Aberrant intracellular targeting and cell cycle-dependent phosphorylation of emerin contribute to the Emery-Dreifuss muscular dystrophy phenotype

1998 ◽  
Vol 111 (6) ◽  
pp. 781-792 ◽  
Author(s):  
J.A. Ellis ◽  
M. Craxton ◽  
J.R. Yates ◽  
J. Kendrick-Jones
Keyword(s):  
Cell Cycle ◽  
Muscular Dystrophy ◽  
Nuclear Membrane ◽  
Nuclear Fraction ◽  
Tissue Samples ◽  
Intracellular Targeting ◽  
Wide Range ◽  
Cycle Dependent ◽  
Mutant Forms

The product of the X-linked Emery-Dreifuss muscular dystrophy gene is a protein called emerin, which is localized to the nuclear membrane. We have expressed full-length recombinant human emerin in an in vitro coupled reticulocyte system; it has a molecular mass of 34 kDa, inserts into microsomes in a type II orientation, and does not exhibit any N-linked glycosylation or cleavage event. Affinity-purified human emerin antiserum cross-reacts with the in vitro-expressed emerin and with a 34 kDa band present in a wide range of human tissue samples. Expression and subcellular distribution of emerin were studied in lymphoblastoid cell lines established from four patients with Emery-Dreifuss muscular dystrophy containing different mutations in the emerin gene. Emerin protein was detected in two of these patients by immunoblotting. In striking contrast to wild-type emerin, which was localized to the nuclear fraction and was insoluble in non-ionic detergents and high salt, emerin from these two patients exhibited a more random subcellular localization and increased solubility. On the basis of the mutations present in these patients, it would appear that emerin possesses two non-overlapping nuclear envelope targeting sequences. We have also demonstrated that emerin can occur in four different phosphorylated forms, three of which appear to be associated with the cell cycle. The mutant forms of emerin taken from the two patients exhibited aberrant cell cycle-dependent phosphorylated forms. This data suggests that for emerin to function normally it must be correctly localized, retained at the nuclear membrane and phosphorylated by cell cycle-mediated events.

The cell cycle dependent mislocalisation of emerin may contribute to the Emery-Dreifuss muscular dystrophy phenotype

2002 ◽  
Vol 115 (2) ◽  
pp. 341-354 ◽  
Author(s):  
Elizabeth A. L. Fairley ◽  
Andrew Riddell ◽  
Juliet A. Ellis ◽  
John Kendrick-Jones
Keyword(s):  
Cell Cycle ◽  
Muscular Dystrophy ◽  
Nuclear Envelope ◽  
Nuclear Membrane ◽  
Transmembrane Domain ◽  
Nuclear Lamina ◽  
Lamin A ◽  
Wild Type ◽  
Cycle Dependent ◽  
Mutant Forms

Emerin is the nuclear membrane protein defective in X-linked Emery-Dreifuss muscular dystrophy (X-EDMD). The majority of X-EDMD patients have no detectable emerin. However, there are cases that produce mutant forms of emerin, which can be used to study its function. Our previous studies have shown that the emerin mutants S54F, P183T, P183H, Del95-99, Del236-241 (identified in X-EDMD patients) are targeted to the nuclear membrane but to a lesser extent than wild-type emerin. In this paper, we have studied how the mislocalisation of these mutant emerins may affect nuclear functions associated with the cell cycle using flow cytometry and immunofluorescence microscopy. We have established that cells expressing the emerin mutant Del236-241 (a deletion in the transmembrane domain), which was mainly localised in the cytoplasm, exhibited an aberrant cell cycle length. Thereafter, by examining the intracellular localisation of endogenously expressed lamin A/C and exogenously expressed wild-type and mutant forms of emerin after a number of cell divisions, we determined that the mutant forms of emerin redistributed endogenous lamin A/C. The extent of lamin A/C redistribution correlated with the amount of EGFP-emerin that was mislocalised. The amount of EGFP-emerin mislocalized, in turn, was associated with alterations in the nuclear envelope morphology. The nuclear morphology and redistribution of lamin A/C was most severely affected in the cells expressing the emerin mutant Del236-241.It is believed that emerin is part of a novel nuclear protein complex consisting of the barrier-to-autointegration factor (BAF), the nuclear lamina, nuclear actin and other associated proteins. The data presented here show that lamin A/C localisation is dominantly directed by its interaction with certain emerin mutants and perhaps wild-type emerin as well. These results suggest that emerin links A-type lamins to the nuclear envelope and that the correct localisation of these nuclear proteins is important for maintaining cell cycle timing.

