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CDK-1 Inhibition in G2 Stabilizes Kinetochore-Microtubules in the following Mitosis.
Gayek AS, Ohi R
(2016) PLoS One 11: e0157491
MeSH Terms: Anaphase, CDC2 Protein Kinase, Cell Line, Transformed, Chromosomes, Human, Cyclin-Dependent Kinases, G2 Phase, Humans, Kinesin, Kinetochores, Microtubules
Show Abstract · Added April 18, 2017
Cell proliferation is driven by cyclical activation of cyclin-dependent kinases (CDKs), which produce distinct biochemical cell cycle phases. Mitosis (M phase) is orchestrated by CDK-1, complexed with mitotic cyclins. During M phase, chromosomes are segregated by a bipolar array of microtubules called the mitotic spindle. The essential bipolarity of the mitotic spindle is established by the kinesin-5 Eg5, but factors influencing the maintenance of spindle bipolarity are not fully understood. Here, we describe an unexpected link between inhibiting CDK-1 before mitosis and bipolar spindle maintenance. Spindles in human RPE-1 cells normally collapse to monopolar structures when Eg5 is inhibited at metaphase. However, we found that inhibition of CDK-1 in the G2 phase of the cell cycle improved the ability of RPE-1 cells to maintain spindle bipolarity without Eg5 activity in the mitosis immediately after release from CDK-1 inhibition. This improved bipolarity maintenance correlated with an increase in the stability of kinetochore-microtubules, the subset of microtubules that link chromosomes to the spindle. The improvement in bipolarity maintenance after CDK-1 inhibition in G2 required both the kinesin-12 Kif15 and increased stability of kinetochore-microtubules. Consistent with increased kinetochore-microtubule stability, we find that inhibition of CDK-1 in G2 impairs mitotic fidelity by increasing the incidence of lagging chromosomes in anaphase. These results suggest that inhibition of CDK-1 in G2 causes unpredicted effects in mitosis, even after CDK-1 inhibition is relieved.
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10 MeSH Terms
Mutation of serine 1333 in the ATR HEAT repeats creates a hyperactive kinase.
Luzwick JW, Nam EA, Zhao R, Cortez D
(2014) PLoS One 9: e99397
MeSH Terms: Amino Acid Sequence, Amino Acid Substitution, Ataxia Telangiectasia Mutated Proteins, Carrier Proteins, Checkpoint Kinase 1, DNA Replication, DNA-Binding Proteins, G2 Phase Cell Cycle Checkpoints, HCT116 Cells, HEK293 Cells, Humans, Hydroxyurea, Molecular Sequence Data, Nuclear Proteins, Phosphorylation, Protein Binding, Protein Kinases, Protein Structure, Secondary, Radiation, Ionizing, Serine, Signal Transduction, Ultraviolet Rays
Show Abstract · Added January 20, 2015
Subcellular localization, protein interactions, and post-translational modifications regulate the DNA damage response kinases ATR, ATM, and DNA-PK. During an analysis of putative ATR phosphorylation sites, we found that a single mutation at S1333 creates a hyperactive kinase. In vitro and in cells, mutation of S1333 to alanine (S1333A-ATR) causes elevated levels of kinase activity with and without the addition of the protein activator TOPBP1. S1333 mutations to glycine, arginine, or lysine also create a hyperactive kinase, while mutation to aspartic acid decreases ATR activity. S1333A-ATR maintains the G2 checkpoint and promotes completion of DNA replication after transient exposure to replication stress but the less active kinase, S1333D-ATR, has modest defects in both of these functions. While we find no evidence that S1333 is phosphorylated in cultured cells, our data indicate that small changes in the HEAT repeats can have large effects on kinase activity. These mutants may serve as useful tools for future studies of the ATR pathway.
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22 MeSH Terms
Nuclear-localized Asunder regulates cytoplasmic dynein localization via its role in the integrator complex.
