scholarly journals Nuclear localization of the ubiquitin-activating enzyme, E1, is cell-cycle-dependent

1994 ◽  
Vol 300 (3) ◽  
pp. 701-708 ◽  
Author(s):  
S J Grenfell ◽  
J S Trausch-Azar ◽  
P M Handley-Gearhart ◽  
A Ciechanover ◽  
A L Schwartz
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Initial Step ◽  
Immunoblot Analysis ◽  
Subcellular Fractionation ◽  
Cell Extract ◽  
Cell Fractionation ◽  
Differential Distribution ◽  
Cycle Dependent ◽  
Substrate Proteins

The mechanisms that regulate ubiquitin-mediated degradation of proteins such as the mitotic cyclins at defined stages of the cell cycle are poorly understood. The initial step in the conjugation of ubiquitin to substrate proteins involves the activation of ubiquitin by the ubiquitin-activating enzyme, E1. Previously we have described the subcellular localization of this enzyme to both nuclear and cytoplasmic compartments. In the present study, we have used the 1C5 anti-E1 monoclonal antibody in immunofluorescent-microscopy and subcellular-fractionation techniques to examine the distribution of E1 during the HeLa cell cycle. E1 is both cytoskeletal and nuclear during the G1-phase. As the cells progress into S-phase, E1 is exclusively cytoskeletal and has a perinuclear distribution. During G2-phase, E1 reappears in the nucleus before breakdown of the nuclear envelope. In mitotic cells, E1 localizes to both the mitotic spindle and the cytosol, but is absent from the chromosomes. Immunoblot analysis reveals multiple forms of E1 in HeLa whole cell extract. This heterogeneity is not a result of polyubiquitination and may represent inactive pools of E1. Only the characteristic E1 doublet is able to activate ubiquitin. Cell-fractionation studies reveal a differential distribution of specific E1 isoforms throughout the cell cycle. Therefore we propose that the subcellular localization of E1 may play a role in regulating cell-cycle-dependent conjugation of ubiquitin to target proteins.

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.

Cell-cycle dependent GATA2 subcellular localization in mouse 2-cell embryos

2021 ◽  
Author(s):  
Masaya Komatsu ◽  
Hayato Tsukahara ◽  
Hanako Bai ◽  
Masashi Takahashi ◽  
Takuya Wakai ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Cycle Dependent

scholarly journals A Novel Mechanism for Mitogen-activated Protein Kinase Localization

2004 ◽  
Vol 15 (10) ◽  
pp. 4457-4466 ◽  
Author(s):  
Eric Bind ◽  
Yelena Kleyner ◽  
Dorota Skowronska-Krawczyk ◽  
Emily Bien ◽  
Brian David Dynlacht ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Fluorescent Protein ◽  
Nuclear Translocation ◽  
Immunoblot Analysis ◽  
Mitogen Activated Protein Kinase ◽  
Carboxy Terminus ◽  
Extracellular Signal ◽  
Mitogen Activated Protein

Mitogen-activated protein kinases/extracellular signal regulated kinases (MAPKs/ERKs) are typically thought to be soluble cytoplasmic enzymes that translocate to the nucleus subsequent to their phosphorylation by their activating kinases or mitogen-activated protein/extracellular signal regulated kinase kinase. We report here the first example of nuclear translocation of a MAPK that occurs via temporally regulated exit from a membranous organelle. Confocal microscopy examining the subcellular localization of ERK3 in several cell lines indicated that this enzyme was targeted to the Golgi/endoplasmic reticulum Golgi intermediate compartment. Deletion analysis of green fluorescent protein (GFP)-ERK3 uncovered a nuclear form that was carboxy-terminally truncated and established a Golgi targeting motif at the carboxy terminus. Immunoblot analysis of cells treated with the proteasome inhibitor MG132 further revealed two cleavage products, suggesting that in vivo, carboxy-terminal cleavage of the full-length protein controls its subcellular localization. In support of this hypothesis, we found that deletion of a small region rich in acidic residues within the carboxy terminus eliminated both the cleavage and nuclear translocation of GFP-ERK3. Finally, cell cycle synchronization studies revealed that the subcellular localization of ERK3 is temporally regulated. These data suggest a novel mechanism for the localization of an MAPK family member, ERK3, in which cell cycle-regulated, site-specific proteolysis generates the nuclear form of the protein.

scholarly journals Human cyclins A and B1 are differentially located in the cell and undergo cell cycle-dependent nuclear transport.

1991 ◽  
Vol 115 (1) ◽  
pp. 1-17 ◽  
Author(s):  
J Pines ◽  
T Hunter
Keyword(s):  
Cell Cycle ◽  
Human Fibroblasts ◽  
Nuclear Lamina ◽  
Cyclin A ◽  
Cyclin B1 ◽  
Cell Fractionation ◽  
Mitotic Apparatus ◽  
Epithelial Tumor ◽  
Mitotic Cyclin ◽  
Cycle Dependent