scholarly journals Biochemical analyses reveal amino acid residues critical for cell cycle-dependent phosphorylation of human Cdc14A phosphatase by cyclin-dependent kinase 1

2018 ◽  
Author(s):  
Sara Ovejero ◽  
Patricia Ayala ◽  
Marcos Malumbres ◽  
Felipe X. Pimentel-Muiños ◽  
Avelino Bueno ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Subcellular Location ◽  
Amino Acid Residues ◽  
Cyclin Dependent Kinase ◽  
Wide Range ◽  
Biochemical Analyses ◽  
Cycle Dependent ◽  
Early Mitosis

ABSTRACTCdc14 enzymes compose a family of highly conserved phosphatases that are present in a wide range of organisms, including yeast and humans, and that preferentially reverse the phosphorylation of Cyclin-Dependent Kinase (Cdk) substrates. The budding yeast Cdc14 orthologue has essential functions in the control of late mitosis and cytokinesis. In mammals, however, the two Cdc14 homologues, Cdc14A and Cdc14B, do not play a prominent role in controlling late mitotic events, suggesting that some Cdc14 functions are not conserved across species. Moreover, in yeast, Cdc14 is regulated by changes in its subcellular location and by phosphorylation events. In contrast, little is known about the regulation of human Cdc14 phosphatases. Here, we have studied how the human Cdc14A orthologue is regulated during the cell cycle. We found that Cdc14A is phosphorylated on Ser411, Ser453 and Ser549 by Cdk1 early in mitosis and becomes dephosphorylated during late mitotic stages. Interestingly, in vivo and in vitro experiments revealed that, unlike in yeast, Cdk1-mediated phosphorylation of human Cdc14A did not control its catalytic activity but likely modulated its interaction with other proteins in early mitosis. These findings point to differences in Cdk1-mediated mechanisms of regulation between human and yeast Cdc14 orthologues.

scholarly journals HIV-1 Vpr activates host CRL4-DCAF1 E3 ligase to degrade histone deacetylase SIRT7

2021 ◽  
Vol 18 (1) ◽  
Author(s):  
Xiaohong Zhou ◽  
Christina Monnie ◽  
Maria DeLucia ◽  
Jinwoo Ahn
Keyword(s):  
Cell Cycle ◽  
Dna Repair ◽  
Cell Cycle Arrest ◽  
Histone Deacetylase ◽  
E3 Ligase ◽  
Cycle Arrest ◽  
Wide Range ◽  
In Vitro Reconstitution ◽  
Hiv 1

