scholarly journals NuSAP, a novel microtubule-associated protein involved in mitotic spindle organization

2003 ◽  
Vol 162 (6) ◽  
pp. 1017-1029 ◽  
Author(s):  
Tim Raemaekers ◽  
Katharina Ribbeck ◽  
Joël Beaudouin ◽  
Wim Annaert ◽  
Mark Van Camp ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Direct Interaction ◽  
Microscopic Analysis ◽  
Mitotic Spindles ◽  
Proliferating Cells ◽  
Microtubule Organization ◽  
Protein Levels ◽  
Microtubule Binding Domain ◽  
Interphase Cells

Here, we report on the identification of nucleolar spindle–associated protein (NuSAP), a novel 55-kD vertebrate protein with selective expression in proliferating cells. Its mRNA and protein levels peak at the transition of G2 to mitosis and abruptly decline after cell division. Microscopic analysis of both fixed and live mammalian cells showed that NuSAP is primarily nucleolar in interphase, and localizes prominently to central spindle microtubules during mitosis. Direct interaction of NuSAP with microtubules was demonstrated in vitro. Overexpression of NuSAP caused profound bundling of cytoplasmic microtubules in interphase cells, and this relied on a COOH-terminal microtubule-binding domain. In contrast, depletion of NuSAP by RNA interference resulted in aberrant mitotic spindles, defective chromosome segregation, and cytokinesis. In addition, many NuSAP-depleted interphase cells had deformed nuclei. Both overexpression and knockdown of NuSAP impaired cell proliferation. These results suggest a crucial role for NuSAP in spindle microtubule organization.

scholarly journals Interaction of Aurora-A and centrosomin at the microtubule-nucleating site in Drosophila and mammalian cells

2003 ◽  
Vol 162 (5) ◽  
pp. 757-764 ◽  
Author(s):  
Yasuhiko Terada ◽  
Yumi Uetake ◽  
Ryoko Kuriyama
Keyword(s):  
Mammalian Cells ◽  
Aurora A Kinase ◽  
Aurora A ◽  
Mitotic Spindles ◽  
Microtubule Nucleation ◽  
Microtubule Organization ◽  
Centrosomal Protein ◽  
Spindle Poles

A mitosis-specific Aurora-A kinase has been implicated in microtubule organization and spindle assembly in diverse organisms. However, exactly how Aurora-A controls the microtubule nucleation onto centrosomes is unknown. Here, we show that Aurora-A specifically binds to the COOH-terminal domain of a Drosophila centrosomal protein, centrosomin (CNN), which has been shown to be important for assembly of mitotic spindles and spindle poles. Aurora-A and CNN are mutually dependent for localization at spindle poles, which is required for proper targeting of γ-tubulin and other centrosomal components to the centrosome. The NH2-terminal half of CNN interacts with γ-tubulin, and induces cytoplasmic foci that can initiate microtubule nucleation in vivo and in vitro in both Drosophila and mammalian cells. These results suggest that Aurora-A regulates centrosome assembly by controlling the CNN's ability to targeting and/or anchoring γ-tubulin to the centrosome and organizing microtubule-nucleating sites via its interaction with the COOH-terminal sequence of CNN.

scholarly journals Raf-1 Physically Interacts with Rb and Regulates Its Function: a Link between Mitogenic Signaling and Cell Cycle Regulation

1998 ◽  
Vol 18 (12) ◽  
pp. 7487-7498 ◽  
Author(s):  
Sheng Wang ◽  
Richik N. Ghosh ◽  
Srikumar P. Chellappan
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Growth Factor ◽  
Cell Surface ◽  
Mammalian Cells ◽  
Direct Interaction ◽  
Physical Interaction ◽  
Proliferating Cells

ABSTRACT Cells initiate proliferation in response to growth factor stimulation, but the biochemical mechanisms linking signals received at the cell surface receptors to the cell cycle regulatory molecules are not yet clear. In this study, we show that the signaling molecule Raf-1 can physically interact with Rb and p130 proteins in vitro and in vivo and that this interaction can be detected in mammalian cells without overexpressing any component. The binding of Raf-1 to Rb occurs subsequent to mitogen stimulation, and this interaction can be detected only in proliferating cells. Raf-1 can inactivate Rb function and can reverse Rb-mediated repression of E2F1 transcription and cell proliferation efficiently. The region of Raf-1 involved in Rb binding spanned residues 1 to 28 at the N terminus, and functional inactivation of Rb required a direct interaction. Serum stimulation of quiescent human fibroblast HSF8 cells led to a partial translocation of Raf-1 into the nucleus, where it colocalized with Rb. Further, Raf-1 was able to phosphorylate Rb in vitro quite efficiently. We believe that the physical interaction of Raf-1 with Rb is a vital step in the growth factor-mediated induction of cell proliferation and that Raf-1 acts as a direct link between cell surface signaling cascades and the cell cycle machinery.

