. How to optimize the checkpoint intervals for each level, however, is an extremely difficult problem. In this paper, we construct an easy-to-use two-level checkpoint model. Checkpoint level 1 deals with errors with low checkpoint/recovery overheads such as transient memory errors, while checkpoint level 2more » deals with hardware crashes such as node failures. Compared with previous optimization work, our new optimal checkpoint solution offers two improvements: (1) it is an online solution without requiring knowledge of the job length in advance, and (2) it shows that periodic patterns are optimal and determines the best pattern. We evaluate the proposed solution and compare it with the most up-to-date related approaches on an extreme-scale simulation testbed constructed based on a real HPC application execution. Simulation results show that our proposed solution outperforms other optimized solutions and can improve the performance significantly in some cases. Specifically, with the new solution the wall-clock time can be reduced by up to 25.3% over that of other state-of-the-art approaches. Lastly, a brute-force comparison with all possible patterns shows that our solution is always within 1% of the best pattern in the experiments.« less

  20. Toward an optimal online checkpoint solution under a two-level HPC checkpoint model

    SciTech Connect

    Di, Sheng; Robert, Yves; Vivien, Frederic; Cappello, Franck

    2016-03-29

    The traditional single-level checkpointing method suffers from significant overhead on large-scale platforms. Hence, multilevel checkpointing protocols have been studied extensively in recent years. The multilevel checkpoint approach allows different levels of checkpoints to be set (each with different checkpoint overheads and recovery abilities), in order to further improve the fault tolerance performance of extreme-scale HPC applications. How to optimize the checkpoint intervals for each level, however, is an extremely difficult problem. In this paper, we construct an easy-to-use two-level checkpoint model. Checkpoint level 1 deals with errors with low checkpoint/recovery overheads such as transient memory errors, while checkpoint level 2 deals with hardware crashes such as node failures. Compared with previous optimization work, our new optimal checkpoint solution offers two improvements: (1) it is an online solution without requiring knowledge of the job length in advance, and (2) it shows that periodic patterns are optimal and determines the best pattern. We evaluate the proposed solution and compare it with the most up-to-date related approaches on an extreme-scale simulation testbed constructed based on a real HPC application execution. Simulation results show that our proposed solution outperforms other optimized solutions and can improve the performance significantly in some cases. Specifically, with the new solution the wall-clock time can be reduced by up to 25.3% over that of other state-of-the-art approaches. Lastly, a brute-force comparison with all possible patterns shows that our solution is always within 1% of the best pattern in the experiments.

  1. Role of phosphoinositide 3-kinase in the aggressive tumor growth of HT1080 human fibrosarcoma cells.

    PubMed

    Gupta, S; Stuffrein, S; Plattner, R; Tencati, M; Gray, C; Whang, Y E; Stanbridge, E J

    2001-09-01

    We have developed a model system of human fibrosarcoma cell lines that do or do not possess and express an oncogenic mutant allele of N-ras. HT1080 cells contain an endogenous mutant allele of N-ras, whereas the derivative MCH603 cell line contains only wild-type N-ras. In an earlier study (S. Gupta et al., Mol. Cell. Biol. 20:9294-9306, 2000), we had shown that HT1080 cells produce rapidly growing, aggressive tumors in athymic nude mice, whereas MCH603 cells produced more slowly growing tumors and was termed weakly tumorigenic. An extensive analysis of the Ras signaling pathways (Raf, Rac1, and RhoA) provided evidence for a potential novel pathway that was critical for the aggressive tumorigenic phenotype and could be activated by elevated levels of constitutively active MEK. In this study we examined the role of phosphoinositide 3-kinase (PI 3-kinase) in the regulation of the transformed and aggressive tumorigenic phenotypes expressed in HT1080 cells. Both HT1080 (mutant N-ras) and MCH603 (wild-type N-ras) have similar levels of constitutively active Akt, a downstream target of activated PI 3-kinase. We find that both cell lines constitutively express platelet-derived growth factor (PDGF) and PDGF receptors. Transfection with tumor suppressor PTEN cDNA into HT1080 and constitutively active PI 3-kinase-CAAX cDNA into MCH603 cells, respectively, resulted in several interesting and novel observations. Activation of the PI 3-kinase/Akt pathway, including NF-kappaB, is not required for the aggressive tumorigenic phenotype in HT1080 cells. Activation of NF-kappaB is complex: in MCH603 cells it is mediated by Akt, whereas in HT1080 cells activation also involves other pathway(s) that are activated by mutant Ras. A threshold level of activation of PI 3-kinase is required in MCH603 cells before stimulatory cross talk to the RhoA, Rac1, and Raf pathways occurs, without a corresponding activation of Ras. The increased levels of activation seen were similar to those observed

  2. A proteomic analysis of ataxia telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) substrates identifies the ubiquitin-proteasome system as a regulator for DNA damage checkpoints.

    PubMed

    Mu, Jung-Jung; Wang, Yi; Luo, Hao; Leng, Mei; Zhang, Jinglan; Yang, Tao; Besusso, Dario; Jung, Sung Yun; Qin, Jun

    2007-06-15

    ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) are proximal checkpoint kinases that regulate DNA damage response (DDR). Identification and characterization of ATM/ATR substrates hold the keys for the understanding of DDR. Few techniques are available to identify protein kinase substrates. Here, we screened for potential ATM/ATR substrates using phospho-specific antibodies against known ATM/ATR substrates. We identified proteins cross-reacting to phospho-specific antibodies in response to DNA damage by mass spectrometry. We validated a subset of the candidate substrates to be phosphorylated in an ATM/ATR-dependent manner in vivo. Combining with a functional checkpoint screen, we identified proteins that belong to the ubiquitin-proteasome system (UPS) to be required in mammalian DNA damage checkpoint control, particularly the G(1) cell cycle checkpoint, thus revealing protein ubiquitylation as an important regulatory mechanism downstream of ATM/ATR activation for checkpoint control.

  3. Sulforaphane prevents human platelet aggregation through inhibiting the phosphatidylinositol 3-kinase/Akt pathway.

    PubMed

    Chuang, Wen-Ying; Kung, Po-Hsiung; Kuo, Chih-Yun; Wu, Chin-Chung

    2013-06-01

    Sulforaphane, a dietary isothiocyanate found in cruciferous vegetables, has been shown to exert beneficial effects in animal models of cardiovascular diseases. However, its effect on platelet aggregation, which is a critical factor in arterial thrombosis, is still unclear. In the present study, we show that sulforaphane inhibited human platelet aggregation caused by different receptor agonists, including collagen, U46619 (a thromboxane A2 mimic), protease-activated receptor 1 agonist peptide (PAR1-AP), and an ADP P2Y12 receptor agonist. Moreover, sulforaphane significantly reduced thrombus formation on a collagen-coated surface under whole blood flow conditions. In exploring the underlying mechanism, we found that sulforaphane specifically prevented phosphatidylinositol 3-kinase (PI3K)/Akt signalling, without markedly affecting other signlaling pathways involved in platelet aggregation, such as protein kinase C activation, calcium mobilisation, and protein tyrosine phosphorylation. Although sulforaphane did not directly inhibit the catalytic activity of PI3K, it caused ubiquitination of the regulatory p85 subunit of PI3K, and prevented PI3K translocation to membranes. In addition, sulforaphane caused ubiquitination and degradation of phosphoinositide-dependent kinase 1 (PDK1), which is required for Akt activation. Therefore, sulforaphane is able to inhibit the PI3K/Akt pathway at two distinct sites. In conclusion, we have demonstrated that sulforaphane prevented platelet aggregation and reduced thrombus formation in flow conditions; our data also support that the inhibition of the PI3K/Akt pathway by sulforaphane contributes it antiplatelet effects.

  4. Atypical Protein Kinase Cι as a human oncogene and therapeutic target

    PubMed Central

    Parker, Peter J.; Justilien, Verline; Riou, Philippe; Linch, Mark; Fields, Alan P.

    2014-01-01

    Protein kinase inhibitors represent a major class of targeted therapeutics that has made a positive impact on treatment of cancer and other disease indications. Among the promising kinase targets for further therapeutic development are members of the Protein Kinase C (PKC) family.The PKCs are central components of many signaling pathways that regulate diverse cellular functions including proliferation, cell cycle, differentiation, survival, cell migration, and polarity. Genetic manipulation of individual PKC isozymes has demonstrated that they often fulfill distinct, nonredundant cellular functions.11 Participation of PKC members in different intracellular signaling pathways reflects responses to varying extracellular stimuli, intracellular localization, tissue distribution, phosphorylation status, and intermolecular interactions. PKC activity, localization, phosphorylation, and/or expression are often altered in human tumors, and PKC isozymes have been implicated in various aspects of transformation, including uncontrolled proliferation, migration, invasion, metastasis, angiogenesis, and resistance to apoptosis. Despite the strong relationship between PKC isozymes and cancer, to date only atypical PKCiota has been shown to function as a bona fide oncogene, and as such is a particularly attractive therapeutic target for cancer treatment. In this review, we discuss the role of PKCiota in transformation and describe mechanism-based approaches to therapeutically target oncogenic PKCiota signaling in cancer. PMID:24231509

  5. Expression and regulation of glycogen synthase kinase 3 in human neutrophils.

    PubMed

    Giambelluca, Miriam S; Cloutier, Nathalie; Rollet-Labelle, Emmanuelle; Boilard, Eric; Pouliot, Marc

    2013-11-01

    Glycogen synthase kinase 3 (GSK-3) is a serine/threonine kinase involved in the regulation of cellular processes ranging from glycogen metabolism to cell cycle regulation. Its two known isoforms, α and β, are differentially expressed in tissues throughout the body and exert distinct but often overlapping functions. GSK-3 is typically active in resting cells, inhibition by phosphorylation of Ser21 (GSK-3α) or Ser9 (GSK-3β) being the most common regulatory mechanism. GSK-3 activity has been linked recently with immune system function, yet little is known about the role of this enzyme in neutrophils, the most abundant leukocyte type. In the present study, we examined GSK-3 expression and regulation in human neutrophils. GSK-3α was found to be the predominant isoform, it was constitutively expressed and cell stimulation with different agonists did not alter its expression. Stimulation by fMLP, LPS, GM-CSF, Fcγ receptor engagement, or adenosine A2A receptor engagement all resulted in phosphorylation of Ser21. The use of metabolic inhibitors revealed that combinations of Src kinase, PKC, PI3K/AKT, ERK/RSK and PKA signaling pathways could mediate phosphorylation, depending on the agonist. Neither PLC nor p38 were involved. We conclude that GSK-3α is the main isoform expressed in neutrophils and that many different pathways can converge to inhibit GSK-3α activity via Ser21-phosphorylation. GSK-3α thus might be a hub of cellular regulation.

  6. Human Placental Lactogen Induces CYP2E1 Expression via PI 3-Kinase Pathway in Female Human Hepatocytes

    PubMed Central

    Lee, Jin Kyung; Chung, Hye Jin; Fischer, Liam; Fischer, James; Gonzalez, Frank J.

    2014-01-01

    The state of pregnancy is known to alter hepatic drug metabolism. Hormones that rise during pregnancy are potentially responsible for the changes. Here we report the effects of prolactin (PRL), placental lactogen (PL), and growth hormone variant (GH-v) on expression of major hepatic cytochromes P450 expression and a potential molecular mechanism underlying CYP2E1 induction by PL. In female human hepatocytes, PRL and GH-v showed either no effect or small and variable effects on mRNA expression of CYP1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1, 3A4, and 3A5. On the other hand, PL increased expression level of CYP2E1 mRNA with corresponding increases in CYP2E1 protein and activity levels. Results from hepatocytes and HepaRG cells indicate that PL does not affect the expression or activity of HNF1α, the known transcriptional activator of basal CYP2E1 expression. Furthermore, transient transfection studies and Western blot results showed that STAT signaling, the previously known mediator of PL actions in certain tissues, does not play a role in CYP2E1 induction by PL. A chemical inhibitor of PI3-kinase signaling significantly repressed the CYP2E1 induction by PL in human hepatocytes, suggesting involvement of PI3-kinase pathway in CYP2E1 regulation by PL. CYP2E1-humanized mice did not exhibit enhanced CYP2E1 expression during pregnancy, potentially because of interspecies differences in PL physiology. Taken together, these results indicate that PL induces CYP2E1 expression via PI3-kinase pathway in human hepatocytes. PMID:24408518

  7. Sphingosine-1-phosphate stimulates human glioma cell proliferation through Gi-coupled receptors: role of ERK MAP kinase and phosphatidylinositol 3-kinase beta.

    PubMed

    Van Brocklyn, James; Letterle, Catherine; Snyder, Pamela; Prior, Thomas

    2002-07-26

    The regulation of glioma cell proliferation by sphingosine-1-phosphate (S1P) was studied using the human glioblastoma cell line U-373 MG. U-373 MG cells responded mitogenically to nanomolar concentrations of S1P, and express mRNA encoding the S1P receptors S1P1/endothelial differentiation gene (EDG)-1, S1P3/EDG-3 and S1P2/EDG-5. S1P-induced proliferation required extracellular signal-regulated kinase activation and was partially sensitive to pertussis toxin and wortmannin, indicating involvement of a Gi-coupled receptor and phosphatidylinositol 3-kinase. Moreover, S1P1, S1P3 and S1P2 receptors are expressed in the majority of human glioblastomas as determined by reverse transcriptase-polymerase chain reaction analysis. Thus, S1P signaling through EDG receptors may contribute to glioblastoma growth in vivo.