Abstract Background Vpr is a virion-associated protein that is encoded by lentiviruses and serves to counteract intrinsic immunity factors that restrict infection. HIV-1 Vpr mediates proteasome-dependent degradation of several DNA repair/modification proteins. Mechanistically, Vpr directly recruits cellular targets onto DCAF1, a substrate receptor of Cullin 4 RING E3 ubiquitin ligase (CRL4) for poly-ubiquitination. Further, Vpr can mediate poly-ubiquitination of DCAF1-interacting proteins by the CRL4. Because Vpr-mediated degradation of its known targets can not explain the primary cell-cycle arrest phenotype that Vpr expression induces, we surveyed the literature for DNA-repair-associated proteins that interact with the CRL4-DCAF1. One such protein is SIRT7, a deacetylase of histone 3 that belongs to the Sirtuin family and regulates a wide range of cellular processes. We wondered whether Vpr can mediate degradation of SIRT7 via the CRL4-DCAF1. Methods HEK293T cells were transfected with cocktails of plasmids expressing DCAF1, DDB1, SIRT7 and Vpr. Ectopic and endogeneous levels of SIRT7 were monitered by immunoblotting and protein–protein interactions were assessed by immunoprecipitation. For in vitro reconstitution assays, recombinant CRL4-DCAF1-Vpr complexes and SIRT7 were prepared and poly-ubiqutination of SIRT7 was monitored with immunoblotting. Results We demonstrate SIRT7 polyubiquitination and degradation upon Vpr expression. Specifically, SIRT7 is shown to interact with the CRL4-DCAF1 complex, and expression of Vpr in HEK293T cells results in SIRT7 degradation, which is partially rescued by CRL inhibitor MNL4924 and proteasome inhibitor MG132. Further, in vitro reconstitution assays show that Vpr induces poly-ubiquitination of SIRT7 by the CRL4-DCAF1. Importantly, we find that Vpr from several different HIV-1 strains, but not HIV-2 strains, mediates SIRT7 poly-ubiquitination in the reconstitution assay and degradation in cells. Finally, we show that SIRT7 degradation by Vpr is independent of the known, distinctive phenotype of Vpr-induced cell cycle arrest at the G2 phase, Conclusions Targeting histone deacetylase SIRT7 for degradation is a conserved feature of HIV-1 Vpr. Altogether, our findings reveal that HIV-1 Vpr mediates down-regulation of SIRT7 by a mechanism that does not involve novel target recruitment to the CRL4-DCAF1 but instead involves regulation of the E3 ligase activity.

The Xenopus protein kinase pEg2 associates with the centrosome in a cell cycle-dependent manner, binds to the spindle microtubules and is involved in bipolar mitotic spindle assembly

1998 ◽  
Vol 111 (5) ◽  
pp. 557-572 ◽  
Author(s):  
C. Roghi ◽  
R. Giet ◽  
R. Uzbekov ◽  
N. Morin ◽  
I. Chartrain ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Mitotic Spindle ◽  
Cultured Cells ◽  
Dependent Manner ◽  
Egg Extracts ◽  
Pericentriolar Material ◽  
Spindle Microtubules ◽  
Cycle Dependent

By differential screening of a Xenopus laevis egg cDNA library, we have isolated a 2,111 bp cDNA which corresponds to a maternal mRNA specifically deadenylated after fertilisation. This cDNA, called Eg2, encodes a 407 amino acid protein kinase. The pEg2 sequence shows significant identity with members of a new protein kinase sub-family which includes Aurora from Drosophila and Ipl1 (increase in ploidy-1) from budding yeast, enzymes involved in centrosome migration and chromosome segregation, respectively. A single 46 kDa polypeptide, which corresponds to the deduced molecular mass of pEg2, is immunodetected in Xenopus oocyte and egg extracts, as well as in lysates of Xenopus XL2 cultured cells. In XL2 cells, pEg2 is immunodetected only in S, G2 and M phases of the cell cycle, where it always localises to the centrosomal region of the cell. In addition, pEg2 ‘invades’ the microtubules at the poles of the mitotic spindle in metaphase and anaphase. Immunoelectron microscopy experiments show that pEg2 is located precisely around the pericentriolar material in prophase and on the spindle microtubules in anaphase. We also demonstrate that pEg2 binds directly to taxol stabilised microtubules in vitro. In addition, we show that the presence of microtubules during mitosis is not necessary for an association between pEg2 and the centrosome. Finally we show that a catalytically inactive pEg2 kinase stops the assembly of bipolar mitotic spindles in Xenopus egg extracts.