Jodoin JN, Sitaram P, Albrecht TR, May SB, Shboul M, Lee E, Reversade B, Wagner EJ, Lee LA
(2013) Mol Biol Cell 24: 2954-65
MeSH Terms: Amino Acid Sequence, Animals, Carrier Proteins, Cell Cycle Proteins, Cell Division, Cell Nucleus, Cytoplasmic Dyneins, Drosophila Proteins, Drosophila melanogaster, G2 Phase, HeLa Cells, Humans, Male, Molecular Sequence Data, Multiprotein Complexes, Nuclear Envelope, Nuclear Localization Signals, Protein Subunits, Protein Transport, RNA, Small Interfering, Spermatocytes, Subcellular Fractions
Show Abstract · Added March 5, 2014
We previously reported that Asunder (ASUN) is essential for recruitment of dynein motors to the nuclear envelope (NE) and nucleus-centrosome coupling at the onset of cell division in cultured human cells and Drosophila spermatocytes, although the mechanisms underlying this regulation remain unknown. We also identified ASUN as a functional component of Integrator (INT), a multisubunit complex required for 3'-end processing of small nuclear RNAs. We now provide evidence that ASUN acts in the nucleus in concert with other INT components to mediate recruitment of dynein to the NE. Knockdown of other individual INT subunits in HeLa cells recapitulates the loss of perinuclear dynein in ASUN-small interfering RNA cells. Forced localization of ASUN to the cytoplasm via mutation of its nuclear localization sequence blocks its capacity to restore perinuclear dynein in both cultured human cells lacking ASUN and Drosophila asun spermatocytes. In addition, the levels of several INT subunits are reduced at G2/M when dynein is recruited to the NE, suggesting that INT does not directly mediate this step. Taken together, our data support a model in which a nuclear INT complex promotes recruitment of cytoplasmic dynein to the NE, possibly via a mechanism involving RNA processing.
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22 MeSH Terms
Human Asunder promotes dynein recruitment and centrosomal tethering to the nucleus at mitotic entry.
Jodoin JN, Shboul M, Sitaram P, Zein-Sabatto H, Reversade B, Lee E, Lee LA
(2012) Mol Biol Cell 23: 4713-24
MeSH Terms: 1-Alkyl-2-acetylglycerophosphocholine Esterase, Animals, Carrier Proteins, Cell Cycle Proteins, Cell Line, Tumor, Cell Nucleus, Centrosome, Chromosomal Proteins, Non-Histone, Drosophila melanogaster, Dyneins, Female, G2 Phase, Genetic Complementation Test, HEK293 Cells, HeLa Cells, Humans, Immunoblotting, Male, Mice, Microfilament Proteins, Microscopy, Fluorescence, Microtubule-Associated Proteins, Mitosis, Mutation, Nuclear Pore, Protein Binding, RNA Interference, Spindle Apparatus
Show Abstract · Added March 5, 2014
Recruitment of dynein motors to the nuclear surface is an essential step for nucleus-centrosome coupling in prophase. In cultured human cells, this dynein pool is anchored to nuclear pore complexes through RanBP2-Bicaudal D2 (BICD2) and Nup133- centromere protein F (CENP-F) networks. We previously reported that the asunder (asun) gene is required in Drosophila spermatocytes for perinuclear dynein localization and nucleus-centrosome coupling at G2/M of male meiosis. We show here that male germline expression of mammalian Asunder (ASUN) protein rescues asun flies, demonstrating evolutionary conservation of function. In cultured human cells, we find that ASUN down-regulation causes reduction of perinuclear dynein in prophase of mitosis. Additional defects after loss of ASUN include nucleus-centrosome uncoupling, abnormal spindles, and multinucleation. Coimmunoprecipitation and overlapping localization patterns of ASUN and lissencephaly 1 (LIS1), a dynein adaptor, suggest that ASUN interacts with dynein in the cytoplasm via LIS1. Our data indicate that ASUN controls dynein localization via a mechanism distinct from that of either BICD2 or CENP-F. We present a model in which ASUN promotes perinuclear enrichment of dynein at G2/M that facilitates BICD2- and CENP-F-mediated anchoring of dynein to nuclear pore complexes.
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28 MeSH Terms
Modulation of Golgi-associated microtubule nucleation throughout the cell cycle.
