scholarly journals The growth-related, translationally controlled protein P23 has properties of a tubulin binding protein and associates transiently with microtubules during the cell cycle

1999 ◽  
Vol 112 (8) ◽  
pp. 1257-1271 ◽  
Author(s):  
Y. Gachet ◽  
S. Tournier ◽  
M. Lee ◽  
A. Lazaris-Karatzas ◽  
T. Poulton ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mutational Analysis ◽  
Binding Protein ◽  
Binding Domain ◽  
Dependent Manner ◽  
Cell Extracts ◽  
Tubulin Binding ◽  
Cycle Dependent ◽  
Almost All

The translationally controlled protein P23 was discovered by the early induction of its rate of synthesis after mitogenic stimulation of mouse fibroblasts. P23 is expressed in almost all mammalian tissues and it is highly conserved between animals, plants and yeast. Based on its amino acid sequence, P23 cannot be attributed to any known protein family, and its cellular function remains to be elucidated. Here, we present evidence that P23 has properties of a tubulin binding protein that associates with microtubules in a cell cycle-dependent manner. (1) P23 is a cytoplasmic protein that occurs in complexes of 100–150 kDa, and part of P23 can be immunoprecipitated from HeLa cell extracts with anti-tubulin antibodies. (2) In immunolocalisation experiments we find P23 associated with microtubules during G1, S, G2 and early M phase of the cell cycle. At metaphase, P23 is also bound to the mitotic spindle, and it is detached from the spindle during metaphase-anaphase transition. (3) A GST-P23 fusion protein interacts with alpha- and beta-tubulin, and recombinant P23 binds to taxol-stabilised microtubules in vitro. The tubulin binding domain of P23 was identified by mutational analysis; it shows similarity to part of the tubulin binding domain of the microtubule-associated protein MAP-1B. (4) Overexpression of P23 results in cell growth retardation and in alterations of cell morphology. Moreover, elevation of P23 levels leads to microtubule rearrangements and to an increase in microtubule mass and stability.

C-terminal domain phosphorylation of ERK3 controlled by Cdk1 and Cdc14 regulates its stability in mitosis

2010 ◽  
Vol 428 (1) ◽  
pp. 103-111 ◽  
Author(s):  
Pierre-Luc Tanguay ◽  
Geneviève Rodier ◽  
Sylvain Meloche
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Mitotic Cell ◽  
Mitogen Activated Protein Kinase ◽  
Phosphorylation Sites ◽  
Dependent Manner ◽  
Cell Extracts ◽  
Cycle Dependent

ERK3 (extracellular-signal-regulated kinase 3) is an atypical MAPK (mitogen-activated protein kinase) that is suggested to play a role in cell-cycle progression and cellular differentiation. However, it is not known whether the function of ERK3 is regulated during the cell cycle. In the present paper, we report that ERK3 is stoichiometrically hyperphosphorylated during entry into mitosis and is dephosphorylated at the M→G1 transition. The phosphorylation of ERK3 is associated with the accumulation of the protein in mitosis. In vitro phosphorylation of a series of ERK3-deletion mutants by mitotic cell extracts revealed that phosphorylation is confined to the unique C-terminal extension of the protein. Using MS analysis, we identified four novel phosphorylation sites, Ser684, Ser688, Thr698 and Ser705, located at the extreme C-terminus of ERK3. All four sites are followed by a proline residue. We have shown that purified cyclin B-Cdk1 (cyclindependent kinase 1) phosphorylates these sites in vitro and demonstrate that Cdk1 acts as a major Thr698 kinase in vivo. Reciprocally, we found that the phosphatases Cdc14A and Cdc14B (Cdc is cell-division cycle) bind to ERK3 and reverse its C-terminal phosphorylation in mitosis. Importantly, alanine substitution of the four C-terminal phosphorylation sites markedly decreased the half-life of ERK3 in mitosis, thereby linking phosphorylation to the stabilization of the kinase. The results of the present study identify a novel regulatory mechanism of ERK3 that operates in a cell-cycle-dependent manner.