Antisense oligonucleotides to CFTR confer a cystic fibrosis phenotype on B lymphocytes

1992 ◽  
Vol 263 (6) ◽  
pp. C1147-C1151 ◽  
Author(s):  
R. D. Krauss ◽  
G. Berta ◽  
T. A. Rado ◽  
J. K. Bubien
Keyword(s):  
Cell Cycle ◽  
Cystic Fibrosis ◽  
Antisense Oligonucleotides ◽  
T84 Cells ◽  
Cftr Protein ◽  
Low Levels ◽  
B Lymphoid ◽  
Cycle Dependent ◽  
Cl Permeability

Cystic fibrosis transmembrane conductance regulator (CFTR) is expressed at low levels in nonepithelial cells. Recently, we demonstrated that CFTR is responsible for cell cycle-dependent adenosine 3',5'-cyclic monophosphate-responsive Cl- permeability in lymphocytes. Agonist responsiveness of cystic fibrosis (CF) lymphocytes was restored by transfection with plasmid containing wild type CFTR cDNA. CFTR mRNA is expressed in the B lymphoid cell line GM03299; however, quantitative reverse transcriptase-polymerase chain reaction indicates that the level of CFTR mRNA is at least 1,000 times lower than in T84 cells. CFTR protein could not be detected by Western blot or by immunoprecipitation of in vitro phosphorylated protein. However, antisense oligonucleotides representing codons 1-12 of CFTR caused a complete inhibition of cell cycle-dependent Cl-permeability [as determined by 6-methoxy-N-(3-sulfopropyl)-quinolinium fluorescence digital-imaging microscopy], thereby inducing normal cells to acquire a "CF phenotype." These studies provide direct evidence that a CFTR-associated Cl- permeability is present and measurable in lymphocytes, even though CFTR mRNA and protein are expressed at low levels.

scholarly journals A U-Rich Element in the 5′ Untranslated Region Is Necessary for the Translation of p27 mRNA

2000 ◽  
Vol 20 (16) ◽  
pp. 5947-5959 ◽  
Author(s):  
S. Sean Millard ◽  
Anxo Vidal ◽  
Maurice Markus ◽  
Andrew Koff
Keyword(s):  
Cell Cycle ◽  
Mrna Stability ◽  
Untranslated Region ◽  
Mrna Processing ◽  
Differentiated Cells ◽  
Specific Subset ◽  
Number Of Factors ◽  
Rnp Complex ◽  
Cycle Dependent

ABSTRACT Increased translation of p27 mRNA correlates with withdrawal of cells from the cell cycle. This raised the possibility that antimitogenic signals might mediate their effects on p27 expression by altering complexes that formed on p27 mRNA, regulating its translation. In this report, we identify a U-rich sequence in the 5′ untranslated region (5′UTR) of p27 mRNA that is necessary for efficient translation in proliferating and nonproliferating cells. We show that a number of factors bind to the 5′UTR in vitro in a manner dependent on the U-rich element, and their availability in the cytosol is controlled in a growth- and cell cycle-dependent fashion. One of these factors is HuR, a protein previously implicated in mRNA stability, transport, and translation. Another is hnRNP C1 and C2, proteins implicated in mRNA processing and the translation of a specific subset of mRNAs expressed in differentiated cells. In lovastatin-treated MDA468 cells, the mobility of the associated hnRNP C1 and C2 proteins changed, and this correlated with increased p27 expression. Together, these data suggest that the U-rich dependent RNP complex on the 5′UTR may regulate the translation of p27 mRNA and may be a target of antimitogenic signals.

scholarly journals Presence of a Poly(A) Binding Protein and Two Proteins with Cell Cycle-Dependent Phosphorylation in Crithidia fasciculata mRNA Cycling Sequence Binding Protein II

2004 ◽  
Vol 3 (5) ◽  
pp. 1185-1197 ◽  
Author(s):  
Bidyottam Mittra ◽  
Dan S. Ray
Keyword(s):  
Cell Cycle ◽  
Binding Protein ◽  
High Specificity ◽  
Binding Activity ◽  
Mrna Levels ◽  
Crithidia Fasciculata ◽  
Target Mrna ◽  
Cycle Dependent