Maia AR, Zhu X, Miller P, Gu G, Maiato H, Kaverina I
(2013) Cytoskeleton (Hoboken) 70: 32-43
MeSH Terms: Cell Cycle, Cell Line, Flow Cytometry, G1 Phase, G2 Phase, Golgi Apparatus, Humans, Microscopy, Fluorescence, Microtubules, Mitosis
Show Abstract · Added February 3, 2014
A microtubule (MT) subpopulation that emanates from Golgi membrane has been recently shown to comprise a significant part of MT network in interphase cells. In this study, we address whether Golgi membrane, which is being extensively remodeled throughout the cell cycle, retains its ability to nucleate MTs at diverse cell cycle stages. Live cell imaging and immunofluorescence microscopy reveals that Golgi-derived MTs form at multiple stages of the cell cycle, including G(1), G(2), and distinct phases of mitosis. However, the capacity of Golgi to nucleate MTs in mitosis is strongly down-regulated as compared with interphase, indicating that this property is cell cycle regulated. We demonstrate that Golgi-derived MTs are indispensable for efficient Golgi assembly in telophase, and speculate that these noncentrosomal MTs may hold specific functions at other cell cycle stages.
Copyright © 2012 Wiley Periodicals, Inc.
1 Communities
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10 MeSH Terms
Analysis of mutations that dissociate G(2) and essential S phase functions of human ataxia telangiectasia-mutated and Rad3-related (ATR) protein kinase.
Nam EA, Zhao R, Cortez D
(2011) J Biol Chem 286: 37320-7
MeSH Terms: Amino Acid Substitution, Ataxia Telangiectasia Mutated Proteins, Cell Cycle Proteins, Cell Line, Cell Survival, DNA-Activated Protein Kinase, DNA-Binding Proteins, G2 Phase, Humans, Mutation, Missense, Phosphorylation, Protein Structure, Secondary, Protein-Serine-Threonine Kinases, S Phase, Tumor Suppressor Proteins
Show Abstract · Added March 5, 2014
ATR (ataxia telangiectasia-mutated and Rad3-related) contains 16 conserved candidate autophosphorylation sites that match its preferred S/TQ consensus. To determine whether any is functionally important, we mutated the 16 candidate residues to alanine in a single cDNA to create a 16A-ATR mutant. The 16A-ATR mutant maintains kinase and G(2) checkpoint activities. However, it fails to rescue the essential function of ATR in maintaining cell viability and fails to promote replication recovery from a transient exposure to replication stress. Further analysis identified T1566A/T1578A/T1589A (3A-ATR) as critical mutations causing this separation of function activity. Secondary structure predictions indicate that these residues occur in a region between ATR HEAT repeats 31R and 32R that aligns with regions of ATM and DNA-PK containing regulatory autophosphorylation sites. Although this region is important for ATR function, the 3A-ATR residues do not appear to be sites of autophosphorylation. Nevertheless, our analysis identifies an important regulatory region of ATR that is shared among the PI3K-related protein kinase family. Furthermore, our data indicate that the essential function of ATR for cell viability is linked to its function in promoting proper replication in the context of replication stress and is independent of G(2) checkpoint activity.
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15 MeSH Terms
Identification of dAven, a Drosophila melanogaster ortholog of the cell cycle regulator Aven.
Zou S, Chang J, LaFever L, Tang W, Johnson EL, Hu J, Wilk R, Krause HM, Drummond-Barbosa D, Irusta PM
(2011) Cell Cycle 10: 989-98
MeSH Terms: Adaptor Proteins, Signal Transducing, Amino Acid Sequence, Animals, Apoptosis Regulatory Proteins, Cell Cycle Proteins, Cell Division, Drosophila Proteins, Drosophila melanogaster, G2 Phase, Histones, Humans, Membrane Proteins, Molecular Sequence Data, Phosphorylation, RNA Interference, Sequence Alignment, Sequence Homology, Amino Acid
Show Abstract · Added March 19, 2017
Aven is a regulator of the DNA-damage response and G2/M cell cycle progression. Overexpression of Aven is associated with poor prognosis in patients with childhood acute lymphoblastic leukemia and acute myeloid leukemia, and altered intracellular Aven distribution is associated with infiltrating ductal carcinoma and papillary carcinoma breast cancer subtypes. Although Aven orthologs have been identified in most vertebrate species, no Aven gene has been reported in invertebrates. Here, we describe a Drosophila melanogaster open reading frame (ORF) that shares sequence and functional similarities with vertebrate Aven genes. The protein encoded by this ORF, which we named dAven, contains several domains that are highly conserved among Aven proteins of fish, amphibian, bird and mammalian origins. In flies, knockdown of dAven by RNA interference (RNAi) resulted in lethality when its expression was reduced either ubiquitously or in fat cells using Gal4 drivers. Animals undergoing moderate dAven knockdown in the fat body had smaller fat cells displaying condensed chromosomes and increased levels of the mitotic marker phosphorylated histone H3 (PHH3), suggesting that dAven was required for normal cell cycle progression in this tissue. Remarkably, expression of dAven in Xenopus egg extracts resulted in G2/M arrest that was comparable to that caused by human Aven. Taken together, these results suggest that, like its vertebrate counterparts, dAven plays a role in cell cycle regulation. Drosophila could be an excellent model for studying the function of Aven and identifying cellular factors that influence its activity, revealing information that may be relevant to human disease.