Phospholipase D1b and D2a generate structurally identical phosphatidic acid species in mammalian cells

2001 ◽  
Vol 360 (3) ◽  
pp. 707-715 ◽  
Author(s):  
Trevor R. PETTITT ◽  
Mark McDERMOTT ◽  
Khalid M. SAQIB ◽  
Neil SHIMWELL ◽  
Michael J. O. WAKELAM
Keyword(s):  
Liquid Chromatography ◽  
Phosphatidic Acid ◽  
Phospholipase D ◽  
Mammalian Cells ◽  
Inducible Promoter ◽  
In Vitro Assays ◽  
Protein Levels ◽  
Intracellular Messenger

Mammalian cells contain different phospholipase D enzymes (PLDs) whose distinct physiological roles are poorly understood and whose products have not been characterized. The development of porcine aortic endothelial (PAE) cell lines able to overexpress PLD-1b or −2a under the control of an inducible promoter has enabled us to characterize both the substrate specificity and the phosphatidic acid (PtdOH) product of these enzymes under controlled conditions. Liquid chromatography–MS analysis showed that PLD1b- and PLD2a-transfected PAE cells, as well as COS7 and Rat1 cells, generate similar PtdOH and, in the presence of butan-1-ol, phosphatidylbutanol (PtdBut) profiles, enriched in mono- and di-unsaturated species, in particular 16:0/18:1. Although PtdBut mass increased, the species profile did not change in cells stimulated with ATP or PMA. Overexpression of PLD made little difference to basal or stimulated PtdBut formation, indicating that activity is tightly regulated in vivo and that factors other than just PLD protein levels limit hydrolytic function. In vitro assays using PLD-enriched lysates showed that the enzyme could utilize both phosphatidylcholine and, much less efficiently, phosphatidylethanolamine, with slight selectivity towards mono- and di-unsaturated species. Phosphatidylinositol was not a substrate. Thus PLD1b and PLD2a hydrolyse a structurally similar substrate pool to generate an identical PtdOH product enriched in mono- and di-unsaturated species that we propose to function as the intracellular messenger forms of this lipid.

Abstract 259: KChIP2 is a Key Transcriptional Regulator of Cardiac Excitability Under Normal and Pathogenic Conditions

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Drew M Nassal ◽  
Xiaoping Wan ◽  
Haiyan Liu ◽  
Danielle Maleski ◽  
Angelina Ramirez-Navarro ◽  
...  
Keyword(s):  
Heart Disease ◽  
Ventricular Myocytes ◽  
Direct Interaction ◽  
Neonatal Rat ◽  
K Channel ◽  
Early Event ◽  
Interacting Protein ◽  
Protein Levels ◽  
Cardiac Excitability

Introduction: Arrhythmogenesis is the primary cause of death in patients with acquired heart disease, and is the consequence of profound dysregulation of both depolarizing and repolarizing currents. Reduction in expression of the auxiliary subunit K+ channel interacting protein 2 (KChIP2), which regulates the transient outward K+ current (Ito), is a common and early event in many cardiac pathologies. Notably, transcriptional changes observed in heart disease can be elicited through direct KChIP2 silencing without disease signaling, suggesting novel transcriptional capacity for KChIP2. Methods and Results: A miRNA microarray was conducted on neonatal rat ventricular myocytes (NRVM) following in vitro silencing of KChIP2, identifying the miR-34 family as a potential transcriptional target of KChIP2. qPCR confirmed reduction in miR-34b/c when over-expressing KChIP2 and increase following silencing. Luciferase assays conducted on the promoter for miR-34b/c reinforced KChIP2 repression on the promoter, while chromatin immunoprecipitation identified direct interaction of KChIP2 on the promoter. Previous studies show modified expression of KChIP2 can lead to changes in several ion channel subunits. Therefore, we investigated if this was the consequence of KChIP2 regulation via miR-34. Expression of miR-34b/c precursors reduced transcript levels of Nav1.5 and Navβ1, and reduced protein levels for Kv4.3, resulting in loss of INa and Ito. To determine the relevance in disease signaling, NRVMs were exposed to 100 μM phenylephrine for 48 hrs, significantly reducing KChIP2, Nav1.5, Navβ1, and Kv4.3, while elevating miR-34b/c. Maintaining KChIP2 expression by adenovirus or blocking miR-34 activity with antagomirs rescued the changes in channel expression. Consequently, miR-34 inhibition rescued the induction of sustained arrhythmias observed in a 2D culture of myocytes through the maintenance of cardiac excitability. Conclusion: Collectively, these observations identify dysregulation of the KChIP2/miR-34 axis as a nodal event in the development of electrical dysfunction that characterize cardiac pathologies, and further identifies miR-34 as a viable therapeutic target for managing arrhythmogenesis in patients with heart disease.