  8. FRMD6 inhibits human glioblastoma growth and progression by negatively regulating activity of receptor tyrosine kinases

    PubMed Central

    Xu, Yin; Wang, Kaiqiang; Yu, Qin

    2016-01-01

    FRMD6 is an Ezrin/Radixin/Moesin (ERM) family protein and a human homologue of Drosophila expanded (ex). Ex functions in parallel of Drosophila merlin at upstream of the Hippo signaling pathway that controls proliferation, apoptosis, tissue regeneration, and tumorigenesis. Even though the core kinase cascade (MST1/2-Lats1/2-YAP/TAZ) of the Hippo pathway has been well established, its upstream regulators are not well understood. Merlin promotes activation of the Hippo pathway. However, the effect of FRMD6 on the Hippo pathway is controversial. Little is known about how FRMD6 functions and the potential role of FRMD in gliomagenesis and glioblastoma (GBM) progression. We demonstrate for the first time that FRMD6 is down-regulated in human GBM cells and tissues and that increased FRMD6 expression inhibits whereas FRMD6 knockdown promotes GBM cell proliferation/invasion in vitro and GBM growth/progression in vivo. Furthermore, we demonstrate that unlike increased expression of merlin, which enhances the stress induced activation of the Hippo pathway, increased FRMD6 expression displays little effect on the pathway. In contrast, we show that FRMD6 inhibits activation of a couple of receptor tyrosine kinases (RTKs) including c-Met and PDGFR and their downstream Erk and AKT kinases. Moreover, we show that expression of constitutively active c-Met, the TPR-Met fusion protein, largely reverses the anti-GBM effect of FRMD6 in vivo, suggesting that FRMD6 functions at least partially through inhibiting activity of RTKs especially c-Met. These results establish a novel function of FRMD6 in inhibiting human GBM growth and progression and uncover a novel mechanism by which FRMD6 exerts its anti-GBM activity. PMID:27661120

  9. Quantitative network mapping of the human kinome interactome reveals new clues for rational kinase inhibitor discovery and individualized cancer therapy

    PubMed Central

    Cheng, Feixiong; Jia, Peilin; Wang, Quan; Zhao, Zhongming

    2014-01-01

    The human kinome is gaining importance through its promising cancer therapeutic targets, yet no general model to address the kinase inhibitor resistance has emerged. Here, we constructed a systems biology-based framework to catalogue the human kinome, including 538 kinase genes, in the broader context of the human interactome. Specifically, we constructed three networks: a kinase-substrate interaction network containing 7,346 pairs connecting 379 kinases to 36,576 phosphorylation sites in 1,961 substrates, a protein-protein interaction network (PPIN) containing 92,699 pairs, and an atomic resolution PPIN containing 4,278 pairs. We identified the conserved regulatory phosphorylation motifs (e.g., Ser/Thr-Pro) using a sequence logo analysis. We found the typical anticancer target selection strategy that uses network hubs as drug targets, might lead to a high adverse drug reaction risk. Furthermore, we found the distinct network centrality of kinases creates a high anticancer drug resistance risk by feedback or crosstalk mechanisms within cellular networks. This notion is supported by the systematic network and pathway analyses that anticancer drug resistance genes are significantly enriched as hubs and heavily participate in multiple signaling pathways. Collectively, this comprehensive human kinome interactome map sheds light on anticancer drug resistance mechanisms and provides an innovative resource for rational kinase inhibitor design. PMID:25003367

  10. Zinc differentially regulates mitogen-activated protein kinases in human T cells.

    PubMed

    Hönscheid, Andrea; Dubben, Svenja; Rink, Lothar; Haase, Hajo

    2012-01-01

    Zinc is an essential nutrient with remarkable importance for immunity, in particular for T-cell function. This is, at least in part, based on an involvement of zinc ions in immune cell signal transduction; dynamic changes of the intracellular free zinc concentration have recently been recognized as signaling events. Because the molecular targets of zinc signals remain incompletely understood, we investigated the impact of elevated intracellular free zinc on mitogen-activated protein kinase (MAPK) activity and MAPK-dependent cytokine production in human T-cells. p38 was activated by treatment with zinc and the ionophore pyrithione, whereas ERK1/2 and c-Jun N-terminal kinases were unaffected. In contrast, after T-cell receptor stimulation with antibodies against CD3, ERK1/2-phosphorylation was selectively suppressed by intracellular zinc. Mechanisms that had been shown to mediate zinc-effects in other cells, such as activation of the Src kinase Lck, inhibition of the protein tyrosine phosphatase CD45 or MAPK phosphatases and cyclic nucleotide/protein kinase A signaling were not involved. This indicates that the differential impact of zinc on the MAPK families in T-cells is mediated by mechanisms that differ from the ones observed in other cell types. Further investigation of the activation of p38 by zinc demonstrated that this MAPK is responsible for the zinc-mediated activation of CREB and mRNA expression of the Th1 cytokines interferon-gamma and interleukin-2. In conclusion, regulation of MAPK activity contributes to the impact of zinc on T-cell function.

  11. Insights into the Phosphoryl Transfer Mechanism of Human Ubiquitous Mitochondrial Creatine Kinase.

    PubMed

    Li, Quanjie; Fan, Shuai; Li, Xiaoyu; Jin, Yuanyuan; He, Weiqing; Zhou, Jinming; Cen, Shan; Yang, ZhaoYong

    2016-12-02

    Human ubiquitous mitochondrial creatine kinase (uMtCK) is responsible for the regulation of cellular energy metabolism. To investigate the phosphoryl-transfer mechanism catalyzed by human uMtCK, in this work, molecular dynamic simulations of uMtCK∙ATP-Mg(2+)∙creatine complex and quantum mechanism calculations were performed to make clear the puzzle. The theoretical studies hereof revealed that human uMtCK utilizes a two-step dissociative mechanism, in which the E227 residue of uMtCK acts as the catalytic base to accept the creatine guanidinium proton. This catalytic role of E227 was further confirmed by our assay on the phosphatase activity. Moreover, the roles of active site residues in phosphoryl transfer reaction were also identified by site directed mutagenesis. This study reveals the structural basis of biochemical activity of uMtCK and gets insights into its phosphoryl transfer mechanism.

  12. Insights into the Phosphoryl Transfer Mechanism of Human Ubiquitous Mitochondrial Creatine Kinase

    PubMed Central

    Li, Quanjie; Fan, Shuai; Li, Xiaoyu; Jin, Yuanyuan; He, Weiqing; Zhou, Jinming; Cen, Shan; Yang, ZhaoYong

    2016-01-01

    Human ubiquitous mitochondrial creatine kinase (uMtCK) is responsible for the regulation of cellular energy metabolism. To investigate the phosphoryl-transfer mechanism catalyzed by human uMtCK, in this work, molecular dynamic simulations of uMtCK∙ATP-Mg2+∙creatine complex and quantum mechanism calculations were performed to make clear the puzzle. The theoretical studies hereof revealed that human uMtCK utilizes a two-step dissociative mechanism, in which the E227 residue of uMtCK acts as the catalytic base to accept the creatine guanidinium proton. This catalytic role of E227 was further confirmed by our assay on the phosphatase activity. Moreover, the roles of active site residues in phosphoryl transfer reaction were also identified by site directed mutagenesis. This study reveals the structural basis of biochemical activity of uMtCK and gets insights into its phosphoryl transfer mechanism. PMID:27909311

  13. Ornithine decarboxylase, mitogen-activated protein kinase and matrix metalloproteinase-2 expressions in human colon tumors

    PubMed Central

    Nemoto, Takahiro; Kubota, Shunichiro; Ishida, Hideyuki; Murata, Nobuo; Hashimoto, Daijo

    2005-01-01

    AIM: To investigate the expressions of ornithine decarboxylase (ODC), MMP-2, and Erk, and their relationship in human colon tumors. METHODS: ODC activity, MMP-2 expression, and mitogen-activated protein (MAP) kinase activity (Erk phosphorylation) were determined in 58 surgically removed human colon tumors and their adjacent normal tissues, using [1-14C]-ornithine as a substrate, ELISA assay, and Western blotting, respectively. RESULTS: ODC activity, MMP-2 expression, and Erk phosphorylation were significantly elevated in colon tumors, compared to those in adjacent normal tissues. A significant correlation was observed between ODC activities and MMP-2 levels. CONCLUSION: This is the first report showing a significant correlation between ODC activities and MMP-2 levels in human colon tumors. As MMP-2 is involved in cancer invasion and metastasis, and colon cancer overexpresses ODC, suppression of ODC expression may be a rational approach to treat colon cancer which overexpresses ODC. PMID:15918191

  14. Dematin, a human erythrocyte cytoskeletal protein, is a substrate for a recombinant FIKK kinase from Plasmodium falciparum.

    PubMed

    Brandt, Gabriel S; Bailey, Scott

    2013-09-01

    P. falciparum causes the most deadly form of malaria, resulting from the adherence of infected red blood cells to blood vessels. During the blood stage of infection, the parasite secretes a large number of proteins into the host erythrocyte. The secretion of a 20-member family of protein kinases known as FIKK kinases, after a conserved Phe-Ile-Lys-Lys sequence motif, is unique to P. falciparum. Identification of physiological substrates of these kinases may provide perspective on the importance of FIKK kinase activity to P. falciparum virulence. We demonstrate, for the first time, the heterologous expression and purification of a FIKK kinase (PfFk4.1, PFD1165w). The recombinant kinase is active against general substrates and phosphorylates itself. Having demonstrated kinase activity, we incubated recombinant Fk4.1 with parasite and human erythrocyte lysates. No parasite-derived substrates were identified. However, treatment of erythrocyte ghosts shows that the FIKK kinase Fk4.1 phosphorylates dematin, a cytoskeletal protein found at the red blood cell spectrin-actin junction.

  15. Molecular docking and NMR binding studies to identify novel inhibitors of human phosphomevalonate kinase

    SciTech Connect

    Boonsri, Pornthip; Neumann, Terrence S.; Olson, Andrew L.; Cai, Sheng; Herdendorf, Timothy J.; Miziorko, Henry M.; Hannongbua, Supa; Sem, Daniel S.

    2013-01-04

    Highlights: Black-Right-Pointing-Pointer Natural and synthetic inhibitors of human phosphomevalonate kinase identified. Black-Right-Pointing-Pointer Virtual screening yielded a hit rate of 15%, with inhibitor K{sub d}'s of 10-60 {mu}M. Black-Right-Pointing-Pointer NMR studies indicate significant protein conformational changes upon binding. -- Abstract: Phosphomevalonate kinase (PMK) phosphorylates mevalonate-5-phosphate (M5P) in the mevalonate pathway, which is the sole source of isoprenoids and steroids in humans. We have identified new PMK inhibitors with virtual screening, using autodock. Promising hits were verified and their affinity measured using NMR-based {sup 1}H-{sup 15}N heteronuclear single quantum coherence (HSQC) chemical shift perturbation and fluorescence titrations. Chemical shift changes were monitored, plotted, and fitted to obtain dissociation constants (K{sub d}). Tight binding compounds with K{sub d}'s ranging from 6-60 {mu}M were identified. These compounds tended to have significant polarity and negative charge, similar to the natural substrates (M5P and ATP). HSQC cross peak changes suggest that binding induces a global conformational change, such as domain closure. Compounds identified in this study serve as chemical genetic probes of human PMK, to explore pharmacology of the mevalonate pathway, as well as starting points for further drug development.

  16. Expression, purification, stability optimization and characterization of human Aurora B kinase domain from E. coli.

    PubMed

    Sheth, Payal R; Ramanathan, Lata; Ranchod, Ashwin; Basso, Andrea D; Barrett, Dianah; Zhao, Jia; Gray, Kimberly; Liu, Yan-Hui; Zhang, Rumin; Le, Hung V

    2010-11-15

    Aurora B kinase plays a critical role in regulating mitotic progression, and its dysregulation has been linked to tumorigenesis. The structure of the kinase domain of human Aurora B and the complementary information of binding thermodynamics of known Aurora inhibitors is lacking. Towards that effort, we sought to identify a human Aurora B construct that would be amenable for large-scale protein production for biophysical and structural studies. Although the designed AurB(69-333) construct expressed at high levels in Escherichia coli, the purified protein was largely unstable and prone to aggregation. We employed thermal-shift assay for high-throughput screening of 192 conditions to identify optimal pH and salt conditions that increased the stability and minimized aggregation of AurB(69-333). Direct ligand binding analyses using temperature-dependent circular dichroism (TdCD) and TR-FRET-based Lanthascreen™ binding assay showed that the purified protein was folded and functional. The affinity rank-order obtained using TdCD and Lanthascreen™ binding assay correlated with enzymatic IC50 values measured using full-length Aurora B protein for all the inhibitors tested except for AZD1152. The direct binding results support the hypothesis that the purified human AurB(69-333) fragment is a good surrogate for its full-length counterpart for biophysical and structural analyses.

  17. Functions of Polo-Like Kinases: A Journey From Yeast To Humans.

    PubMed

    Vaid, Rajni; Sharma, Nitin; Chauhan, Sujata; Deshta, Ankita; Dev, Kamal; Sourirajan, Anuradha

    2016-01-01

    Polo-like kinases (PLKs) belong to the serine/threonine kinase subfamily, characterized by the presence of the signature motif called Polo-box domain. PLK members studied so far have emerged as the conserved regulator of cell cycle and cell division in eukaryotes. The Polo-box domain adds diversity to PLK functions by targeting the enzyme to an array of substrates found at different sub-cellular structures for exquisite regulation of cell cycle. More than a dozen members of PLK subfamily have been identified in the eukaryotic world except in the higher plants. Despite the similarities in governing cell division, PLKs have diverse and unique functions in different organisms. This review summarizes the plethora of functions of PLK in yeast to humans. Along with its classical functions, this review also emphasizes on the role of PLKs in regulating DNA replication, repair, genome integrity, development, and morphogenesis pathways. Perturbations in PLK functions have disease implications, such as cancer in humans, and thus human PLK1 is targeted for cancer therapeutics. PLKs also play a vital role in regulating several stages of meiotic cell division. Thus, PLKs are emerging as a unique class of proteins with multiple and diverse functions in different organisms.