We have used immunofluorescence staining to study the subcellular distribution of cyclin A and B1 during the somatic cell cycle. In both primary human fibroblasts and in epithelial tumor cells, we find that cyclin A is predominantly nuclear from S phase onwards. Cyclin A may associated with condensing chromosomes in prophase, but is not associated with condensed chromosomes in metaphase. By contrast, cyclin B1 accumulates in the cytoplasm of interphase cells and only enters the nucleus at the beginning of mitosis, before nuclear lamina breakdown. In mitotic cells, cyclin B1 associates with condensed chromosomes in prophase and metaphase, and with the mitotic apparatus. Cyclin A is degraded during metaphase and cyclin B1 is precipitously destroyed at the metaphase----anaphase transition. Cell fractionation and immunoprecipitation studies showed that both cyclin A and cyclin B1 are associated with PSTAIRE-containing proteins. The nuclear, but not the cytoplasmic form, of cyclin A is associated with a 33-kD PSTAIRE-containing protein. Cyclin B1 is associated with p34cdc2 in the cytoplasm. Thus we propose that the different localization of cyclin A and cyclin B1 in the cell cycle could be the means by which the two types of mitotic cyclin confer substrate specificity upon their associated PSTAIRE-containing protein kinase subunit.

scholarly journals Cell cycle-dependent expression and subcellular localization of fructose 1,6-bisphosphatase

2011 ◽  
Vol 137 (1) ◽  
pp. 121-136 ◽  
Author(s):  
Piotr Mamczur ◽  
Agnieszka Joanna Sok ◽  
Adam Rzechonek ◽  
Dariusz Rakus
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Fructose 1,6 Bisphosphatase ◽  
Cycle Dependent

scholarly journals Subcellular localization of Cdc42p, a Saccharomyces cerevisiae GTP-binding protein involved in the control of cell polarity.

1993 ◽  
Vol 4 (12) ◽  
pp. 1307-1316 ◽  
Author(s):  
M Ziman ◽  
D Preuss ◽  
J Mulholland ◽  
J M O'Brien ◽  
D Botstein ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Subcellular Localization ◽  
Cell Polarity ◽  
Mutant Strain ◽  
Consensus Sequence ◽  
Restrictive Temperature ◽  
Cell Fractionation ◽  
Gtp Binding ◽  
Bud Emergence

The Saccharomyces cerevisiae Cdc42 protein, a member of the Ras superfamily of low-molecular-weight GTP-binding proteins, is involved in the control of cell polarity during the yeast cell cycle. This protein has a consensus sequence (CAAX) for geranylgeranyl modification and is likely to be associated, at least in part, with cell membranes. Using cell fractionation and immunolocalization techniques, we have investigated the subcellular localization of Cdc42p. Cdc42p was found in both soluble and particulate pools, and neither its abundance nor its distribution varied through the cell cycle. The particulate form of Cdc42p could be solubilized with detergents but not with NaCl or urea, suggesting that it is tightly associated with membranes. An increase in soluble Cdc42p was observed in a geranylgeranyltransferase mutant strain (cdc43-2ts) grown at the restrictive temperature. In addition, Cdc42p from a cdc42C188S mutant strain (that has an alteration at the prenylation consensus site) was almost exclusively in the soluble fraction, suggesting that membrane localization is dependent on geranylgeranyl modification at Cys-188. Immunofluorescence and immunoelectron microscopy experiments demonstrated that Cdc42p localizes to the plasma membrane in the vicinity of secretory vesicles that were found at the site of bud emergence, at the tips and sides of enlarging buds, and within mating projections (shmoo tips) in alpha-factor-arrested cells. These results indicate that Cdc42p is localized to the bud site early in the cell cycle and suggest that this localization is critical for the selection of the proper site for bud emergence and for polarized cell growth.

scholarly journals PTOV1 Enables the Nuclear Translocation and Mitogenic Activity of Flotillin-1, a Major Protein of Lipid Rafts

2005 ◽  
Vol 25 (5) ◽  
pp. 1900-1911 ◽  
Author(s):  
Anna Santamaría ◽  
Elisabeth Castellanos ◽  
Valentí Gómez ◽  
Patricia Benedit ◽  
Jaime Renau-Piqueras ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Nuclear Localization ◽  
Nuclear Translocation ◽  
Subcellular Fractionation ◽  
Major Protein ◽  
Dependent Manner ◽  
Protein Levels ◽  
A Cell ◽  
Cycle Dependent

ABSTRACT PTOV1 is a mitogenic protein that shuttles between the nucleus and the cytoplasm in a cell cycle-dependent manner. It consists of two homologous domains arranged in tandem that constitute a new class of protein modules. We show here that PTOV1 interacts with the lipid raft protein flotillin-1, with which it copurifies in detergent-insoluble floating fractions. Flotillin-1 colocalized with PTOV1 not only at the plasma membrane but, unexpectedly, also in the nucleus, as demonstrated by immunocytochemistry and subcellular fractionation of endogenous and exogenous flotillin-1. Flotillin-1 entered the nucleus concomitant with PTOV1, shortly before the initiation of the S phase. Protein levels of PTOV1 and flotillin-1 oscillated during the cell cycle, with a peak in S. Depletion of PTOV1 significantly inhibited nuclear localization of flotillin-1, whereas depletion of flotillin-1 did not affect nuclear localization of PTOV1. Depletion of either protein markedly inhibited cell proliferation under basal conditions. Overexpression of PTOV1 or flotillin-1 strongly induced proliferation, which required their localization to the nucleus, and was dependent on the reciprocal protein. These observations suggest that PTOV1 assists flotillin-1 in its translocation to the nucleus and that both proteins are required for cell proliferation.

scholarly journals The subcellular localization of E2F-4 is cell-cycle dependent

1997 ◽  
Vol 94 (10) ◽  
pp. 5095-5100 ◽  
Author(s):  
G. J. Lindeman ◽  
S. Gaubatz ◽  
D. M. Livingston ◽  
D. Ginsberg
Keyword(s):  
Cell Cycle ◽  
Subcellular Localization ◽  
Cycle Dependent
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