Noncatalytic Requirement for Cyclin A-cdk2 in p27 Turnover

ABSTRACT Ubiquitin-dependent proteolysis makes a major contribution to decreasing the levels of p27. Ubiquitin-dependent proteolysis of p27kip1 is growth and cell cycle regulated in two ways: first, skp2, a component of the E3-ubiquitin ligase, is growth regulated, and second, a kinase must phosphorylate the threonine-187 position on p27 so that it can be recognized by skp2. In vitro, p27 is phosphorylated by cyclin E- and cyclin A-associated cdk2 as well as by cyclin B1-cdk1. Having analyzed the effect of different cyclin-cyclin-dependent kinase complexes on ubiquitination of p27 in a reconstitution assay system, we now report a noncatalytic requirement for cyclin A-cdk2. Multiparameter flow cytometric analysis also indicates that p27 turnover correlates best with the onset of S phase, once the levels of cyclin A become nearly maximal. Finally, increasing the amount of both cyclin E-cdk2 and skp2 was less efficient at promoting p27 ubiquitination than was increasing the amount of cyclin A-cdk2 alone in extracts prepared from cultures of >93%-purified G1 cells. Together these lines of evidence suggest that cyclin A-cdk2 plays an ancillary noncatalytic role in the ubiquitination of p27 by the SCFskp2 complex.

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An expansion phase precedes terminal erythroid differentiation of hematopoietic progenitor cells from cord blood in vitro and is associated with up-regulation of cyclin E and cyclin-dependent kinase 2

Blood ◽

10.1182/blood.v96.12.3985.h8003985_3985_3987 ◽

2000 ◽

Vol 96 (12) ◽

pp. 3985-3987 ◽

Cited By ~ 1

Author(s):

Mu-Shui Dai ◽

Charlie R. Mantel ◽

Zhen-Biao Xia ◽

Hal E. Broxmeyer ◽

Li Lu

Keyword(s):

Cell Cycle ◽

Cord Blood ◽

Cyclin E ◽

Erythroid Differentiation ◽

S Phase ◽

Cyclin D3 ◽

G1 Phase ◽

Cyclin Dependent Kinase ◽

Terminal Erythroid Differentiation

The dynamics of cell cycle regulation were investigated during in vitro erythroid proliferation and differentiation of CD34+cord blood cells. An unusual cell cycle profile with a majority of cells in S phase (70.2%) and minority of cells in G1 phase (27.4%) was observed in burst-forming unit-erythrocytes (BFU-E)–derived erythroblasts from a 7-day culture of CD34+ cells stimulated with interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), Steel factor, and Epo. Terminal erythroid differentiation was accompanied by a rapid increase of G0/G1 phase cells. Expression of cyclin E and cyclin-dependent kinase 2 (cdk2) correlated with the proportion of S phase cells. Cyclin D3 was moderately up-regulated during the proliferation phase, and both cyclin E and D3 were rapidly down-regulated during terminal differentiation. This suggests that the high proliferation potential of erythroblasts is associated with temporal up-regulation of cyclin E and cdk2.

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Ectopic Expression of Cdc25A Accelerates the G1/S Transition and Leads to Premature Activation of Cyclin E- and Cyclin A-Dependent Kinases

Molecular and Cellular Biology ◽

10.1128/mcb.19.9.6183 ◽

1999 ◽

Vol 19 (9) ◽

pp. 6183-6194 ◽

Cited By ~ 177

Author(s):

Ida Blomberg ◽

Ingrid Hoffmann

Keyword(s):