scholarly journals A Mammalian Ortholog of Saccharomyces cerevisiae Vac14 That Associates with and Up-Regulates PIKfyve Phosphoinositide 5-Kinase Activity

2004 ◽  
Vol 24 (23) ◽  
pp. 10437-10447 ◽  
Author(s):  
Diego Sbrissa ◽  
Ognian C. Ikonomov ◽  
Jana Strakova ◽  
Rajeswari Dondapati ◽  
Krzysztof Mlak ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Kinase Activity ◽  
Ectopic Expression ◽  
Multivesicular Body ◽  
Hek293 Cells ◽  
Lipid Kinase ◽  
Morphological Alterations ◽  
Protein Levels ◽  
Interfering Rna

ABSTRACT Multivesicular body morphology and size are controlled in part by PtdIns(3,5)P2, produced in mammalian cells by PIKfyve-directed phosphorylation of PtdIns(3)P. Here we identify human Vac14 (hVac14), an evolutionarily conserved protein, present in all eukaryotes but studied principally in yeast thus far, as a novel positive regulator of PIKfyve enzymatic activity. In mammalian cells and tissues, Vac14 is a low-abundance 82-kDa protein, but its endogenous levels could be up-regulated upon ectopic expression of hVac14. PIKfyve and hVac14 largely cofractionated, populated similar intracellular locales, and physically associated. A small-interfering RNA-directed gene-silencing approach to selectively eliminate endogenous hVac14 rendered HEK293 cells susceptible to morphological alterations similar to those observed upon expression of PIKfyve mutants deficient in PtdIns(3,5)P2 production. Largely decreased in vitro PIKfyve kinase activity and unaltered PIKfyve protein levels were detected under these conditions. Conversely, ectopic expression of hVac14 increased the intrinsic PIKfyve lipid kinase activity. Concordantly, intracellular PtdIns(3)P-to-PtdIns(3,5)P2 conversion was perturbed by hVac14 depletion and was elevated upon ectopic expression of hVac14. These data demonstrate a major role of the PIKfyve-associated hVac14 protein in activating PIKfyve and thereby regulating PtdIns(3,5)P2 synthesis and endomembrane homeostasis in mammalian cells.

scholarly journals Identification of Three Functions of the Adenovirus E4orf6 Protein That Mediate p53 Degradation by the E4orf6-E1B55K Complex

2001 ◽  
Vol 75 (2) ◽  
pp. 699-709 ◽  
Author(s):  
Emmanuelle Querido ◽  
Megan R. Morisson ◽  
Huan Chu-Pham-Dang ◽  
Sarah W.-L. Thirlwell ◽  
Dominique Boivin ◽  
...  
Keyword(s):  
Nuclear Export ◽  
Mammalian Cells ◽  
Nuclear Export Signal ◽  
Direct Interaction ◽  
26S Proteasome ◽  
Productive Infection ◽  
Nuclear Retention ◽  
Hpv E6 ◽  
Export Signal

ABSTRACT Complexes containing adenovirus E4orf6 and E1B55K proteins play critical roles in productive infection. Both proteins interact directly with the cellular tumor suppressor p53, and in combination they promote its rapid degradation. To examine the mechanism of this process, degradation of exogenously expressed p53 was analyzed in p53-null human cells infected with adenovirus vectors encoding E4orf6 and/or E1B55K. Coexpression of E4orf6 and E1B55K greatly reduced both the level and the half-life of wild-type p53. No effect was observed with the p53-related p73 proteins, which did not appear to interact with E4orf6 or E1B55K. Mutant forms of p53 were not degraded if they could not efficiently bind E1B55K, suggesting that direct interaction between p53 and E1B55K may be required. Degradation of p53 was independent of both MDM2 and p19ARF, regulators of p53 stability in mammalian cells, but required an extended region of E4orf6 from residues 44 to 274, which appeared to possess three separate biological functions. First, residues 39 to 107 were necessary to interact with E1B55K. Second, an overlapping region from about residues 44 to 218 corresponded to the ability of E4orf6 to form complexes with cellular proteins of 19 and 14 kDa. Third, the nuclear retention signal/amphipathic arginine-rich α-helical region from residues 239 to 253 was required. Interestingly, neither the E4orf6 nuclear localization signal nor the nuclear export signal was essential. These results suggested that if nuclear-cytoplasmic shuttling is involved in this process, it must involve another export signal. Degradation was significantly blocked by the 26S proteasome inhibitor MG132, but unlike the HPV E6 protein, E4orf6 and E1B55K were unable to induce p53 degradation in vitro in reticulocyte lysates. Thus, this study implies that the E4orf6-E1B55K complex may direct p53 for degradation by a novel mechanism.