  18. The Late S-Phase Transcription Factor Hcm1 Is Regulated through Phosphorylation by the Cell Wall Integrity Checkpoint

    PubMed Central

    Negishi, Takahiro; Veis, Jiri; Hollenstein, David; Sekiya, Mizuho; Ammerer, Gustav

    2016-01-01

    The cell wall integrity (CWI) checkpoint in the budding yeast Saccharomyces cerevisiae coordinates cell wall construction and cell cycle progression. In this study, we showed that the regulation of Hcm1, a late-S-phase transcription factor, arrests the cell cycle via the cell wall integrity checkpoint. Although the HCM1 mRNA level remained unaffected when the cell wall integrity checkpoint was induced, the protein level decreased. The overproduction of Hcm1 resulted in the failure of the cell wall integrity checkpoint. We identified 39 Hcm1 phosphorylation sites, including 26 novel sites, by tandem mass spectrometry analysis. A mutational analysis revealed that phosphorylation of Hcm1 at S61, S65, and S66 is required for the proper onset of the cell wall integrity checkpoint by regulating the timely decrease in its protein level. Hyperactivation of the CWI mitogen-activated protein kinase (MAPK) signaling pathway significantly reduced the Hcm1 protein level, and the deletion of CWI MAPK Slt2 resulted in a failure to decrease Hcm1 protein levels in response to stress, suggesting that phosphorylation is regulated by CWI MAPK. In conclusion, we suggest that Hcm1 is regulated negatively by the cell wall integrity checkpoint through timely phosphorylation and degradation under stress to properly control budding yeast proliferation. PMID:26729465

  19. Structure of the human autophagy initiating kinase ULK1 in complex with potent inhibitors.

    PubMed

    Lazarus, Michael B; Novotny, Chris J; Shokat, Kevan M

    2015-01-16

    Autophagy is a conserved cellular process that involves the degradation of cellular components for energy maintenance and cytoplasmic quality control that has recently gained interest as a novel target for a variety of human diseases, including cancer. A prime candidate to determine the potential therapeutic benefit of targeting autophagy is the kinase ULK1, whose activation initiates autophagy. Here, we report the first structures of ULK1, in complex with multiple potent inhibitors. These structures show features unique to the enzyme and will provide a path for the rational design of selective compounds as cellular probes and potential therapeutics.

  20. Granule swelling and cleavage of mitogen-activated protein kinases in human neutrophils undergoing apoptosis

    SciTech Connect

    Kato, Takayuki; Ikemoto, Masaru; Hato, Fumihiko; Kitagawa, Seiichi

    2009-04-10

    Extracellular signal-regulated kinase and p38 have been shown to be cleaved in human neutrophils undergoing apoptosis induced by tumor necrosis factor-{alpha} and cycloheximide. However, the cleavage products of these molecules were undetected when apoptotic neutrophils were pretreated with phenylmethylsulfonyl fluoride or disrupted by nitrogen cavitation before preparation of cell lysates. The electron microscopy revealed that granules in apoptotic neutrophils were significantly swollen than those in control cells. These findings suggest that granule membrane may become destabilized during neutrophil apoptosis, leading to rapid proteolysis of these molecules by granule-derived serine proteases during preparation of cell lysates with the conventional lysis buffer.

  1. Role of ERK1/2 kinase in the expression of iNOS by NDMA in human neutrophils.

    PubMed

    Ratajczak-Wrona, Wioletta; Jablonska, Ewa; Garley, Marzena; Jablonski, Jakub; Radziwon, Piotr

    2013-01-01

    Potential role of ERK1/2 kinase in conjunction with p38 in the regulation of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production, and superoxide anion generation by human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was determined. Increased synthesis of NO due to the involvement of iNOS in neutrophils exposed to NDMA was observed. In addition, intensified activation of ERK1/2 and p38 kinases was determined in these cells. Inhibition of kinase regulated by extracellular signals (ERK1/2) pathway, in contrast to p38 pathway, led to an increased production of NO and expression of iNOS in PMNs. Moreover, as a result of inhibition of ERK1/2 pathway, a decreased activation of p38 kinase was observed in neutrophils, while inhibition of p38 kinase did not affect activation of ERK1/2 pathway in these cells. An increased ability to release superoxide anion by the studied PMNs was observed, which decreased after ERK1/2 pathway inhibition. In conclusion, in human neutrophils, ERK1/2 kinase is not directly involved in the regulation of iNOS and NO production induced by NDMA; however, the kinase participates in superoxide anion production in these cells.

  2. Proteins in the Nutrient-Sensing and DNA Damage Checkpoint Pathways Cooperate to Restrain Mitotic Progression following DNA Damage

    PubMed Central

    Searle, Jennifer S.; Wood, Matthew D.; Kaur, Mandeep; Tobin, David V.; Sanchez, Yolanda

    2011-01-01

    Checkpoint pathways regulate genomic integrity in part by blocking anaphase until all chromosomes have been completely replicated, repaired, and correctly aligned on the spindle. In Saccharomyces cerevisiae, DNA damage and mono-oriented or unattached kinetochores trigger checkpoint pathways that bifurcate to regulate both the metaphase to anaphase transition and mitotic exit. The sensor-associated kinase, Mec1, phosphorylates two downstream kinases, Chk1 and Rad53. Activation of Chk1 and Rad53 prevents anaphase and causes inhibition of the mitotic exit network. We have previously shown that the PKA pathway plays a role in blocking securin and Clb2 destruction following DNA damage. Here we show that the Mec1 DNA damage checkpoint regulates phosphorylation of the regulatory (R) subunit of PKA following DNA damage and that the phosphorylated R subunit has a role in restraining mitosis following DNA damage. In addition we found that proteins known to regulate PKA in response to nutrients and stress either by phosphorylation of the R subunit or regulating levels of cAMP are required for the role of PKA in the DNA damage checkpoint. Our data indicate that there is cross-talk between the DNA damage checkpoint and the proteins that integrate nutrient and stress signals to regulate PKA. PMID:21779180

  3. Choreography of the 9-1-1 checkpoint complex: DDK puts a check on the checkpoints.

    PubMed

    Paek, Andrew L; Weinert, Ted

    2010-11-24

    Checkpoint proteins respond to DNA damage by halting the cell cycle until the damage is repaired. In this issue of Molecular Cell, Furuya et al. (2010) provide evidence that checkpoint proteins need to be removed from sites of damage in order to properly repair it.

  4. Acyclic phosphonate nucleotides and human adenylate kinases: impact of a borano group on alpha-P position.

    PubMed

    Topalis, D; Alvarez, K; Barral, K; Munier-Lehmann, H; Schneider, B; Véron, M; Guerreiro, C; Mulard, L; El-Amri, C; Canard, B; Deville-Bonne, D

    2008-04-01

    Adenylate kinases are involved in the activation of antiviral drugs such as the acyclic phosphonates analogs PMEA and (R)PMPA. We examine the in vitro phosphorylation of PMEA and PMPA bearing a borano- or a H- group on the phosphorus atom. The alpha-borano or alpha-H on PMEA and PMPA were detrimental to the activity of recombinant human AMP kinases 1 and 2. Docking PMEA to the active site of AMP kinase 1 indicated that the borano group may prevent two conserved critical Arg interactions with the alpha-phosphate, resulting in substrate bad positioning.

  5. Regulation of AURORA B function by mitotic checkpoint protein MAD2.

    PubMed

    Shandilya, Jayasha; Medler, Kathryn F; Roberts, Stefan G E

    2016-08-17

    Cell cycle checkpoint signaling stringently regulates chromosome segregation during cell division. MAD2 is one of the key components of the spindle and mitotic checkpoint complex that regulates the fidelity of cell division along with MAD1, CDC20, BUBR1, BUB3 and MAD3. MAD2 ablation leads to erroneous attachment of kinetochore-spindle fibers and defective chromosome separation. A potential role for MAD2 in the regulation of events beyond the spindle and mitotic checkpoints is not clear. Together with active spindle assembly checkpoint signaling, AURORA B kinase activity is essential for chromosome condensation as cells enter mitosis. AURORA B phosphorylates histone H3 at serine 10 and serine 28 to facilitate the formation of condensed metaphase chromosomes. In the absence of functional AURORA B cells escape mitosis despite the presence of misaligned chromosomes. In this study we report that silencing of MAD2 results in a drastic reduction of metaphase-specific histone H3 phosphorylation at serine 10 and serine 28. We demonstrate that this is due to mislocalization of AURORA B in the absence of MAD2. Conversely, overexpression of MAD2 concentrated the localization of AURORA B at the metaphase plate and caused hyper-phosphorylation of histone H3. We find that MAD1 plays a minor role in influencing the MAD2-dependent regulation of AURORA B suggesting that the effects of MAD2 on AURORA B are independent of the spindle checkpoint complex. Our findings reveal that, in addition to its role in checkpoint signaling, MAD2 ensures chromosome stability through the regulation of AURORA B.

  6. Mitogen-activated protein kinases and phosphatidylinositol 3-kinase are involved in Prevotella intermedia-induced proinflammatory cytokines expression in human periodontal ligament cells.

    PubMed

    Guan, Su-Min; Zhang, Ming; He, Jian-Jun; Wu, Jun-Zheng

    2009-08-28

    Chronic periodontitis is an inflammatory disease affecting periodontal connective tissues and alveolar bone. Proinflammatory mediators induced by periodontal pathogens play vital roles in the initiation and progression of the disease. In this study, we examined whether Prevotella intermedia induces proinflammatory cytokines expression in human periodontal ligament cells (hPDLs). The mRNA expression and protein production were determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbant assay (ELISA) respectively. P. intermedia treatment dose- and time-dependently increased IL-6, IL-8 and M-CSF, but not IL-1beta and TNF-alpha mRNA expression and protein secretion. Preincubation of hPDLs with extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 kinase and phosphatidylinositol 3-kinase (PI3K) inhibitors PD98059, SP600125, SB203580 and LY294002 resulted in significant reduction in P. intermedia-induced IL-6, IL-8 and M-CSF expression. Blocking the synthesis of prostaglandin E(2) (PGE(2)) by indomethacin also abolished the stimulatory effects of P. intermedia on cytokines expression. Our results indicate that P. intermedia induces proinflammatory cytokines through MAPKs and PI3K signaling pathways, and PGE(2) is involved in the P. intermedia-induced proinflammatory cytokines upregulation.

  7. The MAP kinase-activated protein kinase Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen Candida albicans.

    PubMed

    Li, Xichuan; Du, Wei; Zhao, Jingwen; Zhang, Lilin; Zhu, Zhiyan; Jiang, Linghuo

    2010-06-01

    Rck2p is the Hog1p-MAP kinase-activated protein kinase required for the attenuation of protein synthesis in response to an osmotic challenge in Saccharomyces cerevisiae. Rck2p also regulates rapamycin sensitivity in both S. cerevisiae and Candida albicans. In this study, we demonstrate that the deletion of CaRCK2 renders C. albicans cells sensitive to, and CaRck2p translocates from the cytosol to the nucleus in response to, cell wall stresses caused by Congo red, Calcoflor White, elevated heat and zymolyase. However, the kinase activity of CaRck2p is not required for the cellular response to these cell wall stresses. Furthermore, transcripts of cell wall protein-encoding genes CaBGL2, CaHWP1 and CaXOG1 are reduced in C. albicans cells lacking CaRCK2. The deletion of CaRCK2 also reduces the in vitro filamentation of C. albicans and its virulence in a mouse model of systemic candidasis. The kinase activity of CaRck2p is required for the virulence, but not for the in vitro filamentation, in C. albicans. Therefore, Rck2p regulates cellular responses to cell wall stresses, filamentation and virulence in the human fungal pathogen C. albicans.

  8. Mitogen-activated protein kinase phosphatase-1 inhibition and sustained extracellular signal-regulated kinase 1/2 activation in camptothecin-induced human colon cancer cell death

    PubMed Central

    Lee, Minyoung; Young Kim, Sun; Kim, JongGuk; Kim, Hak-Su; Kim, Sang-Man; Kim, Eun Ju

    2013-01-01

    Camptothecins are commonly used chemotherapeutics; in some models, they enhance signaling via the mitogen-activated protein kinase (MAPK) pathway through effects on upstream kinases. To evaluate the impact of camptothecin (CPT) on MAPKs in human colon cancer, we studied HCT116 and CaCo2 colon cancer cells. We found that HCT116 cells highly express mitogen-activated protein kinase phosphatase-1 (MKP1), which selectively inactivates extracellular signal-regulated kinase (ERK), whereas MKP1 levels were undetectable in CaCo2 cells. CPT did not affect ERK activity in CaCo2 cells, but did induce a striking increase in ERK activity in HCT116 cells in association with a corresponding decrease in MKP1. The reduction in MKP1 expression occurred at a posttranscriptional level and was blocked by the proteasome inhibitor MG132, whereas that CPT-induced downregulation of MKP1 was not due to proteasome-mediated degradation. Treatment of HCT116 cells with CPT induced a sustained activation of nuclear ERK, which was required for CPT-induced apoptosis. P38 and JNK activity were unaffected by CPT, suggesting that the effects of CPT are mediated specifically by ERK. These results suggest that targeting dual-specificity MAPK phosphatases in colon cancer cells may be a viable strategy for optimizing camptothecin-based therapeutic protocols. PMID:24005240

  9. N-acetylcysteine attenuates TNF-α-induced p38 MAP kinase activation and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells

    PubMed Central

    Hashimoto, Shu; Gon, Yasuhiro; Matsumoto, Ken; Takeshita, Ikuko; Horie, Takashi

    2001-01-01

    We have previously shown that tumour necrosis factor-α (TNF-α) activates p38 mitogen-activated protein (MAP) kinase to produce interleukin-8 (IL-8) by human pulmonary vascular endothelial cells. Reactive oxygen species (ROS) including H2O2 generated by TNF-α can act as signalling intermediates for cytokine induction; therefore, scavenging ROS by anti-oxidants is important for the regulation of cytokine production. However, the effect of N-acetylcysteine (NAC), which acts as a precursor of glutathione (GSH) synthesis, on TNF-α-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells has not been determined. To clarify these issues, we examined the effect of NAC on TNF-α-induced activation of p38 MAP kinase, MAP kinase kinase (MKK) 3 and MKK6 which are upstream regulators of p38 MAP kinase, and p38 MAP kinase-mediated IL-8 production. Human pulmonary vascular endothelial cells that had been preincubated with NAC were stimulated with TNF-α and then the activation of p38 MAP kinase and MKK3/MKK6 in the cells and IL-8 concentrations in the culture supernatants were determined. Intracellular GSH levels increased in NAC-treated cells. NAC attenuated TNF-α-induced activation of p38 MAP kinase and MKK3/MKK6. NAC attenuated p38 MAP kinase-mediated IL-8 production by TNF-α-stimulated cells. These results indicate that the cellular reduction and oxidation (redox) regulated by intracellular GSH is critical for TNF-α-induced activation of p38 MAP kinase pathway and p38 MAP kinase-mediated IL-8 production by human pulmonary vascular endothelial cells, and we emphasize that anti-oxidant therapy is an important strategy for the treatment of acute lung injury. PMID:11156586

  10. Lazy Checkpointing : Exploiting Temporal Locality in Failures to Mitigate Checkpointing Overheads on Extreme-Scale Systems

    SciTech Connect

    Tiwari, Devesh; Gupta, Saurabh; Vazhkudai, Sudharshan S

    2014-01-01

    Continuing increase in the computational power of supercomputers has enabled large-scale scientific applications in the areas of astrophysics, fusion, climate and combustion to run larger and longer-running simulations, facilitating deeper scientific insights. However, these long-running simulations are often interrupted by multiple system failures. Therefore, these applications rely on ``checkpointing'' as a resilience mechanism to store application state to permanent storage and recover from failures. \\\\ \\indent Unfortunately, checkpointing incurs excessive I/O overhead on supercomputers due to large size of checkpoints, resulting in a sub-optimal performance and resource utilization. In this paper, we devise novel mechanisms to show how checkpointing overhead can be mitigated significantly by exploiting the temporal characteristics of system failures. We provide new insights and detailed quantitative understanding of the checkpointing overheads and trade-offs on large-scale machines. Our prototype implementation shows the viability of our approach on extreme-scale machines.

  11. Asynchronous Checkpoint Migration with MRNet in the Scalable Checkpoint / Restart Library

    SciTech Connect

    Mohror, K; Moody, A; de Supinski, B R

    2012-03-20

    Applications running on today's supercomputers tolerate failures by periodically saving their state in checkpoint files on stable storage, such as a parallel file system. Although this approach is simple, the overhead of writing the checkpoints can be prohibitive, especially for large-scale jobs. In this paper, we present initial results of an enhancement to our Scalable Checkpoint/Restart Library (SCR). We employ MRNet, a tree-based overlay network library, to transfer checkpoints from the compute nodes to the parallel file system asynchronously. This enhancement increases application efficiency by removing the need for an application to block while checkpoints are transferred to the parallel file system. We show that the integration of SCR with MRNet can reduce the time spent in I/O operations by as much as 15x. However, our experiments exposed new scalability issues with our initial implementation. We discuss the sources of the scalability problems and our plans to address them.

  12. Assignment of the protein kinase C delta polypeptide gene (PRKCD) to human chromosome 3 and mouse chromosome 14.

    PubMed

    Huppi, K; Siwarski, D; Goodnight, J; Mischak, H

    1994-01-01

    The protein kinase C (pkc) enzymes are a family of serine-threonine protein kinases, each encoded by a distinct and separate gene. The chromosomal locations of human PRKCA, PRKCB, and PRKCG have previously been established. We now report that PRKCD, a novel member of the pkc gene family, maps to human chromosome 3. The chromosomal location of Pkcd has also been determined in the mouse by analysis of recombination frequency in an interspecific panel of backcross mice. We find that the locus encoding pkcd resides proximal to nucleoside phosphorylase (Np-2) and Tcra on mouse chromosome 14 in a region syntenic with human 3p.

  13. Localization of a novel human A-kinase-anchoring protein, hAKAP220, during spermatogenesis.

    PubMed

    Reinton, N; Collas, P; Haugen, T B; Skâlhegg, B S; Hansson, V; Jahnsen, T; Taskén, K

    2000-07-01

    Using a combination of protein kinase A type II overlay screening, rapid amplification of cDNA ends, and database searches, a contig of 9923 bp was assembled and characterized in which the open reading frame encoded a 1901-amino-acid A-kinase-anchoring protein (AKAP) with an apparent SDS-PAGE mobility of 220 kDa, named human AKAP220 (hAKAP220). The hAKAP220 amino acid sequence revealed high similarity to rat AKAP220 in the 1167 C-terminal residues, but contained 727 residues in the N-terminus not present in the reported rat AKAP220 sequence. The hAKAP220 mRNA was expressed at high levels in human testis and in isolated human pachytene spermatocytes and round spermatids. The hAKAP220 protein was present in human male germ cells and mature sperm. Immunofluorescent labeling with specific antibodies indicated that hAKAP220 was localized in the cytoplasm of premeiotic pachytene spermatocytes and in the centrosome of developing postmeiotic germ cells, while a midpiece/centrosome localization was found in elongating spermatocytes and mature sperm. The hAKAP220 protein together with a fraction of PKA types I and II and protein phosphatase I was resistant to detergent extraction of sperm tails, suggesting an association with cytoskeletal structures. In contrast, S-AKAP84/D-AKAP1, which is also present in the midpiece, was extracted under the same conditions. Anti-hAKAP220 antisera coimmunoprecipitated both type I and type II regulatory subunits of PKA in human testis lysates, indicating that hAKAP220 interacts with both classes of R subunits, either through separate or through a common binding motif(s).

  14. Characterization of cyclin-dependent kinases and Cdc2/Cdc28 kinase subunits in Trichomonas vaginalis.

    PubMed

    Amador, Erick; López-Pacheco, Karla; Morales, Nataly; Coria, Roberto; López-Villaseñor, Imelda

    2017-04-01

    Cyclin-dependent kinases (CDKs) have important roles in regulating key checkpoints between stages of the cell cycle. Their activity is tightly regulated through a variety of mechanisms, including through binding with cyclin proteins and the Cdc2/Cdc28 kinase subunit (CKS), and their phosphorylation at specific amino acids. Studies of the components involved in cell cycle control in parasitic protozoa are limited. Trichomonas vaginalis is the causative agent of trichomoniasis in humans and is therefore important in public health; however, some of the basic biological processes used by this organism have not been defined. Here, we characterized proteins potentially involved in cell cycle regulation in T. vaginalis. Three genes encoding protein kinases were identified in the T. vaginalis genome, and the corresponding recombinant proteins (TvCRK1, TvCRK2, TvCRK5) were studied. These proteins displayed similar sequence features to CDKs. Two genes encoding CKSs were also identified, and the corresponding recombinant proteins were found to interact with TvCRK1 and TvCRK2 by a yeast two-hybrid system. One putative cyclin B protein from T. vaginalis was found to bind to and activate the kinase activities of TvCRK1 and TvCRK5, but not TvCRK2. This work is the first characterization of proteins involved in cell cycle control in T. vaginalis.

  15. Comparative Analysis of Human Nucleoside Kinase-Based Reporter Systems for PET Imaging

    PubMed Central

    Lee, Jason T.; Zhang, Hanwen; Moroz, Maxim A.; Likar, Yury; Shenker, Larissa; Sumzin, Nikita; Lobo, Jose; Zurita, Juan; Collins, Jeffrey; van Dam, R. Michael; Ponomarev, Vladimir

    2017-01-01

    Purpose Radionuclide-based reporter gene imaging has the sensitivity to monitor gene- and cell-based therapies in human subjects. Potential immunogenicity of current viral transgenes warrants development of human-based reporter systems. We compared human nucleoside kinase reporters to a panel of nucleoside analogs of FEAU, FMAU, and FIAU, including the first in vivo assessment of L-[18F]FEAU. Procedures Human isogenic U87 cell lines were transduced to express different human reporter genes including dCK-R104M/D133A (dCKDM), dCK-R104Q/D133N (dCKep16A), dCK-A100V/R104M/D133A (dCK3M), and TK2-N93D/L109F (TK2DM), and wild-type dCK (dCK) and herpes simplex virus type-1 (HSVTK) reporter gene as references. In vitro cell uptake assays were performed with [18F]FEAU, L-[18F]FEAU, [14C]FMAU, L-[18F]FMAU, and [124I]FIAU. Micro-positron emission tomography/X-ray computed tomography imaging of xenograft-bearing nu/nu mice was conducted with [18F]FEAU, L-[18F]FEAU, L-[18F]FMAU, and [124I]FIAU on consecutive days. A cell viability assay was also performed to assess sensitivities to gemcitabine and bromovinyldeoxyuridine (BVdU). Results In vitro, dCKep16A and dCKDM with [18F]FEAU exhibited the highest sensitivity and selectivity of the human reporters, second only to HSVTK/[18F]FEAU. L-[18F]FEAU biodistribution in mice was on par with [18F]FEAU and L-[18F]FMAU. L-[18F]FMAU uptake in isogenic xenografts was highest for all human reporter genes. However, [18F]FEAU was the most selective of the short half-life reporter probes due to its minimal recognition by human dCK and relative sensitivity, whereas [124I]FIAU permitted imaging at a later time point, improving signal-to-background ratio. Of the human reporter genes, dCKep16A consistently outperformed the other tested reporters. Reporter genes of interest increased potency to the nucleoside analog prodrugs gemcitabine and BVdU. Conclusions We demonstrate that human nucleoside kinase reporter systems vary significantly in their

  16. A novel microfluidic assay reveals a key role for protein kinase C δ in regulating human neutrophil-endothelium interaction.

    PubMed

    Soroush, Fariborz; Zhang, Ting; King, Devon J; Tang, Yuan; Deosarkar, Sudhir; Prabhakarpandian, Balabhaskar; Kilpatrick, Laurie E; Kiani, Mohammad F

    2016-11-01

    A key step in neutrophil-mediated tissue damage is the migration of activated neutrophils across the vascular endothelium. Previously, we identified protein kinase C δ as a critical regulator of neutrophil migration in sepsis but did not identify specific steps in migration. In this study, we used our novel biomimetic microfluidic assay to delineate systematically the mechanism by which protein kinase C δ regulates individual steps in human neutrophil-endothelial interaction during inflammation. The biomimetic microfluidic assay includes a network of vascular channels, produced from in vivo images connected to a tissue compartment through a porous barrier. HUVECs cultured in vascular channels formed a complete lumen under physiologic shear flow. HUVECs were pretreated with TNF-α ± a protein kinase C δ inhibitor, and the tissue compartment was filled with a chemoattractant (fMLP or IL-8). Under physiologic shear flow, the role of protein kinase C δ on spatial and temporal neutrophil adherence/migration was quantified. Protein kinase C δ inhibition significantly reduced neutrophil adhesion in response to fMLP and IL-8 only under low shear rate and near bifurcations. Protein kinase C δ inhibition also decreased adherence to nonactivated HUVECs in response to fMLP or IL-8. Protein kinase C δ inhibition reduced neutrophil migration into the tissue compartment in response to fMLP and to a lesser degree, to IL-8. Antibody-coated microparticles demonstrated that protein kinase C δ inhibition down-regulated E-selectin and ICAM-1 but not VCAM-1 expression. With the use of a physiologically relevant in vitro model system, we demonstrate that protein kinase C δ plays an important role in the regulation of neutrophil adherence/migration during inflammation and identifies key steps regulated by protein kinase C δ in neutrophil-endothelial interactions.

  17. Role of Phosphoinositide 3-Kinase in the Aggressive Tumor Growth of HT1080 Human Fibrosarcoma Cells

    PubMed Central

    Gupta, Swati; Stuffrein, Selma; Plattner, Rina; Tencati, Michael; Gray, Christa; Whang, Young E.; Stanbridge, Eric J.