Phase Transition ◽

Cyclin E ◽

Ectopic Expression ◽

Cyclin A ◽

S Phase ◽

Important Regulator ◽

Cycle Regulation ◽

Cdc25a Phosphatase ◽

Rate Limiting

ABSTRACT Human Cdc25 phosphatases play important roles in cell cycle regulation by removing inhibitory phosphates from tyrosine and threonine residues of cyclin-dependent kinases. Three human Cdc25 isoforms, A, B, and C, have been discovered. Cdc25B and Cdc25C play crucial roles at the G2/M transition. In the present study, we have investigated the function of human Cdc25A phosphatase. Cell lines that express human Cdc25A in an inducible manner have been generated. Ectopic expression of Cdc25A accelerates the G1/S-phase transition, indicating that Cdc25A controls an event(s) that is rate limiting for entry into S phase. Furthermore, we carried out a detailed analysis of the expression and activation of human Cdc25A. Activation of endogenous Cdc25A occurs during late G1 phase and increases in S and G2 phases. We further demonstrate that Cdc25A is activated at the same time as cyclin E- and cyclin A-dependent kinases. In vitro, Cdc25A dephosphorylates and activates the cyclin-Cdk complexes that are active during G1. Overexpression of Cdc25A in the inducible system, however, leads to a premature activation of both cyclin E-Cdk2 and cyclin A-Cdk2 complexes, while no effect of cyclin D-dependent kinases is observed. Furthermore, Cdc25A overexpression induces a tyrosine dephosphorylation of Cdk2. These results suggest that Cdc25A is an important regulator of the G1/S-phase transition and that cyclin E- and cyclin A-dependent kinases act as direct targets.

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Maturation of Human Cyclin E Requires the Function of Eukaryotic Chaperonin CCT

Molecular and Cellular Biology ◽

10.1128/mcb.18.12.7584 ◽

1998 ◽

Vol 18 (12) ◽

pp. 7584-7589 ◽

Cited By ~ 93

Author(s):

Kwang-Ai Won ◽

Robert J. Schumacher ◽

George W. Farr ◽

Arthur L. Horwich ◽

Steven I. Reed

Keyword(s):

Cyclin E ◽

Positive Control ◽

S Phase ◽

Cyclin Dependent Kinase ◽

Native State ◽

Human Proteins ◽

Reticulocyte Lysate ◽

Dependent Processes

ABSTRACT Cyclin E, a partner of the cyclin-dependent kinase Cdk2, has been implicated in positive control of the G1/S phase transition. Whereas degradation of cyclin E has been shown to be exquisitely regulated by ubiquitination and proteasomal action, little is known about posttranscriptional aspects of its biogenesis. In a yeast-based screen designed to identify human proteins that interact with human cyclin E, we identified components of the eukaryotic cytosolic chaperonin CCT. We found that the endogenous CCT complex in yeast was essential for the maturation of cyclin E in vivo. Under conditions of impaired CCT function, cyclin E failed to accumulate. Furthermore, newly translated cyclin E, both in vitro in reticulocyte lysate and in vivo in human cells in culture, is efficiently bound and processed by the CCT. In vitro, in the presence of ATP, the bound protein is folded and released in order to become associated with Cdk2. Thus, both the acquisition of the native state and turnover of cyclin E involve ATP-dependent processes mediated by large oligomeric assemblies.

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An expansion phase precedes terminal erythroid differentiation of hematopoietic progenitor cells from cord blood in vitro and is associated with up-regulation of cyclin E and cyclin-dependent kinase 2

Blood ◽

10.1182/blood.v96.12.3985 ◽

2000 ◽

Vol 96 (12) ◽

pp. 3985-3987 ◽

Cited By ~ 24

Author(s):

Mu-Shui Dai ◽

Charlie R. Mantel ◽

Zhen-Biao Xia ◽

Hal E. Broxmeyer ◽

Li Lu

Keyword(s):

Cell Cycle ◽

Cord Blood ◽

Cyclin E ◽

Erythroid Differentiation ◽

S Phase ◽

Cyclin D3 ◽

G1 Phase ◽

Cyclin Dependent Kinase ◽

Terminal Erythroid Differentiation

Abstract The dynamics of cell cycle regulation were investigated during in vitro erythroid proliferation and differentiation of CD34+cord blood cells. An unusual cell cycle profile with a majority of cells in S phase (70.2%) and minority of cells in G1 phase (27.4%) was observed in burst-forming unit-erythrocytes (BFU-E)–derived erythroblasts from a 7-day culture of CD34+ cells stimulated with interleukin 3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), Steel factor, and Epo. Terminal erythroid differentiation was accompanied by a rapid increase of G0/G1 phase cells. Expression of cyclin E and cyclin-dependent kinase 2 (cdk2) correlated with the proportion of S phase cells. Cyclin D3 was moderately up-regulated during the proliferation phase, and both cyclin E and D3 were rapidly down-regulated during terminal differentiation. This suggests that the high proliferation potential of erythroblasts is associated with temporal up-regulation of cyclin E and cdk2.