Regulation of HDM2 E3-ubiquitin ligase in esophageal adenocarcinoma cells by AURKA.

2013 ◽  
Vol 31 (4_suppl) ◽  
pp. 35-35
Author(s):  
Vikas Sehdev ◽  
Abbes Belkhiri ◽  
Mohammed Soutto ◽  
Ahmed M. Katsha ◽  
Wael El-Rifai
Keyword(s):  
Cell Lines ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Direct Interaction ◽  
Kinase Assay ◽  
Intrinsic Resistance ◽  
Tissue Samples ◽  
Protein Levels ◽  
Eac Cells

35 Background: Esophageal adenocarcinomas (EAC) exhibit intrinsic resistance against chemotherapy. AURKA regulates cell cycle progression and its overexpression is associated with oncogenic transformation. We have recently reported that AURKA is significantly overexpressed in about 70% of human EAC tissue samples and EAC cell lines. We have previously shown that AURKA inhibits p53- and p73-mediated apoptotic pathways in GI adenocarcinomas. HDM2 is an E3-ubiquitin ligase which is closely involved in regulating p53 and p73 protein stability and activity. In this study we demonstrate that AURKA directly interacts with HDM2 and regulates HDM2 protein expression and phosphorylation in both FLO-1 and OE33 EAC cells. Methods and Results: Western blot analyses were done following AURKA overexpression with adenovirus, knockdown with si-RNA or inhibition with MLN 8237 (0.5µM) in FLO-1 and OE33 EAC cell lines. The data indicated that overexpression of AURKA induced both total and phospho-HDM2-(Ser166) protein levels. Knockdown or inhibition of AURKA significantly decreased expression of both total and phospho-HDM2-(Ser166) protein levels in FLO-1 and OE33 EAC cells. Additionally, following adenovirus mediated overexpression of AURKA, co-immunoprecipitaion (Co-IP) was done for AURKA and HDM2 in FLO-1 and OE33 EAC cells. The two-way Co-IP data indicated the presence of HDM2 in a complex associated with AURKA and vice-versa. The data from in vitro protein kinase assay indicated that recombinant AURKA directly phosphorylates recombinant HDM2 at Ser166 site. To confirm direct interaction between recombinant AURKA and HDM2 proteins we performed IP following the in vitro kinase assay. The in vitro kinase IP data indicates that kinase intact recombinant AURKA directly interacts and phosphorylates recombinant HDM2 protein. Conclusions: Our data indicate that AURKA regulates HDM2 expression and phosphorylation in both FLO-1 and OE33 EAC cells. Additionally, we also report for the first time that AURKA directly interacts with HDM2 and phosphorylates it at Ser166 site. Therefore, our study suggests that AURKA-mediated regulation of HDM2 could be the major underlying mechanism for induction of apoptosis in p53-negative EAC.

scholarly journals Identification of an NTF2-Related Factor That Binds Ran-GTP and Regulates Nuclear Protein Export

1999 ◽  
Vol 19 (12) ◽  
pp. 8616-8624 ◽  
Author(s):  
Ben E. Black ◽  
Lyne Lévesque ◽  
James M. Holaska ◽  
Todd C. Wood ◽  
Bryce M. Paschal
Keyword(s):  
Mammalian Cells ◽  
Nuclear Protein ◽  
Direct Interaction ◽  
Effector Proteins ◽  
Protein Export ◽  
Nuclear Pore ◽  
Related Factor ◽  
Transport Reaction ◽  
Ran Gtp