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.

scholarly journals E2F activity is regulated by cell cycle-dependent changes in subcellular localization.

1997 ◽  
Vol 17 (12) ◽  
pp. 7268-7282 ◽  
Author(s):  
R Verona ◽  
K Moberg ◽  
S Estes ◽  
M Starz ◽  
J P Vernon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Nuclear Localization ◽  
Mammalian Cells ◽  
Critical Role ◽  
Biological Properties ◽  
Dependent Manner ◽  
Restriction Point ◽  
Cycle Dependent ◽  
Almost All

E2F directs the cell cycle-dependent expression of genes that induce or regulate the cell division process. In mammalian cells, this transcriptional activity arises from the combined properties of multiple E2F-DP heterodimers. In this study, we show that the transcriptional potential of individual E2F species is dependent upon their nuclear localization. This is a constitutive property of E2F-1, -2, and -3, whereas the nuclear localization of E2F-4 is dependent upon its association with other nuclear factors. We previously showed that E2F-4 accounts for the majority of endogenous E2F species. We now show that the subcellular localization of E2F-4 is regulated in a cell cycle-dependent manner that results in the differential compartmentalization of the various E2F complexes. Consequently, in cycling cells, the majority of the p107-E2F, p130-E2F, and free E2F complexes remain in the cytoplasm. In contrast, almost all of the nuclear E2F activity is generated by pRB-E2F. This complex is present at high levels during G1 but disappears once the cells have passed the restriction point. Surprisingly, dissociation of this complex causes little increase in the levels of nuclear free E2F activity. This observation suggests that the repressive properties of the pRB-E2F complex play a critical role in establishing the temporal regulation of E2F-responsive genes. How the differential subcellular localization of pRB, p107, and p130 contributes to their different biological properties is also discussed.

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.

Repetitive sperm-induced Ca2+ transients in mouse oocytes are cell cycle dependent

Development ◽  
1995 ◽  
Vol 121 (10) ◽  
pp. 3259-3266 ◽  
Author(s):  
K.T. Jones ◽  
J. Carroll ◽  
J.A. Merriman ◽  
D.G. Whittingham ◽  
T. Kono
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Dependent Manner ◽  
Nuclear Envelope Breakdown ◽  
Mouse Oocytes ◽  
A Cell ◽  
Mature Mouse ◽  
Cycle Dependent ◽  
Stage 1

Mature mouse oocytes are arrested at metaphase of the second meiotic division. Completion of meiosis and a block to polyspermy is caused by a series of repetitive Ca2+ transients triggered by the sperm at fertilization. These Ca2+ transients have been widely reported to last for a number of hours but when, or why, they cease is not known. Here we show that Ca2+ transients cease during entry into interphase, at the time when pronuclei are forming. In fertilized oocytes arrested at metaphase using colcemid, Ca2+ transients continued for as long as measurements were made, up to 18 hours after fertilization. Therefore sperm is able to induce Ca2+ transients during metaphase but not during interphase. In addition metaphase II oocytes, but not pronuclear stage 1-cell embryos showed highly repetitive Ca2+ oscillations in response to microinjection of inositol trisphosphate. This was explored further by treating in vitro maturing oocytes at metaphase I for 4–5 hours with cycloheximide, which induced nuclear progression to interphase (nucleus formation) and subsequent re-entry to metaphase (nuclear envelope breakdown). Fertilization of cycloheximide-treated oocytes revealed that continuous Ca2+ oscillations in response to sperm were observed after nuclear envelope breakdown but not during interphase. However interphase oocytes were able to generate Ca2+ transients in response to thimerosal. This data suggests that the ability of the sperm to trigger repetitive Ca2+ transients in oocytes is modulated in a cell cycle-dependent manner.