    2001-01-01

    We have developed a model system of human fibrosarcoma cell lines that do or do not possess and express an oncogenic mutant allele of N-ras. HT1080 cells contain an endogenous mutant allele of N-ras, whereas the derivative MCH603 cell line contains only wild-type N-ras. In an earlier study (S. Gupta et al., Mol. Cell. Biol. 20:9294–9306, 2000), we had shown that HT1080 cells produce rapidly growing, aggressive tumors in athymic nude mice, whereas MCH603 cells produced more slowly growing tumors and was termed weakly tumorigenic. An extensive analysis of the Ras signaling pathways (Raf, Rac1, and RhoA) provided evidence for a potential novel pathway that was critical for the aggressive tumorigenic phenotype and could be activated by elevated levels of constitutively active MEK. In this study we examined the role of phosphoinositide 3-kinase (PI 3-kinase) in the regulation of the transformed and aggressive tumorigenic phenotypes expressed in HT1080 cells. Both HT1080 (mutant N-ras) and MCH603 (wild-type N-ras) have similar levels of constitutively active Akt, a downstream target of activated PI 3-kinase. We find that both cell lines constitutively express platelet-derived growth factor (PDGF) and PDGF receptors. Transfection with tumor suppressor PTEN cDNA into HT1080 and constitutively active PI 3-kinase–CAAX cDNA into MCH603 cells, respectively, resulted in several interesting and novel observations. Activation of the PI 3-kinase/Akt pathway, including NF-κB, is not required for the aggressive tumorigenic phenotype in HT1080 cells. Activation of NF-κB is complex: in MCH603 cells it is mediated by Akt, whereas in HT1080 cells activation also involves other pathway(s) that are activated by mutant Ras. A threshold level of activation of PI 3-kinase is required in MCH603 cells before stimulatory cross talk to the RhoA, Rac1, and Raf pathways occurs, without a corresponding activation of Ras. The increased levels of activation seen were similar to those observed

  18. Overexpression of polo-like kinase 1 and its clinical significance in human non-small cell lung cancer.

    PubMed

    Wang, Zhao-Xia; Xue, Dong; Liu, Zhi-Li; Lu, Bin-Bin; Bian, Hai-Bo; Pan, Xuan; Yin, Yong-Mei

    2012-01-01

    Polo-like kinase 1 is a serine/threonine kinase which plays an essential role in mitosis and malignant transformation. The aim of this study was to investigate the prognostic significance of polo-like kinase 1 expression and determine its possibility as a therapeutic target in non-small cell lung cancer. Semi-quantitative RT-PCR assay was performed to detect polo-like kinase 1 mRNA expression in non-small cell lung cancer cells or tissues. Immunohistochemistry was performed to detect polo-like kinase 1 protein expression in 100 non-small cell lung cancer tissue samples, and the associations of polo-like kinase 1 expression with clinicopathological factors or prognosis of non-small cell lung cancer patients were evaluated. RNA interference was employed to inhibit endogenous polo-like kinase 1 expression and analyzed the effects of polo-like kinase 1 inhibition on the malignant phenotypes of non-small cell lung cancer cells including growth, apoptosis, radio- or chemoresistance. Also, the possible molecular mechanisms were also investigated. The levels of polo-like kinase 1 mRNA expression in non-small cell lung cancer cell lines or tissues were significantly higher than those in normal human bronchial epithelial cell line or corresponding non-tumor tissues. High polo-like kinase 1 expression was significantly correlated with advanced clinical stage, higher tumor classification and lymph node metastasis of non-small cell lung cancer patients (P=0.001, 0.004 and 0.001, respectively). Meanwhile, high polo-like kinase 1 protein expression was also an independent prognostic molecular marker for non-small cell lung cancer patients (hazard ratio: 2.113; 95% confidence interval: 1.326-3.557; P=0.017). Polo-like kinase 1 inhibition could significantly inhibit in vitro and in vivo proliferation, induce cell arrest of G(2)/M phase and apoptosis enhancement in non-small cell lung cancer cells, which might be activation of the p53 pathway and the Cdc25C/cdc2/cyclin B1 feedback

  19. TAM Receptor Tyrosine Kinases: Biologic Functions, Signaling, and Potential Therapeutic Targeting in Human Cancer

    PubMed Central

    Linger, Rachel M. A.; Keating, Amy K.; Earp, H. Shelton; Graham, Douglas K.

    2011-01-01

    Tyro-3, Axl, and Mer constitute the TAM family of receptor tyrosine kinases (RTKs) characterized by a conserved sequence within the kinase domain and adhesion molecule-like extracellular domains. This small family of RTKs regulates an intriguing mix of processes, including cell proliferation/survival, cell adhesion and migration, blood clot stabilization, and regulation of inflammatory cytokine release. Genetic or experimental alteration of TAM receptor function can contribute to a number of disease states, including coagulopathy, autoimmune disease, retinitis pigmentosa, and cancer. In this chapter, we first provide a comprehensive review of the structure, regulation, biologic functions, and down-stream signaling pathways of these receptors. In addition, we discuss recent evidence which suggests a role for TAM receptors in oncogenic mechanisms as family members are over-expressed in a spectrum of human cancers and have prognostic significance in some. Possible strategies for targeted inhibition of the TAM family in the treatment of human cancer are described. Further research will be necessary to evaluate the full clinical implications of TAM family expression and activation in cancer. PMID:18620092

  20. Doublecortin-like kinase 1-positive enterocyte - a new cell type in human intestine.

    PubMed

    Leppänen, Joni; Helminen, Olli; Huhta, Heikki; Kauppila, Joonas H; Miinalainen, Ilkka; Ronkainen, Veli-Pekka; Saarnio, Juha; Lehenkari, Petri P; Karttunen, Tuomo J

    2016-11-01

    Doublecortin-like kinase 1 (DCLK1) is a microtubule-associated kinase. In murine intestine, DCLK1 marks tuft cells with characteristic microvilli, features of neuroendocrine cells and also quiescent stem cell-like properties. The occurrence and pathological role of DCLK1-positive cells in human intestinal mucosa is unknown. We analysed DCLK1 expression in healthy duodenal, jejunal and colorectal mucosa samples (n = 35), and in duodenal specimens from patients with coeliac disease (n = 20). The samples were immunohistochemically double-stained with DCLK1, and synaptophysin, chromogranin A and Ki-67. Ultrastructure of DCLK1-expressing duodenal cells was assessed using correlative light and electron microscopy. DCLK1 expression was seen in about 1% of epithelial cells diffusely scattered through the intestinal epithelium. Electron microscopy showed that the duodenal DCLK1-positive cells had short apical microvilli similar to neighbouring enterocytes and cytoplasmic granules on the basal side. DCLK1-positive cells were stained with synaptophysin. The number of DCLK1-positive cells was decreased in villus atrophy in coeliac disease. Our findings indicate that in human intestinal epithelium, DLCK1-positive cells form a subpopulation of non-proliferating neuroendocrine cells with apical brush border similar to that in enterocytes, and their number is decreased in untreated coeliac disease.

  1. X-ray structure and characterization of carbamate kinase from the human parasite Giardia lamblia.

    PubMed

    Galkin, Andrey; Kulakova, Liudmila; Wu, Rui; Nash, Theodore E; Dunaway-Mariano, Debra; Herzberg, Osnat

    2010-04-01

    Carbamate kinase catalyzes the reversible conversion of carbamoyl phosphate and ADP to ATP and ammonium carbamate, which is hydrolyzed to ammonia and carbonate. The three-dimensional structure of carbamate kinase from the human parasite Giardia lamblia (glCK) has been determined at 3 A resolution. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 69.77, b = 85.41, c = 102.1 A, beta = 106.8 degrees . The structure was refined to a final R factor of 0.227. The essentiality of glCK together with its absence in humans makes the enzyme an attractive candidate for anti-Giardia drug development. Steady-state kinetic rate constants have been determined. The k(cat) for ATP formation is 319 +/- 9 s(-1). The K(m) values for carbamoyl phosphate and ADP are 85 +/- 6 and 70 +/- 5 microM, respectively. The structure suggests that three invariant lysine residues (Lys131, Lys216 and Lys278) may be involved in the binding of substrates and phosphoryl transfer. The structure of glCK reveals that a glycerol molecule binds in the likely carbamoyl phosphate-binding site.

  2. Assignment of the human diacylglycerol kinase 4 (DAGK4) gene to chromosome 4p16.3

    SciTech Connect

    Endele, S.; Zabel, B.; Winterpacht, A.

    1996-04-01

    This report describes the localization of the human gene for diacylglycerol kinase 4 (DAGK4) to human chromosome 4p16.3 using an exon amplification scheme. It also discusses the possible implications of the chromosomal location of this gene in certain hereditary malignancies. 9 refs., 1 fig.

  3. Mesenchymal Stromal Cells Prevent Allostimulation In Vivo and Control Checkpoints of Th1 Priming: Migration of Human DC to Lymph Nodes and NK Cell Activation.

    PubMed

    Consentius, C; Akyüz, L; Schmidt-Lucke, J A; Tschöpe, C; Pinzur, L; Ofir, R; Reinke, P; Volk, H-D; Juelke, K

    2015-10-01

    Although the immunomodulatory potency of mesenchymal stromal cells (MSC) is well established, the mechanisms behind are still not clear. The crosstalk between myeloid dendritic cells (mDC) and natural killer (NK) cells and especially NK cell-derived interferon-gamma (IFN-γ) play a pivotal role in the development of type 1 helper (Th1) cell immune responses. While many studies explored the isolated impact of MSC on either in vitro generated DC, NK, or T cells, there are only few data available on the complex interplay between these cells. Here, we investigated the impact of MSC on the functionality of human mDC and the consequences for NK cell and Th1 priming in vitro and in vivo. In critical limb ischemia patients, who have been treated with allogeneic placenta-derived mesenchymal-like stromal cells (PLX-PAD), no in vivo priming of Th1 responses toward the major histocompatibility complex (MHC) mismatches could be detected. Further in vitro studies revealed that mDC reprogramming could play a central role for these effects. Following crosstalk with MSC, activated mDC acquired a tolerogenic phenotype characterized by reduced migration toward CCR7 ligand and impaired ability to stimulate NK cell-derived IFN-γ production. These effects, which were strongly related to an altered interleukin (IL)-12/IL-10 production by mDC, were accompanied by an effective prevention of Th1 priming in vivo. Our findings provide novel evidence for the regulation of Th1 priming by MSC via modulation of mDC and NK cell crosstalk and show that off-the-shelf produced MHC-mismatched PLX-PAD can be used in patients without any sign of immunogenicity.

  4. Induction of human pancreatic beta cell replication by inhibitors of dual specificity tyrosine regulated kinase

    PubMed Central

    Wang, Peng; Alvarez-Perez, Juan-Carlos; Felsenfeld, Dan P.; Liu, Hongtao; Sivendran, Sharmila; Bender, Aaron; Kumar, Anil; Sanchez, Roberto; Scott, Donald K.; Garcia-Ocaña, Adolfo; Stewart, Andrew F.

    2015-01-01

    Types 1 and 2 diabetes affect some 380 million people worldwide. Both result ultimately from a deficiency of functional pancreatic insulin-producing beta cells. Beta cells proliferate in humans during a brief temporal window beginning around the time of birth, with peak beta cell labeling indices achieving approximately 2% in first year of life1-4. In embryonic life and after early childhood, beta cell replication rates are very low. While beta cell expansion seems an obvious therapeutic approach to beta cell deficiency, adult human beta cells have proven recalcitrant to such efforts1-8. Hence, there remains an urgent need for diabetes therapeutic agents that can induce regeneration and expansion of adult human beta cells in vivo or ex vivo. Here, we report the results of a high-throughput small molecule screen (HTS) revealing a novel class of human beta cell mitogenic compounds, analogues of the small molecule, harmine. We also define dual specificity tyrosine-regulated kinase-1a (DYRK1A) as the likely target of harmine, and the Nuclear Factors of activated T-cells (NFAT) family of transcription factors as likely mediators of human beta cell proliferation as well as beta cell differentiation. These observations suggest that harmine analogues (“harmalogs”) may have unique therapeutic promise for human diabetes therapy. Enhancing potency and beta cell specificity are important future challenges. PMID:25751815

  5. Molecular Docking and NMR Binding Studies to Identify Novel Inhibitors of Human Phosphomevalonate Kinase

    PubMed Central

    Boonsri, Pornthip; Neumann, Terrence S.; Olson, Andrew L.; Cai, Sheng; Herdendorf, Timothy J.; Miziorko, Henry M.; Hannongbua, Supa; Sem, Daniel S.

    2012-01-01

    Phosphomevalonate kinase (PMK) phosphorylates mevalonate-5-phosphate (M5P) in the mevalonate pathway, which is the sole source of isoprenoids and steroids in humans. We have identified new PMK inhibitors with virtual screening, using Autodock. Promising hits were verified and their affinity measured using NMR-based 1H-15N Heteronuclear Single Quantum Coherence (HSQC) chemical shift perturbation and fluorescence titrations. Chemical shift changes were monitored, plotted, and fitted to obtain dissociation constants (Kd). Tight binding compounds with Kd’s ranging from 6–60 µM were identified. These compounds tended to have significant polarity and negative charge, similar to the natural substrates (M5P and ATP). HSQC crosspeak changes suggest that binding induces a global conformational change, such as domain closure. Compounds identified in this study serve as chemical genetic probes of human PMK, to explore pharmacology of the mevalonate pathway, as well as starting points for further drug development. PMID:23146631

  6. Biguanides and thiazolidinediones inhibit stimulated lipolysis in human adipocytes through activation of AMP-activated protein kinase.

    PubMed

    Bourron, O; Daval, M; Hainault, I; Hajduch, E; Servant, J M; Gautier, J F; Ferré, P; Foufelle, F

    2010-04-01

    In rodent adipocytes, activated AMP-activated protein kinase reduces the lipolytic rate. As the hypoglycaemic drugs metformin and thiazolidinediones activate this enzyme in rodents, we tested the hypothesis that in addition to their known actions they could have an anti-lipolytic effect in human adipocytes. Adipose tissue was obtained from individuals undergoing plastic surgery. Adipocytes were isolated and incubated with lipolytic agents (isoprenaline, atrial natriuretic peptide) and biguanides or thiazolidinediones. Lipolysis was quantified by the glycerol released in the medium. AMP-activated protein kinase activity and phosphorylation state were determined using standard procedures. In human adipocytes, isoprenaline and atrial natriuretic peptide stimulated the lipolytic rate three- to fourfold. Biguanides and thiazolidinediones activated AMP-activated protein kinase and inhibited lipolysis by 30-40%, at least in part by inhibiting hormone-sensitive lipase translocation to the lipid droplet. Inhibition of AMP-activated protein kinase by compound C precluded this inhibitory effect on lipolysis. Stimulation of lipolysis also induced an activation of AMP-activated protein kinase concomitant with a drop in ATP concentration. We show for the first time in human adipocytes that biguanides and thiazolidinediones activate AMP-activated protein kinase, thus counteracting lipolysis induced by lipolytic agents. In addition, beta-agonist- or ANP-stimulated lipolysis increases AMP-activated protein kinase activity. This is because of an increase in the AMP/ATP ratio, linked to activation of some of the released fatty acids into acyl-CoA. AMP-activated protein kinase activation could represent a physiological means of avoiding a deleterious drain of energy during lipolysis but could be used to restrain pharmacological release of fatty acids.

  7. Inner nuclear membrane protein Lem2 facilitates Rad3-mediated checkpoint signaling under replication stress induced by nucleotide depletion in fission yeast.

