The mitotic checkpoint prevents cells with unaligned chromosomes from prematurely exiting mitosis by inhibiting the anaphase-promoting complex/cyclosome (APC/C) targeting key proteins for ubiquitin-mediated proteolysis. We have examined mechanism which inhibits APC/C purifying an inhibitory factor HeLa cells. call this complex (MCC) as it consists of hBUBR1, hBUB3, CDC20, and MAD2 in near equal stoichiometry. MCC activity is 3,000-fold greater than that recombinant MAD2, has also been shown...

Human cells express two kinases that are related to the yeast mitotic checkpoint kinase BUB1. hBUB1 and hBUBR1 bind kinetochores where they postulated be components of monitors kinetochore activities determine if chromosomes have achieved alignment at spindle equator (Jablonski, S.A., G.K.T. Chan, C.A. Cooke, W.C. Earnshaw, T.J. Yen. 1998. Chromosoma. 107:386–396). In support this, homologous mouse BUB1 been shown important for (Cahill, D.P., C. Lengauer, J. Yu, G.J. Riggins, J.K. Willson,...

CENP-E is a kinesin-like protein that binds to kinetochores and may provide functions are critical for normal chromosome motility during mitosis. To directly test the in vivo function of CENP-E, we microinjected affinity-purified antibodies block assembly onto then examined behavior these chromosomes. Chromosomes lacking at their consistently exhibited two types defects blocked alignment spindle equator. positioned near pole remained mono-oriented as they were unable establish bipolar...

We have identified a 350–amino acid domain in the kinetochore motor CENP-E that specifies binding mitosis but not during interphase. The was used yeast two-hybrid screen to isolate interacting proteins included CENP-E, CENP-F, and hBUBR1, BUB1-related kinase found be mutated some colorectal carcinomas (Cahill, D.P., C. Lengauer, J. Yu, G.J. Riggins, J.K. Wilson, S.D. Markowitz, K.W. Kinzler, B. Vogelstein. 1998. Nature. 392:300–303). assembled onto kinetochores sequential order late stages...

CENP-E is a kinesin-like protein that when depleted from mammalian kinetochores leads to mitotic arrest with mixture of aligned and unaligned chromosomes. In the present study, we used immunofluorescence, video, electron microscopy demonstrate depletion via antibody microinjection reduces kinetochore microtubule binding by 23% at chromosomes, severely Disruption function also tension across centromere, increases incidence spindle pole fragmentation, results in monooriented chromosomes...

The receptor for hyaluronan-mediated motility (RHAMM), an acidic coiled coil protein, has previously been characterized as a cell surface hyaluronan, and microtubule-associated intracellular hyaluronan binding protein. In this study, we demonstrate that subset of cellular RHAMM localizes to the centrosome functions in maintenance spindle integrity. We confirm previous study showing amino terminus interacts with microtubules further separate carboxy-terminal domain is required centrosomal...

The mitotic checkpoint (or spindle assembly checkpoint) is a fail-safe mechanism to prevent chromosome missegregation by delaying anaphase onset in the presence of defective kinetochore-microtubule attachment. target E3 ubiquitin ligase anaphase-promoting complex/cyclosome. Once all chromosomes are properly attached and bioriented at metaphase plate, needs be silenced. Previously, we others have reported that TRIP13 AAA-ATPase binds checkpoint-silencing protein p31comet. Here show endogenous...

We used the cancer-intrinsic property of oncogene-induced DNA damage as base for a conditional synthetic lethality approach. To target mechanisms important cancer cell adaptation to genotoxic stress and thereby achieve cell-specific killing, we combined inhibition kinases ATR Wee1. Wee1 regulates cycle progression, whereas is an apical kinase in DNA-damage response. In orthotopic breast model, tumor-selective combination bioavailable inhibitors led tumor remission inhibited metastasis with...

Ataxia telangiectasia–mutated gene (ATM) is a 350-kDa protein whose function defective in the autosomal recessive disorder ataxia telangiectasia (AT). Affinity-purified polyclonal antibodies were used to characterize ATM. Steady-state levels of ATM varied from undetectable most AT cell lines highly expressed HeLa, U2OS, and normal human fibroblasts. Subcellular fractionation showed that predominantly nuclear associated with chromatin matrix. remained constant throughout cycle did not change...