Two proteins that cycle asynchronously between centrosomes and nuclear structures: Drosophila CP60 and CP190

1997 ◽  
Vol 110 (14) ◽  
pp. 1573-1583 ◽  
Author(s):  
K. Oegema ◽  
W.F. Marshall ◽  
J.W. Sedat ◽  
B.M. Alberts
Keyword(s):  
Cell Cycle ◽  
Nuclear Envelope ◽  
Wide Field ◽  
Dependent Manner ◽  
Independent Manner ◽  
Nuclear Envelope Breakdown ◽  
Nuclear Structures ◽  
Dynamic Structures ◽  
Cycle Dependent

Both the nucleus and the centrosome are complex, dynamic structures whose architectures undergo cell cycle-specific rearrangements. CP190 and CP60 are two Drosophila proteins of unknown function that shuttle between centrosomes and nuclei in a cell cycle-dependent manner. These two proteins are associated in vitro, and localize to centrosomes in a microtubule independent manner. We injected fluorescently labeled, bacterially expressed CP190 and CP60 into living Drosophila embryos and followed their behavior during the rapid syncytial blastoderm divisions (nuclear cycles 10–13). Using quantitative 3-D wide-field fluorescence microscopy, we show that CP190 and CP60 cycle between nuclei and centrosomes asynchronously with the accumulation of CP190 leading that of CP60 both at centrosomes and in nuclei. During interphase, CP190 is found in nuclei. Immediately following nuclear envelope breakdown, CP190 localizes to centrosomes where it remains until telophase, thereafter accumulating in reforming nuclei. Unlike CP190, CP60 accumulates at centrosomes primarily during anaphase, where it remains into early interphase. During nuclear cycles 10 and 11, CP60 accumulates in nuclei simultaneous with nuclear envelope breakdown, suggesting that CP60 binds to an unknown nuclear structure that persists into mitosis. During nuclear cycles 12 and 13, CP60 accumulates gradually in nuclei during interphase, reaching peak levels just before nuclear envelope breakdown. Once in the nucleus, both CP190 and CP60 appear to form fibrous intranuclear networks that remain coherent even after nuclear envelope breakdown. The CP190 and CP60 networks do not co-localize extensively with each other or with DNA. This work provides direct evidence, in living cells, of a coherent protein network that may represent a nuclear skeleton.

scholarly journals Effect of Sterols Isolated fromMyrtillocactus geometrizanson Growth Inhibition of Colon and Breast Cancer Cells

2015 ◽  
Vol 2015 ◽  
pp. 1-9 ◽  
Author(s):  
Mario Augusto Bolaños-Carrillo ◽  
Jose Luis Ventura-Gallegos ◽  
Arturo David Saldivar-Jiménez ◽  
Alejandro Zentella-Dehesa ◽  
Mariano Martínez-Vázquez
Keyword(s):  
Cell Cycle ◽  
Growth Inhibition ◽  
Cancer Cells ◽  
Biological Activities ◽  
Annexin V ◽  
Parp Cleavage ◽  
Dependent Manner ◽  
Cycle Dependent ◽  
Mcf 7

Objective. To explore the effect of peniocerol and macdougallin on HCT-15 and MCF-7 cells proliferation, cell cycle, apoptosis, and PARP cleavage.Methods. HCT-15 and MCF-7 cells were treated with various concentrations of peniocerol and macdougallin (10–80 μM) during 24 or 48 h. Crystal Violet Assay was used to evaluate the inhibition effect. Cell cycle regulation was examined by a propidium iodide method. Cell apoptosis was detected through both Annexin–V FLUOS/PI double-labeled cytometry assays and Western blot was applied to assess PARP cleavage.Results. Peniocerol and macdougallin induced growth inhibition and apoptosisin vitroin a time- and dose-dependent manner. Moreover, peniocerol and macdougallin induced arrest of cell cycle-dependent manner and increased the proportion of cells in G0/G1phase. PARP cleavage in HCT-15 and MCF-7 cells was induced by treatment with peniocerol and macdougallin after 36 hours.Conclusions. Our results showed that the mechanism of cytotoxicity displayed by peniocerol and macdougallin is related to cell cycle arrest and apoptosis in both cell lines. This is a significant observation because it helps to understand the way some oxysterols isolated fromMyrtillocactus geometrizansdevelop their biological activities against cancer cells.

scholarly journals The cell cycle-dependent nuclear import of v-Jun is regulated by phosphorylation of a serine adjacent to the nuclear localization signal.