    PubMed

    Xu, Yong-Jie

    2016-04-01

    DNA replication checkpoint is a highly conserved cellular signaling pathway critical for maintaining genome integrity in eukaryotes. It is activated when DNA replication is perturbed. In Schizosaccharomyces pombe, perturbed replication forks activate the sensor kinase Rad3 (ATR/Mec1), which works cooperatively with mediator Mrc1 and the 9-1-1 checkpoint clamp to phosphorylate the effector kinase Cds1 (CHK2/Rad53). Phosphorylation of Cds1 promotes autoactivation of the kinase. Activated Cds1 diffuses away from the forks and stimulates most of the checkpoint responses under replication stress. Although this signaling pathway has been well understood in fission yeast, how the signaling is initiated and thus regulated remains incompletely understood. Previous studies have shown that deletion of lem2(+) sensitizes cells to the inhibitor of ribonucleotide reductase, hydroxyurea. However, the underlying mechanism is still not well understood. This study shows that in the presence of hydroxyurea, Lem2 facilitates Rad3-mediated checkpoint signaling for Cds1 activation. Without Lem2, all known Rad3-dependent phosphorylations critical for replication checkpoint signaling are seriously compromised, which likely causes the aberrant mitosis and drug sensitivity observed in this mutant. Interestingly, the mutant is not very sensitive to DNA damage and the DNA damage checkpoint remains largely intact, suggesting that the main function of Lem2 is to facilitate checkpoint signaling in response to replication stress. Since Lem2 is an inner nuclear membrane protein, these results also suggest that the replication checkpoint may be spatially regulated inside the nucleus, a previously unknown mechanism.

  8. The sensitivity of the DNA damage checkpoint prevents oocyte maturation in endometriosis

    PubMed Central

    Hamdan, Mukhri; Jones, Keith T.; Cheong, Ying; Lane, Simon I. R.

    2016-01-01

    Mouse oocytes respond to DNA damage by arresting in meiosis I through activity of the Spindle Assembly Checkpoint (SAC) and DNA Damage Response (DDR) pathways. It is currently not known if DNA damage is the primary trigger for arrest, or if the pathway is sensitive to levels of DNA damage experienced physiologically. Here, using follicular fluid from patients with the disease endometriosis, which affects 10% of women and is associated with reduced fertility, we find raised levels of Reactive Oxygen Species (ROS), which generate DNA damage and turn on the DDR-SAC pathway. Only follicular fluid from patients with endometriosis, and not controls, produced ROS and damaged DNA in the oocyte. This activated ATM kinase, leading to SAC mediated metaphase I arrest. Completion of meiosis I could be restored by ROS scavengers, showing this is the primary trigger for arrest and offering a novel clinical therapeutic treatment. This study establishes a clinical relevance to the DDR induced SAC in oocytes. It helps explain how oocytes respond to a highly prevalent human disease and the reduced fertility associated with endometriosis. PMID:27841311

  9. Tyrosine-specific phosphorylation of calmodulin by the insulin receptor kinase purified from human placenta.

    PubMed Central

    Sacks, D B; Fujita-Yamaguchi, Y; Gale, R D; McDonald, J M

    1989-01-01

    It has previously been demonstrated that calmodulin can be phosphorylated in vitro and in vivo by both tyrosine-specific and serine/threonine protein kinase. We demonstrate here that the insulin receptor tyrosine kinase purified from human placenta phosphorylates calmodulin. The highly purified receptors (prepared by insulin-Sepharose chromatography) were 5-10 times more effective in catalysing the phosphorylation of calmodulin than an equal number of partially purified receptors (prepared by wheat-germ agglutinin-Sepharose chromatography). Phosphorylation occurred exclusively on tyrosine residues, up to a maximum of 1 mol [0.90 +/- 0.14 (n = 5)] of phosphate incorporated/mol of calmodulin. Phosphorylation of calmodulin was dependent on the presence of certain basic proteins and divalent cations. Some of these basic proteins, i.e. polylysine, polyarginine, polyornithine, protamine sulphate and histones H1 and H2B, were also able to stimulate the phosphorylation of calmodulin via an insulin-independent activation of the receptor tyrosine kinase. Addition of insulin further increased incorporation of 32P into calmodulin. The magnitude of the effect of insulin was dependent on the concentration and type of basic protein used, ranging from 0.5- to 9.0-fold stimulation. Maximal phosphorylation of calmodulin was obtained at an insulin concentration of 10(-10) M, with half-maximal effect at 10(-11) M. Either Mg2+ or Mn2+ was necessary to obtain phosphorylation, but Mg2+ was far more effective than Mn2+. In contrast, maximal phosphorylation of calmodulin was observed in the absence of Ca2+. Inhibition of phosphorylation was observed as free Ca2+ concentration exceeded 0.1 microM, with almost complete inhibition at 30 microM free Ca2+. The Km for calmodulin was approx. 0.1 microM. To gain further insight into the effects of basic proteins in this system, we examined the binding of calmodulin to the insulin receptor and the polylysine. Calmodulin binds to the insulin

  10. Osteoarthritis-associated basic calcium phosphate crystals activate membrane proximal kinases in human innate immune cells.

    PubMed

    Corr, Emma M; Cunningham, Clare C; Helbert, Laura; McCarthy, Geraldine M; Dunne, Aisling

    2017-02-07

    Osteoarthritis (OA) is a chronic debilitating joint disorder of particularly high prevalence in the elderly population. Intra-articular basic calcium phosphate (BCP) crystals are present in the majority of OA joints and are associated with severe degeneration. They are known to activate macrophages, synovial fibroblasts, and articular chondrocytes, resulting in increased cell proliferation and the production of pro-inflammatory cytokines and matrix metalloproteases (MMPs). This suggests a pathogenic role in OA by causing extracellular matrix degradation and subchondral bone remodelling. There are currently no disease-modifying drugs available for crystal-associated OA; hence, the aim of this study was to explore the inflammatory pathways activated by BCP crystals in order to identify potential therapeutic targets to limit crystal-induced inflammation. Primary human macrophages and dendritic cells were stimulated with BCP crystals, and activation of spleen tyrosine kinase (Syk), phosphoinositide-3 kinase (PI3K), and mitogen-activated protein kinases (MAPKs) was detected by immunoblotting. Lipopolysaccharide (LPS)-primed macrophages were pre-treated with inhibitors of Syk, PI3K, and MAPKs prior to BCP stimulation, and cytokine production was quantified by enzyme-linked immunosorbent assay (ELISA). Aa an alternative, cells were treated with synovial fluid derived from osteoarthritic knees in the presence or absence of BCP crystals, and gene induction was assessed by real-time polymerase chain reaction (PCR). We demonstrate that exposure of primary human macrophages and dendritic cells to BCP crystals leads to activation of the membrane-proximal tyrosine kinases Syk and PI3K. Furthermore, we show that production of the pro-inflammatory cytokines interleukin (IL)-1α and IL-1β and phosphorylation of downstream MEK and ERK MAPKs is suppressed following treatment with inhibitors of Syk or PI3K. Finally, we demonstrate that treatment of macrophages with BCP crystals

  11. Timeless Links Replication Termination to Mitotic Kinase Activation

    PubMed Central

    Dheekollu, Jayaraju; Wiedmer, Andreas; Hayden, James; Speicher, David; Gotter, Anthony L.; Yen, Tim; Lieberman, Paul M.

    2011-01-01

    The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim) associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1). Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication. PMID:21573113

  12. Timeless links replication termination to mitotic kinase activation.

    PubMed

    Dheekollu, Jayaraju; Wiedmer, Andreas; Hayden, James; Speicher, David; Gotter, Anthony L; Yen, Tim; Lieberman, Paul M

    2011-05-06

    The mechanisms that coordinate the termination of DNA replication with progression through mitosis are not completely understood. The human Timeless protein (Tim) associates with S phase replication checkpoint proteins Claspin and Tipin, and plays an important role in maintaining replication fork stability at physical barriers, like centromeres, telomeres and ribosomal DNA repeats, as well as at termination sites. We show here that human Tim can be isolated in a complex with mitotic entry kinases CDK1, Auroras A and B, and Polo-like kinase (Plk1). Plk1 bound Tim directly and colocalized with Tim at a subset of mitotic structures in M phase. Tim depletion caused multiple mitotic defects, including the loss of sister-chromatid cohesion, loss of mitotic spindle architecture, and a failure to exit mitosis. Tim depletion caused a delay in mitotic kinase activity in vivo and in vitro, as well as a reduction in global histone H3 S10 phosphorylation during G2/M phase. Tim was also required for the recruitment of Plk1 to centromeric DNA and formation of catenated DNA structures at human centromere alpha satellite repeats. Taken together, these findings suggest that Tim coordinates mitotic kinase activation with termination of DNA replication.

  13. Normal ABL1 is a tumor suppressor and therapeutic target in human and mouse leukemias expressing oncogenic ABL1 kinases.

    PubMed

    Dasgupta, Yashodhara; Koptyra, Mateusz; Hoser, Grazyna; Kantekure, Kanchan; Roy, Darshan; Gornicka, Barbara; Nieborowska-Skorska, Margaret; Bolton-Gillespie, Elisabeth; Cerny-Reiterer, Sabine; Müschen, Markus; Valent, Peter; Wasik, Mariusz A; Richardson, Christine; Hantschel, Oliver; van der Kuip, Heiko; Stoklosa, Tomasz; Skorski, Tomasz

    2016-04-28

    Leukemias expressing constitutively activated mutants of ABL1 tyrosine kinase (BCR-ABL1, TEL-ABL1, NUP214-ABL1) usually contain at least 1 normal ABL1 allele. Because oncogenic and normal ABL1 kinases may exert opposite effects on cell behavior, we examined the role of normal ABL1 in leukemias induced by oncogenic ABL1 kinases. BCR-ABL1-Abl1(-/-) cells generated highly aggressive chronic myeloid leukemia (CML)-blast phase-like disease in mice compared with less malignant CML-chronic phase-like disease from BCR-ABL1-Abl1(+/+) cells. Additionally, loss of ABL1 stimulated proliferation and expansion of BCR-ABL1 murine leukemia stem cells, arrested myeloid differentiation, inhibited genotoxic stress-induced apoptosis, and facilitated accumulation of chromosomal aberrations. Conversely, allosteric stimulation of ABL1 kinase activity enhanced the antileukemia effect of ABL1 tyrosine kinase inhibitors (imatinib and ponatinib) in human and murine leukemias expressing BCR-ABL1, TEL-ABL1, and NUP214-ABL1. Therefore, we postulate that normal ABL1 kinase behaves like a tumor suppressor and therapeutic target in leukemias expressing oncogenic forms of the kinase. © 2016 by The American Society of Hematology.

  14. Studies on adenosine triphosphate transphosphorylases. Human isoenzymes of adenylate kinase: isolation and physicochemical comparison of the crystalline human ATP-AMP transphosphorylases from muscle and liver.

    PubMed

    Kuby, S A; Fleming, G; Frischat, A; Cress, M C; Hamada, M

    1983-02-10

    Procedures are described for the isolation, in crystalline form, of the adenylate kinases from autopsy samples of human muscle and from human liver. Weight average molecular weights were determined by sedimentation equilibrium to be 22,000 (+/- 700) and 25,450 (+/- 160) for the human muscle and liver isoenzymes, respectively. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, their molecular weights were estimated to be 21,700 and 26,500 for the muscle and liver enzymes, respectively. Both isoenzymes are accordingly monomeric proteins in their native state. Amino acid analyses are reported here for the normal human liver, calf liver, and rabbit liver adenylate kinases and compared with the normal human muscle, calf muscle, and rabbit muscle myokinases. The liver types as a group and the muscle types as a group show a great deal of homology, but some distinct differences are evident between the liver and muscle enzyme groups, especially in the number of residues of His, Pro, half-cystine, and the presence of tryptophan in the liver enzymes. The normal human liver adenylate kinase, as isolated in this report, has proved to be similar in its properties, if not identical, to the adenylate kinase isolated directly from human liver mitochondria (Hamada, M., Sumida, M., Okuda, H., Watanabe, T., Nojima, M., and Kuby, S. A. (1982) J. Biol. Chem. 257, 13120-13128). Therefore, the liver-type adenylate kinase may be considered a mitochondrial type.

  15. A Synthetic Human Kinase Can Control Cell Cycle Progression in Budding Yeast

    PubMed Central

    Davey, Megan J.; Andrighetti, Heather J.; Ma, Xiaoli; Brandl, Christopher J.

    2011-01-01

    The DDK kinase complex, composed of Cdc7 and Dbf4, is required for S-phase progression. The two component proteins show different degrees of sequence conservation between human and yeast. Here, we determine that Saccharomyces cerevisiae bearing human CDC7 and DBF4 grows comparably to cells with yeast DDK under standard growth conditions. HsDrf1 (a second human Dbf4-like protein) does not support growth, suggesting that HsDbf4 is the true ortholog of ScDbf4. Both human subunits are required to complement yeast cdc7Δ or dbf4Δ due to the inability of human Cdc7 or Dbf4 to interact with the corresponding yeast protein. Flow cytometry indicates normal cell cycle progression for yeast containing human DDK. However, yeast containing human DDK is sensitive to long-term exposure to hydroxyurea and fails to sporulate, suggesting that human DDK substitutes for some, but not all, of yeast DDK’s functions. We mapped the region of Cdc7 required for species-specific function of DDK to the C-terminus of Cdc7 by substituting the yeast C-terminal 55 amino acid residues in place of the equivalent human residues. The resulting hybrid protein supported growth of a cdc7Δ strain only in the presence of ScDBF4. The strain supported by the hybrid CDC7 was not sensitive to HU and formed tetrads. Together, our data indicate that DDK’s targeting of its essential substrate is conserved between species, whereas the interactions within DDK are species specific. PMID:22384342

  16. Protective features of resveratrol on human spermatozoa cryopreservation may be mediated through 5' AMP-activated protein kinase activation.