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17 MeSH Terms
Topoisomerase IIalpha binding domains of adenomatous polyposis coli influence cell cycle progression and aneuploidy.
Wang Y, Coffey RJ, Osheroff N, Neufeld KL
(2010) PLoS One 5: e9994
MeSH Terms: Adenomatous Polyposis Coli, Adenomatous Polyposis Coli Protein, Aneuploidy, Antigens, Neoplasm, Binding Sites, Cell Cycle, Cell Line, Tumor, Codon, Nonsense, DNA Topoisomerases, Type II, DNA-Binding Proteins, Epithelial Cells, G2 Phase, Humans, Repetitive Sequences, Nucleic Acid, beta Catenin
Show Abstract · Added August 12, 2010
BACKGROUND - Truncating mutations in the tumor suppressor gene APC (Adenomatous Polyposis Coli) are thought to initiate the majority of colorectal cancers. The 15- and 20-amino acid repeat regions of APC bind beta-catenin and have been widely studied for their role in the negative regulation of canonical Wnt signaling. However, functions of APC in other important cellular processes, such as cell cycle control or aneuploidy, are only beginning to be studied. Our previous investigation implicated the 15-amino acid repeat region of APC (M2-APC) in the regulation of the G2/M cell cycle transition through interaction with topoisomerase IIalpha (topo IIalpha).
METHODOLOGY/PRINCIPAL FINDINGS - We now demonstrate that the 20-amino acid repeat region of APC (M3-APC) also interacts with topo IIalpha in colonic epithelial cells. Expression of M3-APC in cells with full-length endogenous APC causes cell accumulation in G2. However, cells with a mutated topo IIalpha isoform and lacking topo IIbeta did not arrest, suggesting that the cellular consequence of M2- or M3-APC expression depends on functional topoisomerase II. Both purified recombinant M2- and M3-APC significantly enhanced the activity of topo IIalpha. Of note, although M3-APC can bind beta-catenin, the G2 arrest did not correlate with beta-catenin expression or activity, similar to what was seen with M2-APC. More importantly, expression of either M2- or M3-APC also led to increased aneuploidy in cells with full-length endogenous APC but not in cells with truncated endogenous APC that includes the M2-APC region.
CONCLUSIONS/SIGNIFICANCE - Together, our data establish that the 20-amino acid repeat region of APC interacts with topo IIalpha to enhance its activity in vitro, and leads to G2 cell cycle accumulation and aneuploidy when expressed in cells containing full-length APC. These findings provide an additional explanation for the aneuploidy associated with many colon cancers that possess truncated APC.
1 Communities
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15 MeSH Terms
Growth inhibition and radiosensitization of glioblastoma and lung cancer cells by small interfering RNA silencing of tumor necrosis factor receptor-associated factor 2.