1995 ◽  
Vol 130 (2) ◽  
pp. 255-263 ◽  
Author(s):  
T Tagawa ◽  
T Kuroki ◽  
P K Vogt ◽  
K Chida
Keyword(s):  
Cell Cycle ◽  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Protein Kinase Inhibitors ◽  
Nuclear Accumulation ◽  
Dependent Manner ◽  
Localization Signal ◽  
Cycle Dependent

Cell cycle-dependent phosphorylation and nuclear import of the tumorigenic transcription factor viral Jun (v-Jun) were investigated in chicken embryo fibroblasts. Nuclear accumulation of v-Jun but not of cellular Jun (c-Jun) is cell cycle dependent, decreasing in G1 and increasing in G2. The cell cycle-dependent regulation of v-Jun was mapped to a single serine residue at position 248 (Ser248), adjacent to the nuclear localization signal (NLS). Ser248 of v-Jun represents an amino acid substitution, replacing cysteine of c-Jun. It was shown by peptidase digestion and immunoprecipitation with antibody to the NLS that v-Jun is phosphorylated at Ser248 in the cytoplasm but not in the nucleus. This phosphorylation is high in G1 and low in G2. Nuclear accumulation of v-Jun is correlated with underphosphorylation at Ser248. The regulation of nuclear import by phosphorylation was also examined using NLS peptides with Ser248 of v-Jun. Phosphorylation of the serine inhibited nuclear import mediated by the NLS peptide in vivo and in vitro. The protein kinase inhibitors staurosporine and H7 stimulated but the phosphatase inhibitor okadaic acid inhibited nuclear import mediated by the NLS peptide. The cytosolic activity of protein kinases phosphorylating Ser248 increased in G0 and decreased during cell cycle progression, reaching a minimum in G2, whereas phosphatase activity dephosphorylating Ser248 was not changed. These results show that nuclear import of v-Jun is negatively regulated by phosphorylation at Ser248 in the cytoplasm in a cell cycle-dependent manner.

scholarly journals Poly(A)-binding protein binds to the non-polyadenylated 3′ untranslated region of dengue virus and modulates translation efficiency

2009 ◽  
Vol 90 (3) ◽  
pp. 687-692 ◽  
Author(s):  
Charlotta Polacek ◽  
Peter Friebe ◽  
Eva Harris
Keyword(s):  
Dengue Virus ◽  
Translation Initiation ◽  
Untranslated Region ◽  
Binding Protein ◽  
Initiation Factor ◽  
Translation Efficiency ◽  
Dependent Manner ◽  
Stem Loop ◽  
Cell Extracts

Poly(A)-binding protein (PABP) is a key player in mRNA circularization and translation initiation of polyadenylated mRNAs. It simultaneously binds the 3′ poly(A) tail of an mRNA and eukaryotic initiation factor 4G (eIF4G), which forms part of the translation initiation complex assembling at the 5′end, thus circularizing the RNA molecule and enhancing translation initiation. Here, we report the binding of PABP to the non-polyadenylated 3′end of dengue virus (DENV) RNA. PABP binds the DENV 3′ untranslated region (3′UTR) internally, upstream of the conserved 3′stem–loop near the two dumb-bell structures, and can be displaced by poly(A) RNA. The PABP-specific translation inhibitor PABP-interacting protein 2 (Paip2) interferes with the DENV 3′UTR–PABP interaction, and in vitro translation of DENV reporter RNAs in baby hamster kidney cell extracts is inhibited by Paip2 in a dose-dependent manner. Our findings show an expanded translation mechanism for PABP, binding to a viral RNA lacking a terminal poly(A) tail.

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