ABSTRACT Active transport of macromolecules between the nucleus and cytoplasm requires signals for import and export and their recognition by shuttling receptors. Each class of macromolecule is thought to have a distinct receptor that mediates the transport reaction. Assembly and disassembly reactions of receptor-substrate complexes are coordinated by Ran, a GTP-binding protein whose nucleotide state is regulated catalytically by effector proteins. Ran function is modulated in a noncatalytic fashion by NTF2, a protein that mediates nuclear import of Ran-GDP. Here we characterize a novel component of the Ran system that is 26% identical to NTF2, which based on its function we refer to as NTF2-related export protein 1 (NXT1). In contrast to NTF2, NXT1 preferentially binds Ran-GTP, and it colocalizes with the nuclear pore complex (NPC) in mammalian cells. These properties, together with the fact that NXT1 shuttles between the nucleus and the cytoplasm, suggest an active role in nuclear transport. Indeed, NXT1 stimulates nuclear protein export of the NES-containing protein PKI in vitro. The export function of NXT1 is blocked by the addition of leptomycin B, a compound that selectively inhibits the NES receptor Crm1. Thus, NXT1 regulates the Crm1-dependent export pathway through its direct interaction with Ran-GTP.

scholarly journals Drosophila EB1 is important for proper assembly, dynamics, and positioning of the mitotic spindle

2002 ◽  
Vol 158 (5) ◽  
pp. 873-884 ◽  
Author(s):  
Stephen L. Rogers ◽  
Gregory C. Rogers ◽  
David J. Sharp ◽  
Ronald D. Vale
Keyword(s):  
Cell Attachment ◽  
Cell Cortex ◽  
Mitotic Spindles ◽  
Microtubule Organization ◽  
Chromosomal Segregation ◽  
Spindle Poles ◽  
Evolutionarily Conserved ◽  
Interphase Cells ◽  
Spindle Elongation ◽  
Drosophila Embryos

EB1 is an evolutionarily conserved protein that localizes to the plus ends of growing microtubules. In yeast, the EB1 homologue (BIM1) has been shown to modulate microtubule dynamics and link microtubules to the cortex, but the functions of metazoan EB1 proteins remain unknown. Using a novel preparation of the Drosophila S2 cell line that promotes cell attachment and spreading, we visualized dynamics of single microtubules in real time and found that depletion of EB1 by RNA-mediated inhibition (RNAi) in interphase cells causes a dramatic increase in nondynamic microtubules (neither growing nor shrinking), but does not alter overall microtubule organization. In contrast, several defects in microtubule organization are observed in RNAi-treated mitotic cells, including a drastic reduction in astral microtubules, malformed mitotic spindles, defocused spindle poles, and mispositioning of spindles away from the cell center. Similar phenotypes were observed in mitotic spindles of Drosophila embryos that were microinjected with anti-EB1 antibodies. In addition, live cell imaging of mitosis in Drosophila embryos reveals defective spindle elongation and chromosomal segregation during anaphase after antibody injection. Our results reveal crucial roles for EB1 in mitosis, which we postulate involves its ability to promote the growth and interactions of microtubules within the central spindle and at the cell cortex.

scholarly journals Direct Interaction of Pericentrin with Cytoplasmic Dynein Light Intermediate Chain Contributes to Mitotic Spindle Organization

1999 ◽  
Vol 147 (3) ◽  
pp. 481-492 ◽  
Author(s):  
Aruna Purohit ◽  
Sharon H. Tynan ◽  
Richard Vallee ◽  
Stephen J. Doxsey
Keyword(s):  
Molecular Motor ◽  
Physiological Role ◽  
Direct Interaction ◽  
Cytoplasmic Dynein ◽  
Mitotic Spindles ◽  
Microtubule Organization ◽  
Cellular Targets ◽  
Spindle Poles ◽  
And Function

Pericentrin is a conserved protein of the centrosome involved in microtubule organization. To better understand pericentrin function, we overexpressed the protein in somatic cells and assayed for changes in the composition and function of mitotic spindles and spindle poles. Spindles in pericentrin-overexpressing cells were disorganized and mispositioned, and chromosomes were misaligned and missegregated during cell division, giving rise to aneuploid cells. We unexpectedly found that levels of the molecular motor cytoplasmic dynein were dramatically reduced at spindle poles. Cytoplasmic dynein was diminished at kinetochores also, and the dynein-mediated organization of the Golgi complex was disrupted. Dynein coimmunoprecipitated with overexpressed pericentrin, suggesting that the motor was sequestered in the cytoplasm and was prevented from associating with its cellular targets. Immunoprecipitation of endogenous pericentrin also pulled down cytoplasmic dynein in untransfected cells. To define the basis for this interaction, pericentrin was coexpressed with cytoplasmic dynein heavy (DHCs), intermediate (DICs), and light intermediate (LICs) chains, and the dynamitin and p150Glued subunits of dynactin. Only the LICs coimmunoprecipitated with pericentrin. These results provide the first physiological role for LIC, and they suggest that a pericentrin–dynein interaction in vivo contributes to the assembly, organization, and function of centrosomes and mitotic spindles.

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