    PubMed

    Shabani Nashtaei, M; Amidi, F; Sedighi Gilani, M A; Aleyasin, A; Bakhshalizadeh, Sh; Naji, M; Nekoonam, S

    2017-03-01

    Biochemical and physical modifications during the freeze-thaw process adversely influence the restoration of energy-dependent sperm functions required for fertilization. Resveratrol, a phytoalexin, has been introduced to activate 5' AMP-activated protein kinase which is a cell energy sensor and a cell metabolism regulator. The cryoprotection of resveratrol on sperm cryoinjury via activation of AMP-activated protein kinase also remains to be elucidated. Our aim, thus, was to investigate: (i) the presence and intracellular localization of AMP-activated protein kinase protein; (ii) whether resveratrol may exert a protective effect on certain functional properties of fresh and post-thaw human spermatozoa through modulation of AMP-activated protein kinase. Spermatozoa from normozoospermic men were incubated with or without different concentrations of Compound C as an AMP-activated protein kinase inhibitor or resveratrol as an AMP-activated protein kinase activator for different lengths of time and were then cryopreserved. AMP-activated protein kinase is expressed essentially in the entire flagellum and the post-equatorial region. Viability of fresh spermatozoa was not significantly affected by the presence of Compound C or resveratrol. However, although Compound C caused a potent inhibition of spermatozoa motility parameters, resveratrol did not induce negative effect, except a significant reduction in motility at 25 μm for 1 h. Furthermore, resveratrol significantly increased AMP-activated protein kinase phosphorylation and mitochondrial membrane potential and decreased reactive oxygen species and apoptosis-like changes in frozen-thawed spermatozoa. Nevertheless, it was not able to compensate decreased sperm viability and motility parameters following cryopreservation. In contrast, Compound C showed opposite effects to resveratrol on AMP-activated protein kinase phosphorylation, reactive oxygen species, apoptosis-like changes, mitochondrial membrane potential, and

  17. Robust cell size checkpoint from spatiotemporal positive feedback loop in fission yeast.

    PubMed

    Yan, Jie; Ni, Xin; Yang, Ling

    2013-01-01

    Cells must maintain appropriate cell size during proliferation. Size control may be regulated by a size checkpoint that couples cell size to cell division. Biological experimental data suggests that the cell size is coupled to the cell cycle in two ways: the rates of protein synthesis and the cell polarity protein kinase Pom1 provide spatial information that is used to regulate mitosis inhibitor Wee1. Here a mathematical model involving these spatiotemporal regulations was developed and used to explore the mechanisms underlying the size checkpoint in fission yeast. Bifurcation analysis shows that when the spatiotemporal regulation is coupled to the positive feedback loops (active Cdc2 promotes its activator, Cdc25, and suppress its inhibitor, Wee1), the mitosis-promoting factor (MPF) exhibits a bistable steady-state relationship with the cell size. The switch-like response from the positive feedback loops naturally generates the cell size checkpoint. Further analysis indicated that the spatial regulation provided by Pom1 enhances the robustness of the size checkpoint in fission yeast. This was consistent with experimental data.

  18. SKI-606 (bosutinib), a novel Src kinase inhibitor, suppresses migration and invasion of human breast cancer cells.

    PubMed

    Vultur, Adina; Buettner, Ralf; Kowolik, Claudia; Liang, Wei; Smith, David; Boschelli, Frank; Jove, Richard

    2008-05-01

    Src family kinase activity is elevated in many human tumors, including breast cancer, and is often associated with aggressive disease. We examined the effects of SKI-606 (bosutinib), a selective Src family kinase inhibitor, on human cancer cells derived from breast cancer patients to assess its potential for breast cancer treatment. Our results show that SKI-606 caused a decrease in cell motility and invasion of breast cancer cell lines with an IC50 of approximately 250 nmol/L, which was also the IC50 for inhibition of cellular Src kinase activity in intact tumor cells. These changes were accompanied by an increase in cell-to-cell adhesion and membrane localization of beta-catenin. By contrast, cell proliferation and survival were unaffected by SKI-606 at concentrations sufficient to block cell migration and invasion. Analysis of downstream effectors of Src revealed that SKI-606 inhibits the phosphorylation of focal adhesion kinase (FAK), proline-rich tyrosine kinase 2 (Pyk2), and Crk-associated substrate (p130Cas), with an IC50 similar to inhibition of cellular Src kinase. Our findings indicate that SKI-606 inhibits signaling pathways involved in controlling tumor cell motility and invasion, suggesting that SKI-606 is a promising therapeutic for breast cancer.

  19. Molecular Mechanisms of DNA Replication Checkpoint Activation

    PubMed Central

    Recolin, Bénédicte; van der Laan, Siem; Tsanov, Nikolay; Maiorano, Domenico

    2014-01-01

    The major challenge of the cell cycle is to deliver an intact, and fully duplicated, genetic material to the daughter cells. To this end, progression of DNA synthesis is monitored by a feedback mechanism known as replication checkpoint that is untimely linked to DNA replication. This signaling pathway ensures coordination of DNA synthesis with cell cycle progression. Failure to activate this checkpoint in response to perturbation of DNA synthesis (replication stress) results in forced cell division leading to chromosome fragmentation, aneuploidy, and genomic instability. In this review, we will describe current knowledge of the molecular determinants of the DNA replication checkpoint in eukaryotic cells and discuss a model of activation of this signaling pathway crucial for maintenance of genomic stability. PMID:24705291

  20. [Therapeutic Cancer Vaccine and Immune Checkpoint Inhibitor].

    PubMed

    Mimura, Kousaku; Kono, Koji

    2017-09-01

    Therapeutic cancer vaccine enhances a specific immune response against tumor cells in vivo, resulting in exertion of antitumor effects. On the other hand, immune checkpoint inhibitors promote the induction of tumor-specific T cells and also enhance the cytotoxic abilityof these T cells in tumor microenvironment. There is a possibilitythat immune checkpoint inhibitors enhance tumor immune responses induced bytherapeutic cancer vaccine, and it is expected that additive or synergistic effects will be obtained bythe combination of them. Moreover, according to previous reports, we should use an immune checkpoint inhibitor to enhance the cytotoxic ability of tumor-specific T cells as the combination for therapeutic cancer vaccine. Furthermore, the combination of a specific antibodyagainst newlyidentified co-inhibitoryreceptors (Lag-3, Tim-3, TIGIT, etc)and a therapeutic cancer vaccine is also one of newlyexpected treatments in the future.

  1. Checkpoint triggering in a computer system

    SciTech Connect

    Cher, Chen-Yong

    2016-09-06

    According to an aspect, a method for triggering creation of a checkpoint in a computer system includes executing a task in a processing node of the computer system and determining whether it is time to read a monitor associated with a metric of the task. The monitor is read to determine a value of the metric based on determining that it is time to read the monitor. A threshold for triggering creation of the checkpoint is determined based on the value of the metric. Based on determining that the value of the metric has crossed the threshold, the checkpoint including state data of the task is created to enable restarting execution of the task upon a restart operation.

  2. Immune checkpoint inhibitors for cancer treatment.

    PubMed

    Park, Junsik; Kwon, Minsuk; Shin, Eui-Cheol

    2016-11-01

    During immune responses antigen-specific T cells are regulated by several mechanisms, including through inhibitory receptors and regulatory T cells, to avoid excessive or persistent immune responses. These regulatory mechanisms, which are called 'immune checkpoints', suppress T cell responses, particularly in patients with chronic viral infections and cancer where viral antigens or tumor antigens persist for a long time and contribute to T cell exhaustion. Among these regulatory mechanisms, cytotoxic T lymphocyte associated protein-4 (CTLA-4) and programmed cell death 1 (PD-1) are the most well-known receptors and both have been targeted for drug development. As a result, anti-CTLA-4 and anti-PD-1 (or anti-PD-L1) antibodies were recently developed as immune checkpoint inhibitors for use in cancer treatments. In this review we describe several receptors that function as immunological checkpoints as well as the pharmaceuticals that target them.

  3. Bir1 deletion causes malfunction of the spindle assembly checkpoint and apoptosis in yeast.

    PubMed

    Ren, Qun; Liou, Liang-Chun; Gao, Qiuqiang; Bao, Xiaoming; Zhang, Zhaojie

    2012-01-01

    Cell division in yeast is a highly regulated and well studied event. Various checkpoints are placed throughout the cell cycle to ensure faithful segregation of sister chromatids. Unexpected events, such as DNA damage or oxidative stress, cause the activation of checkpoint(s) and cell cycle arrest. Malfunction of the checkpoints may induce cell death. We previously showed that under oxidative stress, the budding yeast cohesin Mcd1, a homolog of human Rad21, was cleaved by the caspase-like protease Esp1. The cleaved Mcd1 C-terminal fragment was then translocated to mitochondria, causing apoptotic cell death. In the present study, we demonstrated that Bir1 plays an important role in spindle assembly checkpoint and cell death. Similar to H(2)O(2) treatment, deletion of BIR1 using a BIR1-degron strain caused degradation of the securin Pds1, which binds and inactivates Esp1 until metaphase-anaphase transition in a normal cell cycle. BIR1 deletion caused an increase level of ROS and mis-location of Bub1, a major protein for spindle assembly checkpoint. In wild type, Bub1 was located at the kinetochores, but was primarily in the cytoplasm in bir1 deletion strain. When BIR1 was deleted, addition of nocodazole was unable to retain the Bub1 localization on kinetochores, further suggesting that Bir1 is required to activate and maintain the spindle assembly checkpoint. Our study suggests that the BIR1 function in cell cycle regulation works in concert with its anti-apoptosis function.

  4. Human CD180 Transmits Signals via the PIM-1L Kinase

    PubMed Central

    Egli, Nicole; Zajonz, Alexandra; Burger, Matthew T.; Schweighoffer, Tamas

    2015-01-01

    Toll-like receptors (TLRs) are important sensors of the innate immune system that recognize conserved structural motifs and activate cells via a downstream signaling cascade. The CD180/MD1 molecular complex is an unusual member of the TLR family, since it lacks the components that are normally required for signal transduction by other TLRs. Therefore the CD180/MD 1 complex has been considered of being incapable of independently initiating cellular signals. Using chemogenetic approaches we identified specifically the membrane bound long form of PIM-1 kinase, PIM-1L as the mediator of CD180-dependent signaling. A dominant negative isoform of PIM-1L, but not of other PIM kinases, inhibited signaling elicited by cross-linking of CD180, and this effect was phenocopied by PIM inhibitors. PIM-1L was directed to the cell membrane by its N-terminal extension, where it colocalized and physically associated with CD180. Triggering CD180 also induced increased phosphorylation of the anti-apoptotic protein BAD in a PIM kinase-dependent fashion. Also in primary human B cells, which are the main cells expressing CD180 in man, cross-linking of CD180 by monoclonal antibodies stimulated cell survival and proliferation that was abrogated by specific inhibitors. By associating with PIM-1L, CD180 can thus obtain autonomous signaling capabilities, and this complex is then channeling inflammatory signals into B cell survival programs. Pharmacological inhibition of PIM-1 should therefore provide novel therapeutic options in diseases that respond to innate immune stimulation with subsequently increased B cell activity, such as lupus erythematosus or myasthenia gravis. PMID:26555723

  5. Fructosamine 3-kinase is involved in an intracellular deglycation pathway in human erythrocytes.

    PubMed Central

    Delpierre, Ghislain; Collard, François; Fortpied, Juliette; Van Schaftingen, Emile

    2002-01-01

    Fructosamine 3-kinase, which phosphorylates low-molecular-mass and protein-bound fructosamines on the third carbon of their deoxyfructose moiety, is quite active in erythrocytes, and was proposed to initiate a process removing fructosamine residues from proteins. In the present study, we show that incubation of human erythrocytes with 200 mM glucose not only caused the progressive formation of glycated haemoglobin, but also increased the level of an anionic form of haemoglobin containing alkali-labile phosphate, to approx. 5% of total haemoglobin. 1-Deoxy-1-morpholinofructose (DMF), a substrate and competitive inhibitor of fructosamine 3-kinase, doubled the rate of accumulation of glycated haemoglobin, but markedly decreased the amount of haemoglobin containing alkali-labile phosphate. The latter corresponds therefore to haemoglobin bound to a fructosamine 3-phosphate group (FN3P-Hb). Returning erythrocytes incubated with 200 mM glucose and DMF to a low-glucose medium devoid of DMF caused a decrease in the amount of glycated haemoglobin, a transient increase in FN3P-Hb and a net decrease in the sum (glycated haemoglobin+FN3P-Hb). These effects were prevented by DMF, indicating that fructosamine 3-kinase is involved in the removal of fructosamine residues. The second step of this 'deglycation' process is most likely a spontaneous decomposition of the fructosamine 3-phosphate residues to a free amine, 3-deoxyglucosone and P(i). This is consistent with the findings that 2-oxo-3-deoxygluconate, the product of 3-deoxyglucosone oxidation, is formed in erythrocytes incubated for 2 days with 200 mM glucose in a sufficient amount to account for the removal of fructosamine residues from proteins, and that DMF appears to inhibit the formation of 2-oxo-3-deoxygluconate from elevated glucose concentrations. PMID:11975663

  6. Cadmium activates extracellular signal-regulated kinase 5 in HK-2 human renal proximal tubular cells

    SciTech Connect

    Kondo, Mio; Inamura, Hisako; Matsumura, Ken-ichi; Matsuoka, Masato

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Cadmium exposure induces ERK5 phosphorylation in HK-2 renal proximal tubular cells. Black-Right-Pointing-Pointer BIX02189 treatment suppresses cadmium-induced ERK5 but not ERK1/2 phosphorylation. Black-Right-Pointing-Pointer BIX02189 treatment suppresses cadmium-induced CREB and c-Fos phosphorylation. Black-Right-Pointing-Pointer ERK5 activation by cadmium exposure may play an anti-apoptotic role in HK-2 cells. -- Abstract: We examined the effects of cadmium chloride (CdCl{sub 2}) exposure on the phosphorylation and functionality of extracellular signal-regulated kinase 5 (ERK5), a recently identified member of the mitogen-activated protein kinase (MAPK) family, in HK-2 human renal proximal tubular cells. Following exposure to CdCl{sub 2}, ERK5 phosphorylation increased markedly, but the level of total ERK5 was unchanged. ERK5 phosphorylation following CdCl{sub 2} exposure was rapid and transient, similar to the time course of ERK1/2 phosphorylation. Treatment of HK-2 cells with the MAPK/ERK kinase 5 inhibitor, BIX02189, suppressed CdCl{sub 2}-induced ERK5 but not ERK1/2 phosphorylation. The CdCl{sub 2}-induced increase of phosphorylated cAMP response element-binding protein (CREB) and activating transcription factor-1 (ATF-1), as well as the accumulation of mobility-shifted c-Fos protein, were suppressed by BIX02189 treatment. Furthermore, BIX02189 treatment enhanced cleavage of poly(ADP-ribose) polymerase and increased the level of cytoplasmic nucleosomes in HK-2 cells exposed to CdCl{sub 2}. These findings suggest that ERK5 pathway activation by CdCl{sub 2} exposure might induce the phosphorylation of cell survival-transcription factors, such as CREB, ATF-1, and c-Fos, and may exert a partial anti-apoptotic role in HK-2 cells.