ABSTRACT Crithidia fasciculata cycling sequence binding proteins (CSBP) have been shown to bind with high specificity to sequence elements present in several mRNAs that accumulate periodically during the cell cycle. The first described CSBP has subunits of 35.6 (CSBPA) and 42 kDa (CSBPB). A second distinct binding protein termed CSBP II has been purified from CSBPA null mutant cells, lacking both CSBPA and CSBPB proteins, and contains three major polypeptides with predicted molecular masses of 63, 44.5, and 33 kDa. Polypeptides of identical size were radiolabeled in UV cross-linking assays performed with purified CSBP II and 32P-labeled RNA probes containing six copies of the cycling sequence. The CSBP II binding activity was found to cycle in parallel with target mRNA levels during progression through the cell cycle. We have cloned genes encoding these three CSBP II proteins, termed RBP63, RBP45, and RBP33, and characterized their binding properties. The RBP63 protein is a member of the poly(A) binding protein family. Homologs of RBP45 and RBP33 proteins were found only among the kinetoplastids. Both RBP45 and RBP33 proteins and their homologs have a conserved carboxy-terminal half that contains a PSP1-like domain. All three CSBP II proteins show specificity for binding the wild-type cycling sequence in vitro. RBP45 and RBP33 are phosphoproteins, and RBP45 has been found to bind in vivo specifically to target mRNA containing cycling sequences. The levels of phosphorylation of both RBP45 and RBP33 were found to cycle during the cell cycle.

scholarly journals Cell Cycle Synchrony in Giardia intestinalis Cultures Achieved by Using Nocodazole and Aphidicolin

2008 ◽  
Vol 7 (4) ◽  
pp. 569-574 ◽  
Author(s):  
Marianne K. Poxleitner ◽  
Scott C. Dawson ◽  
W. Zacheus Cande
Keyword(s):  
Cell Cycle ◽  
Model Organism ◽  
Protozoan Parasite ◽  
Giardia Intestinalis ◽  
Mitotic Spindles ◽  
Intestinal Protozoan ◽  
Cycle Arrest ◽  
Cycle Dependent ◽  
Dependent Processes

ABSTRACT Giardia intestinalis is a ubiquitous intestinal protozoan parasite and has been proposed to represent the earliest diverging lineage of extant eukaryotes. Despite the importance of Giardia as a model organism, research on Giardia has been hampered by an inability to achieve cell cycle synchrony for in vitro cultures. This report details successful methods for attaining cell cycle synchrony in Giardia cultures. The research presented here demonstrates reversible cell cycle arrest in G1/S and G2/M with aphidicolin and nocodazole, respectively. Following synchronization, cells were able to recover completely from drug treatment and remained viable and maintained synchronous growth for 6 h. These techniques were used to synchronize Giardia cultures to increase the percentages of mitotic spindles in the cultures. This method of synchronization will enhance our ability to study cell cycle-dependent processes in G. intestinalis.

scholarly journals Gold Nanoparticles Promote Oxidant-Mediated Activation of NF-κB and 53BP1 Recruitment-Based Adaptive Response in Human Astrocytes

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Jennifer Mytych ◽  
Anna Lewinska ◽  
Jacek Zebrowski ◽  
Maciej Wnuk
Keyword(s):  
Oxidative Stress ◽  
Cell Cycle ◽  
Gold Nanoparticles ◽  
Adaptive Response ◽  
Inverse Correlation ◽  
Galactosidase Activity ◽  
Promising Candidate ◽  
Human Astrocytes ◽  
Wide Range

Nanogold-based materials are promising candidate tools for nanobased medicine. Nevertheless, no conclusive information on their cytotoxicity is available. In the present study, we investigated the effects of gold nanoparticles (AuNPs) on human astrocytesin vitro. Nanogold treatment in a wide range of concentrations did not result in cytotoxicity. In contrast, nanogold provoked changes in the astrocyte cell cycle and induced senescence-associatedβ-galactosidase activity. AuNPs promoted oxidative stress and caused activation of NF-κB pathway. After nanogold treatment, an inverse correlation between the formation of 53BP1 foci and micronuclei generation was observed. The robust 53BP1 recruitment resulted in reduced micronuclei production. Thus, nanogold treatment stimulated an adaptive response in a human astrocyte cell.

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