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Liganded ERα Stimulates the E3 Ubiquitin Ligase Activity of UBE3C to Facilitate Cell Proliferation

Molecular Endocrinology ◽

10.1210/me.2015-1125 ◽

2015 ◽

Vol 29 (11) ◽

pp. 1646-1657 ◽

Cited By ~ 5

Author(s):

Maiko Okada ◽

Fumiaki Ohtake ◽

Hiroyuki Nishikawa ◽

Wenwen Wu ◽

Yasushi Saeki ◽

...

Keyword(s):

Cell Proliferation ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Cyclin B1 ◽

Dependent Manner ◽

Ubiquitin Ligase Activity ◽

Dependent Cell ◽

Estrogen Antagonist

Abstract Estrogen receptor (ER)α is a well-characterized ligand-dependent transcription factor. However, the global picture of its nongenomic functions remains to be illustrated. Here, we demonstrate a novel function of ERα during mitosis that facilitates estrogen-dependent cell proliferation. An E3 ubiquitin ligase, UBE3C, was identified in an ERα complex from estrogen-treated MCF-7 breast cancer cells arrested at mitosis. UBE3C interacts with ERα during mitosis in an estrogen-dependent manner. In vitro, estrogen dramatically stimulates the E3 activity of UBE3C in the presence of ERα. This effect was inhibited by the estrogen antagonist tamoxifen. Importantly, estrogen enhances the ubiquitination of cyclin B1 (CCNB1) and destabilizes CCNB1 during mitosis in a manner dependent on endogenous UBE3C. ERα, UBE3C, and CCNB1 colocalize in prophase nuclei and at metaphase spindles before CCNB1 is degraded in anaphase. Depletion of UBE3C attenuates estrogen-dependent cell proliferation without affecting the transactivation function of ERα. Collectively, these results demonstrate a novel ligand-dependent action of ERα that stimulates the activity of an E3 ligase. The mitotic role of estrogen may contribute to its effects on proliferation in addition to its roles in target gene expression.

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p130 and p107 use a conserved domain to inhibit cellular cyclin-dependent kinase activity.

Molecular and Cellular Biology ◽

10.1128/mcb.17.7.3566 ◽

1997 ◽

Vol 17 (7) ◽

pp. 3566-3579 ◽

Cited By ~ 73

Author(s):

M S Woo ◽

I Sánchez ◽

B D Dynlacht

Keyword(s):

Cell Cycle ◽

Transcription Factor ◽

Kinase Activity ◽

Cyclin E ◽

Critical Role ◽

Cyclin A ◽

Terminal Region ◽

Cyclin Dependent Kinase ◽

Amino Terminal

The pRB-related proteins p107 and p130 are thought to suppress growth in part through their associations with two important cell cycle kinases, cyclin A-cdk2 and cyclin E-cdk2, and transcription factor E2F. Although each protein plays a critical role in cell proliferation, the functional consequences of the association among growth suppressor, cyclin-dependent kinase, and transcription factor have remained elusive. In an attempt to understand the biochemical properties of such complexes, we reconstituted each of the p130-cyclin-cdk2 and p107-cyclin-cdk2 complexes found in vivo with purified, recombinant proteins. Strikingly, stoichiometric association of p107 or p130 with either cyclin E-cdk2 or cyclin A-cdk2 negated the activities of these kinases. The results of our experiments suggest that inhibition does not result from substrate competition or loss of cdk2 activation. Kinase inhibitory activity was dependent upon an amino-terminal region of p107 that is highly conserved with p130. Further, a role for this amino-terminal region in growth suppression was uncovered by using p107 mutants unable to bind E2F. To determine whether cellular complexes might display similar regulatory properties, we purified p130-cyclin A-cdk2 complexes from human cells and found that such complexes exist in two forms, one that contains E2F-4-DP-1 and one that lacks the heterodimer. These endogenous complexes behaved like the in vitro-reconstituted complexes, exhibiting low levels of associated kinase activity that could be significantly augmented by dissociation of p130. The results of these experiments suggest a mechanism whereby p130 and p107 suppress growth by inhibiting important cell cycle kinases.