  7. Detection of mutations in the mitogen-activated protein kinase pathway in human melanoma.

    PubMed

    Alsina, Janivette; Gorsk, David H; Germino, F Joseph; Shih, Weichung; Lu, Shou-En; Zhang, Zhi-Gang; Yang, Jin-Ming; Hait, William N; Goydos, James S

    2003-12-15

    Recent studies suggest that activating point mutations in B-RAF may commonly occur in melanoma. We devised a method to detect point mutations in heterogeneous tissues containing both wild-type and mutant B-RAF and N-RAS genes by using site-directed mutagenesis to introduce new restrictions sites in the cDNA sequence when the specific point mutations are present. We used this technique to determine the incidence of mitogen-activated protein kinase (MAPK) mutations in human melanoma. We screened 85 melanoma samples for the most common B-RAF and N-RAS mutations found in melanoma using a site-directed mutagenesis-based detection technique. Western blotting was used to evaluate downstream up-regulation of the mitogen-activated protein kinase pathway in these tissues. Thirty-three samples (7 of 25 primaries, 15 of 25 regional metastases, 5 of 25 nodal metastases, and 6 of 10 distant metastases) harbored the V599E B-RAF mutation (39%), 12 contained a Q61R N-RAS mutation and 5 a Q61K N-RAS mutation. Western blotting with antiphosphorylated extracellular signal-regulated kinase 1/2 antibodies demonstrated up-regulation of the MAPK pathway in samples containing activating B-RAF or N-RAS mutations compared with wild-type samples. This method of detection was sensitive and specific with no false positives. Activating mutations of the MAPK pathway were present in approximately 60% of samples tested and caused activation of this cellular pathway that appears to be important in the pathogenesis of melanoma.

  8. Crystal structure of human protein kinase CK2: insights into basic properties of the CK2 holoenzyme

    PubMed Central

    Niefind, Karsten; Guerra, Barbara; Ermakowa, Inessa; Issinger, Olaf-Georg

    2001-01-01

    The crystal structure of a fully active form of human protein kinase CK2 (casein kinase 2) consisting of two C-terminally truncated catalytic and two regulatory subunits has been determined at 3.1 Å resolution (Protein Data Bank code: 1JWH). In the CK2 complex the regulatory subunits form a stable dimer linking the two catalytic subunits, which make no direct contact with one another. Each catalytic subunit interacts with both regulatory chains, predominantly via an extended C-terminal tail of the regulatory subunit. The CK2 structure is consistent with its constitutive activity and with a flexible role of the regulatory subunit as a docking partner for various protein kinases. Furthermore it shows an inter-domain mobility in the catalytic subunit known to be functionally important in protein kinases and detected here for the first time directly within one crystal structure. PMID:11574463

  9. A diacylglycerol kinase inhibitor, R59022, potentiates secretion by and aggregation of thrombin-stimulated human platelets.

    PubMed Central

    Nunn, D L; Watson, S P

    1987-01-01

    The diacylglycerol kinase inhibitor R59022 (10 microM) potentiates secretion and aggregation responses in human platelets challenged with sub-maximal concentrations of thrombin. Potentiation correlates closely with increased formation of diacylglycerol, increased phosphorylation of a 40 kDa protein, a known substrate for protein kinase C, and with decreased formation of phosphatidic acid, the product of diacylglycerol kinase. Phosphorylation of myosin light chains, formation of inositol phosphates and the mobilization of Ca2+ by thrombin are not affected by R59022 (10 microM). These data support a role for protein kinase C in platelet aggregation and secretion, and provide further evidence that endogenous diacylglycerols bring about the activation of this enzyme. These data also add further argument against a role for phosphatidic acid in platelet activation. PMID:2821994

  10. Multifunctional human transcriptional coactivator protein PC4 is a substrate of Aurora kinases and activates the Aurora enzymes.

    PubMed

    Dhanasekaran, Karthigeyan; Kumari, Sujata; Boopathi, Ramachandran; Shima, Hiroki; Swaminathan, Amrutha; Bachu, Mahesh; Ranga, Udaykumar; Igarashi, Kazuhiko; Kundu, Tapas K

    2016-03-01

    Positive coactivator 4 (PC4), a human transcriptional coactivator, is involved in diverse processes like chromatin organization and transcription regulation. It is hyperphosphorylated during mitosis, with unknown significance. For the first time, we demonstrate the function of PC4 outside the nucleus upon nuclear envelope breakdown. A fraction of PC4 associates with Aurora A and Aurora B and undergoes phosphorylation, following which PC4 activates both Aurora A and B to sustain optimal kinase activity to maintain the phosphorylation gradient for the proper functioning of the mitotic machinery. This mitotic role is evident in PC4 knockdown cells where the defects are rescued only by the catalytically active Aurora kinases, but not the kinase-dead mutants. Similarly, the PC4 phosphodeficient mutant failed to rescue such defects. Hence, our observations establish a novel mitotic function of PC4 that might be dependent on Aurora kinase-mediated phosphorylation.

  11. Mitochondrial localization of human PANK2 and hypotheses of secondary iron accumulation in pantothenate kinase-associated neurodegeneration.

    PubMed

    Johnson, Monique A; Kuo, Yien Ming; Westaway, Shawn K; Parker, Susan M; Ching, Katherine H L; Gitschier, Jane; Hayflick, Susan J

    2004-03-01

    Mutations in the pantothenate kinase 2 gene (PANK2) lead to pantothenate kinase-associated neurodegeneration (PKAN, formerly Hallervorden-Spatz syndrome). This neurodegenerative disorder is characterized by iron accumulation in the basal ganglia. Pantothenate kinase is the first enzyme in the biosynthesis of coenzyme A from pantothenate (vitamin B(5)). PANK2, one of four human pantothenate kinase genes, is uniquely predicted to be targeted to mitochondria. We demonstrate mitochondrial localization of PANK2 and speculate on mechanisms of secondary iron accumulation in PKAN. Furthermore, PANK2 uses an unconventional translational start codon, CUG, which is polymorphic in the general population. The variant sequence, CAG (allele frequency: 0.05), leads to skipping of the mitochondrial targeting signal and cytosolic localization of PANK2. This common variant may cause mitochondrial dysfunction and impart susceptibility to late-onset neurodegenerative disorders with brain iron accumulation, including Parkinson's disease.

  12. Inhibition of cyclin-dependent kinase 1 (CDK1) by indirubin derivatives in human tumour cells

    PubMed Central

    Marko, D; Schätzle, S; Friedel, A; Genzlinger, A; Zankl, H; Meijer, L; Eisenbrand, G

    2001-01-01

    The bisindole indirubin has been described, more than 30 years ago, as being clinically active in the treatment of human chronic myelocytic leukaemia. However, the underlying mechanism of action has remained unclear. We have reported previously that indirubin and its analogues are potent and selective inhibitors of cyclin-dependent kinases (CDK). In this study, we investigated the influence of indirubin and derivatives on CDK1/cyclin B kinase in human tumour cells at concentrations known to induce growth inhibition. Cells of the mammary carcinoma cell line MCF-7, synchronized by serum deprivation, after serum repletion stay arrested in the G1/G0 phase of the cell cycle in the presence of 2 μM indirubin-3′-monoxime. At higher drug concentrations (≥ 5 μM) an increase of the cell population in the G2/M phase is additionally observed. Cells synchronized in G2/M phase by nocodazole remain arrested in the G2/M phase after release, in the presence of indirubin-3′-monoxime (≥5 μM). After 24 h treatment with 10 μM indirubin-3′-monoxime a sub-G2 peak appears, indicative for the onset of apoptotic cell death. Treatment of MCF-7 cells with growth inhibitory concentrations of indirubin-3′-monoxime induces dose-dependent inhibition of the CDK1 activity in the cell. After 24 h treatment, a strong decrease of the CDK1 protein level along with a reduction of cyclin B in complex with CDK1 is observed. Taken together, the results of this study strongly suggest that inhibition of CDK activity in human tumour cells is a major mechanism by which indirubin derivatives exert their potent antitumour efficacy. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11161389

  13. Optimal message log reclamation for independent checkpointing

    NASA Technical Reports Server (NTRS)

    Wang, Yi-Min; Fuchs, W. Kent

    1993-01-01

    Independent (uncoordinated) check pointing for parallel and distributed systems allows maximum process autonomy but suffers from possible domino effects and the associated storage space overhead for maintaining multiple checkpoints and message logs. In most research on check pointing and recovery, it was assumed that only the checkpoints and message logs older than the global recovery line can be discarded. It is shown how recovery line transformation and decomposition can be applied to the problem of efficiently identifying all discardable message logs, thereby achieving optimal garbage collection. Communication trace-driven simulation for several parallel programs is used to show the benefits of the proposed algorithm for message log reclamation.

  14. Granulocyte macrophage-colony stimulating factor stimulates both association and activation of phosphoinositide 3OH-kinase and src-related tyrosine kinase(s) in human myeloid derived cells.

    PubMed Central

    Corey, S; Eguinoa, A; Puyana-Theall, K; Bolen, J B; Cantley, L; Mollinedo, F; Jackson, T R; Hawkins, P T; Stephens, L R

    1993-01-01

    The signalling pathways used by the GM-CSF receptor are currently unknown. Here we show that in human myeloid derived cells GM-CSF can stimulate; (i) the accumulation of PtdIns(3,4,5)P3; (ii) increases in p53/p56lyn and p62c-yes directed protein tyrosine kinase activities in anti-lyn and anti-c-yes antibody directed immunoprecipitates, respectively and; (iii) increases in phosphoinositide 3OH-kinase activity in antiphosphotyrosine, anti-p53/p56lyn and anti-p62c-yes antibody directed immunoprecipitates. These results suggest that GM-CSF can stimulate formation of protein tyrosine kinase co-ordinated signalling complexes, that contain p53/p56lyn, p62c-yes and an activated PtdInsP2 directed phosphoinositide 3OH-kinase, which can drive the accumulation of the putative second-messenger PtdIns(3,4,5)P3. Images PMID:8392933

  15. Inhibition of Aurora B kinase sensitizes a subset of human glioma cells to TRAIL concomitant with induction of TRAIL-R2.

    PubMed

    Li, J; Anderson, M G; Tucker, L A; Shen, Y; Glaser, K B; Shah, O J

    2009-03-01

    Small-molecule inhibitors of the Aurora A and B kinases interfere with mitotic centrosome function and disrupt the mitotic spindle assembly checkpoint resulting in polyploidization and apoptosis of proliferating cells. As such, several Aurora kinase inhibitors are at various stages of clinical development as anticancer agents. To identify candidate apoptosis-sensitizing genes that could be exploited in combination with Aurora kinase inhibitors in malignant glioma, we have carried out global gene expression analysis in a D54MG glioma cell derivative treated with three Aurora kinase inhibitors chosen for their distinctive selectivities: MLN8054 (Aurora A-selective), AZD1152 (Aurora B-selective), and VX-680 (Aurora A/B). The modulation of apoptotic gene expression by p53 under these conditions was ascertained, as p53 expression can be toggled on and off in this D54MG derivative by virtue of a stable, inducible, p53-targeting short hairpin RNA (D54MG(shp53)). This analysis identified the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) death receptor, TRAIL receptor 2 (TRAIL-R2), as an apoptosis-sensitizing gene induced selectively following inhibition of Aurora B. In glioma cell lines where TRAIL-R2 was induced following polyploidization, the sensitivity, kinetics, and magnitude of TRAIL-mediated apoptosis were enhanced. Our data shed light on the apoptotic program induced during polyploidization and suggest that TRAIL-R2 activation is a putative point of therapeutic intervention in combination with inhibitors of Aurora B.

  16. A cDNA clone encoding human cAMP-dependent protein kinase catalytic subunit C. alpha

    SciTech Connect

    Maldonado, F.; Hanks, S.K. )

    1988-08-25

    The authors have determined the nucleotide sequence from both complementary strands of a human cDNA coding for cAMP-dependent protein kinase catalytic subunit type {alpha} (cAPK-C{alpha}). This cDNA was one of many protein kinase cDNAs isolated from a HeLa cell library by screening with oligonucleotide probes designed to recognize target sequences encoding highly conserved segments within the catalytic domains. The deduced human cAPK-C{alpha} amino acid sequence of 350 residues differs from the bovine and murine sequences at 3 and 7 positions, respectively.

  17. Metabolomic analysis of <