Zheng M, Morgan-Lappe SE, Yang J, Bockbrader KM, Pamarthy D, Thomas D, Fesik SW, Sun Y
(2008) Cancer Res 68: 7570-8
MeSH Terms: Animals, Apoptosis, Carcinoma, Non-Small-Cell Lung, Cell Cycle Proteins, Cell Division, Cell Growth Processes, Cell Line, Tumor, G2 Phase, Gene Silencing, Glioblastoma, Humans, I-kappa B Proteins, Lung Neoplasms, Mice, NF-KappaB Inhibitor alpha, NF-kappa B, Nuclear Pore Complex Proteins, RNA, Small Interfering, RNA-Binding Proteins, Radiation Tolerance, TNF Receptor-Associated Factor 2
Show Abstract · Added March 5, 2014
Radiotherapy combined with chemotherapy is the treatment of choice for glioblastoma and locally advanced lung cancer, but radioresistance of these two types of cancer remains a significant therapeutic hindrance. To identify molecular target(s) for radiosensitization, we screened a small interfering RNA (siRNA) library targeting all protein kinases and E3 ubiquitin ligases in the human genome and identified tumor necrosis factor receptor-associated factor 2 (TRAF2). Silencing of TRAF2 using siRNA caused a significant growth suppression of glioblastoma U251 cells and moderately sensitized these radioresistant cells to radiation. Overexpression of a really interesting new gene (RING)-deleted dominant-negative TRAF2 mutant also conferred radiosensitivity, whereas overexpression of wild-type (WT) TRAF2 significantly protected cells from radiation-induced killing. Likewise, siRNA silencing of TRAF2 in radioresistant lung cancer H1299 cells caused growth suppression and radiosensitization, whereas overexpression of WT TRAF2 enhanced radioresistance in a RING ligase-dependent manner. Moreover, siRNA silencing of TRAF2 in UM-SCC-1 head and neck cancer cells also conferred radiosensitization. Further support for the role of TRAF2 in cancer comes from the observations that TRAF2 is overexpressed in both lung adenocarcinoma tissues and multiple lung cancer cell lines. Importantly, TRAF2 expression was very low in normal bronchial epithelial NL20 cells, and TRAF2 silencing had a minimal effect on NL20 growth and radiation sensitivity. Mechanistically, TRAF2 silencing blocks the activation of the nuclear factor-kappaB signaling pathway and down-regulates several G(2)-M cell cycle control proteins, resulting in enhanced G(2)-M arrest, growth suppression, and radiosensitization. Our studies suggest that TRAF2 is an attractive drug target for anticancer therapy and radiosensitization.
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21 MeSH Terms
Interaction between tumor suppressor adenomatous polyposis coli and topoisomerase IIalpha: implication for the G2/M transition.
Wang Y, Azuma Y, Moore D, Osheroff N, Neufeld KL
(2008) Mol Biol Cell 19: 4076-85
MeSH Terms: Adenomatous Polyposis Coli Protein, Antigens, Neoplasm, Cell Division, Cell Line, Tumor, Cell Nucleus, DNA Topoisomerases, Type II, DNA-Binding Proteins, Flow Cytometry, Fluorescence Resonance Energy Transfer, G2 Phase, Green Fluorescent Proteins, Humans, Microscopy, Fluorescence, Protein Binding, Signal Transduction, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, beta Catenin
Show Abstract · Added March 5, 2014
The tumor suppressor adenomatous polyposis coli (APC) is implicated in regulating multiple stages of the cell cycle. APC participation in G1/S is attributed to its recognized role in Wnt signaling. APC function in the G2/M transition is less well established. To identify novel protein partners of APC that regulate the G2/M transition, APC was immunoprecipitated from colon cell lysates and associated proteins were analyzed by matrix-assisted laser desorption ionization/time of flight (MALDI-TOF). Topoisomerase IIalpha (topo IIalpha) was identified as a potential binding partner of APC. Topo IIalpha is a critical regulator of G2/M transition. Evidence supporting an interaction between endogenous APC and topo IIalpha was obtained by coimmunoprecipitation, colocalization, and Förster resonance energy transfer (FRET). The 15-amino acid repeat region of APC (M2-APC) interacted with topo IIalpha when expressed as a green fluorescent protein (GFP)-fusion protein in vivo. Although lacking defined nuclear localization signals (NLS) M2-APC predominantly localized to the nucleus. Furthermore, cells expressing M2-APC displayed condensed or fragmented nuclei, and they were arrested in the G2 phase of the cell cycle. Although M2-APC contains a beta-catenin binding domain, biochemical studies failed to implicate beta-catenin in the observed phenotype. Finally, purified recombinant M2-APC enhanced topo IIalpha activity in vitro. Together, these data support a novel role for APC in the G2/M transition, potentially through association with topo IIalpha.
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17 MeSH Terms