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Analysis of a Drosophila cyclin E Hypomorphic Mutation Suggests a Novel Role for Cyclin E in Cell Proliferation Control During Eye Imaginal Disc Development

Genetics ◽

10.1093/genetics/149.4.1867 ◽

1998 ◽

Vol 149 (4) ◽

pp. 1867-1882 ◽

Cited By ~ 3

Author(s):

Julie Secombe ◽

Johanna Pispa ◽

Robert Saint ◽

Helena Richardson

Keyword(s):

Imaginal Disc ◽

Cyclin E ◽

Cyclin A ◽

Cyclin B1 ◽

S Phase ◽

P Element ◽

Pigment Cells ◽

Hypomorphic Mutation ◽

Eye Imaginal Disc ◽

Number Of Cells

Abstract We have generated and characterized a Drosophila cyclin E hypomorphic mutation, DmcycEJP, that is homozygous viable and fertile, but results in adults with rough eyes. The mutation arose from an internal deletion of an existing P[w+lacZ] element inserted 14 kb upstream of the transcription start site of the DmcycE zygotic mRNA. The presence of this deleted P element, but not the P[w+lacZ] element from which it was derived, leads to a decreased level of DmcycE expression during eye imaginal disc development. Eye imaginal discs from DmcycEJP larvae contain fewer S phase cells, both anterior and posterior to the morphogenetic furrow. This results in adults with small rough eyes, largely due to insufficient numbers of pigment cells. Altering the dosage of the Drosophila cdk2 homolog, cdc2c, retinoblastoma, or p21CIP1 homolog dacapo, which encode proteins known to physically interact with Cyclin E, modified the DmcycEJP rough eye phenotype as expected. Decreasing the dosage of the S phase transcription factor gene, dE2F, enhanced the DmcycEJP rough eye phenotype. Surprisingly, mutations in G2/M phase regulators cyclin A and string (cdc25), but not cyclin B1, B3, or cdc2, enhanced the DmcycE JP phenotype without affecting the number of cells entering S phase, but by decreasing the number of cells entering mitosis. Our analysis establishes the DmcycE JP allele as an excellent resource for searching for novel cyclin E genetic interactors. In addition, this analysis has identified cyclin A and string as DmcycEJP interactors, suggesting a novel role for cyclin E in the regulation of Cyclin A and String function during eye development.

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The PP2A-B56 Phosphatase Opposes Cyclin E Autocatalytic Degradation via Site-Specific Dephosphorylation

Molecular and Cellular Biology ◽

10.1128/mcb.00657-16 ◽

2017 ◽

Vol 37 (8) ◽

Cited By ~ 5

Author(s):

Ryan J. Davis ◽

Jherek Swanger ◽

Bridget T. Hughes ◽

Bruce E. Clurman

Keyword(s):

Cell Cycle ◽

Ubiquitin Ligase ◽

Cell Cycle Progression ◽

Cyclin E ◽

Phosphorylation Site ◽

S Phase ◽

Cyclin Dependent Kinase ◽

Cycle Progression ◽

Cdk2 Activity ◽

Interphase Cells

ABSTRACT Cyclin E, in conjunction with its catalytic partner cyclin-dependent kinase 2 (CDK2), regulates cell cycle progression as cells exit quiescence and enter S-phase. Multiple mechanisms control cyclin E periodicity during the cell cycle, including phosphorylation-dependent cyclin E ubiquitylation by the SCFFbw7 ubiquitin ligase. Serine 384 (S384) is the critical cyclin E phosphorylation site that stimulates Fbw7 binding and cyclin E ubiquitylation and degradation. Because S384 is autophosphorylated by bound CDK2, this presents a paradox as to how cyclin E can evade autocatalytically induced degradation in order to phosphorylate its other substrates. We found that S384 phosphorylation is dynamically regulated in cells and that cyclin E is specifically dephosphorylated at S384 by the PP2A-B56 phosphatase, thereby uncoupling cyclin E degradation from cyclin E-CDK2 activity. Furthermore, the rate of S384 dephosphorylation is high in interphase but low in mitosis. This provides a mechanism whereby interphase cells can oppose autocatalytic cyclin E degradation and maintain cyclin E-CDK2 activity while also enabling cyclin E destruction in mitosis, when inappropriate cyclin E expression is genotoxic.

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E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

International Journal of Molecular Sciences ◽

10.3390/ijms22115712 ◽

2021 ◽

Vol 22 (11) ◽

pp. 5712

Author(s):

Michał Tracz ◽

Ireneusz Górniak ◽

Andrzej Szczepaniak ◽

Wojciech Białek

Keyword(s):

Ubiquitin Ligase ◽

Conformational Changes ◽

Protein Interactions ◽

E3 Ubiquitin Ligase ◽

Lanthanide Ions ◽

E3 Ligases ◽

Fluorescence Measurements ◽

Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

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