Mad1 was first identified in budding yeast as an essential component of the checkpoint system that monitors spindle assembly mitosis and prevents premature anaphase onset. Using antibodies to human homologue (HsMAD1), we have begun characterize this protein mammalian cells. HsMad1 is found localized at kinetochores mitosis. The labeling brightest prometaphase absent from metaphase anaphase. In cells where most chromosomes reached plate, those aligned plate show no while remaining, unaligned...

We have determined that the previously identified dual-specificity protein kinase TTK is human orthologue of yeast MPS1 kinase. Yeast (monopolar spindle) required for spindle pole duplication and checkpoint. Consistent with recently vertebrate homologues, we found hMPS1 localized to centrosomes kinetochores. In addition, part a growing list kinetochore proteins are nuclear pores. by cells arrest in mitosis response defects resulting from loss kinesin-like protein, CENP-E. The pattern...

Histone deacetylase 6 (HDAC6) contains tandem catalytic domains and a ubiquitin-binding zinc finger displays activity toward acetylated microtubules. Here we show that unlike its orthologs from Caenorhabditis elegans, Drosophila, mouse, human HDAC6 possesses tetradecapeptide repeat domain located between the second C-terminal motif. Related to this structural difference, cytoplasmic localization of human, but not murine, is resistant treatment with leptomycin B (LMB). Although it dispensable...

hSgo2 (previously annotated as Tripin) was recently reported to be a new inner centromere protein that is essential for cohesion (Kitajima et al., 2006). In this study, we show exhibits dynamic distribution pattern, and its localization depends on the BUB1 Aurora B kinases. concentrated at of unattached kinetochores, but extends toward kinetochores are under tension. This pattern reminiscent MCAK, which microtubule depolymerase believed key component error correction mechanism kinetochores....

Cytoplasmic dynein functions at several sites during mitosis; however, the basis of targeting to each site remains unclear. Tandem mass spectrometry analysis mitotic revealed a phosphorylation in intermediate chains (ICs) that mediates binding kinetochores. IC directs zw10 rather than dynactin, and this interaction is needed for kinetochore localization. Phosphodynein associates with kinetochores from nuclear envelope breakdown metaphase, but bioriented microtubule (MT) attachment chromosome...

Kinetochore (KT) localization of mitotic checkpoint proteins is essential for their function during mitosis. hSpindly KT dependent on the RZZ complex and recruits dynein–dynactin to KTs mitosis, but mechanism recruitment unknown. Through domain-mapping studies we characterized domain discovered it undergoes farnesylation at C-terminal cysteine residue. The N-terminal 293 residues are dispensable its localization. Inhibition using a farnesyl transferase inhibitor (FTI) abrogated without...

// Cody W. Lewis 1, 2, 3 , Zhigang Jin Dawn Macdonald Wenya Wei 1 Xu Jing Qian Won Shik Choi Ruicen He Xuejun Sun and Gordon Chan Department of Oncology, University Alberta, Edmonton, Canada T6G 1Z2 2 Experimental Cross Cancer Institute, Research Institute Northern 2J7 Correspondence to: Chan, email: gkc@ualberta.ca gordonch@ahs.ca Keywords: cell cycle checkpoint, Wee1 kinase, paclitaxel, mitotic catastrophe, breast cancer Received: October 21, 2016 Accepted: April 27, 2017 Published: May...

Abstract Adavosertib (also known as AZD1775 or MK1775) is a small-molecule inhibitor of the protein kinase Wee1, with single-agent activity in multiple solid tumors, including sarcoma, glioblastoma, and head neck cancer. also shows promising results combination genotoxic agents such ionizing radiation chemotherapy. Previous studies have investigated molecular mechanisms primary resistance to Wee1 inhibition. Here, we acquired inhibition, focusing on role Wee1-related Myt1. Myt1 kinases were...

Multinucleated cells have been noted in pathophysiological states of the liver including infection with hepatitis B virus (HBV), status which is also closely associated genomic instability cancer. Here, we showed that X oncoprotein (HBx) expression Chang results a multinuclear phenotype and an abnormal number centrosomes (n >or=3). Regulation centrosome duplication HBx-expressing ChangX-34 was defective uncoupled from cell cycle. HBx induced amplification centrosomes, multipolar spindle...