scholarly journals Preventing the Degradation of Mps1 at Centrosomes Is Sufficient to Cause Centrosome Reduplication in Human Cells

2007 ◽  
Vol 18 (11) ◽  
pp. 4457-4469 ◽  
Author(s):  
Christopher Kasbek ◽  
Ching-Hui Yang ◽  
Adlina Mohd Yusof ◽  
Heather M. Chapman ◽  
Mark Winey ◽  
...  
Keyword(s):  
Phosphorylation Site ◽  
Proteasome Inhibition ◽  
Cyclin A ◽  
S Phase ◽  
Human Cells ◽  
Centrosome Duplication ◽  
Cyclin Dependent Kinase ◽  
Mitotic Spindles ◽  
Cdk2 Activity ◽  
Necessary And Sufficient

Supernumerary centrosomes promote the assembly of abnormal mitotic spindles in many human tumors. In human cells, overexpression of the cyclin-dependent kinase (Cdk)2 partner cyclin A during a prolonged S phase produces extra centrosomes, called centrosome reduplication. Cdk2 activity protects the Mps1 protein kinase from proteasome-mediated degradation, and we demonstrate here that Mps1 mediates cyclin A-dependent centrosome reduplication. Overexpression of cyclin A or a brief proteasome inhibition increases the centrosomal levels of Mps1, whereas depletion of Cdk2 leads to the proteasome-dependent loss of Mps1 from centrosomes only. When a Cdk2 phosphorylation site within Mps1 (T468) is mutated to alanine, Mps1 cannot accumulate at centrosomes or participate in centrosome duplication. In contrast, phosphomimetic mutations at T468 or deletion of the region surrounding T468 prevent the proteasome-dependent removal of Mps1 from centrosomes in the absence of Cdk2 activity. Moreover, cyclin A-dependent centrosome reduplication requires Mps1, and these stabilizing Mps1 mutations cause centrosome reduplication, bypassing cyclin A. Together, our data demonstrate that the region surrounding T468 contains a motif that regulates the accumulation of Mps1 at centrosomes. We suggest that phosphorylation of T468 attenuates the degradation of Mps1 at centrosomes and that preventing this degradation is necessary and sufficient to cause centrosome reduplication in human cells.

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

2017 ◽  
Vol 37 (8) ◽  
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.

scholarly journals Periodic Expression of the Cyclin-dependent Kinase Inhibitor p57Kip2 in Trophoblast Giant Cells Defines a G2-like Gap Phase of the Endocycle

2000 ◽  
Vol 11 (3) ◽  
pp. 1037-1045 ◽  
Author(s):  
Naka Hattori ◽  
Tyler C. Davies ◽  
Lynn Anson-Cartwright ◽  
James C. Cross
Keyword(s):  
Dna Replication ◽  
Protein Targeting ◽  
Kinase Inhibitor ◽  
Phosphorylation Site ◽  
Giant Cells ◽  
S Phase ◽  
Stable Form ◽  
Cdk Inhibitor ◽  
Cyclin Dependent Kinase ◽  
Trophoblast Giant Cells

Endoreduplication is an unusual form of cell cycle in which rounds of DNA synthesis repeat in the absence of intervening mitoses. How G1/S cyclin-dependent kinase (Cdk) activity is regulated during the mammalian endocycle is poorly understood. We show here that expression of the G1/S Cdk inhibitor p57Kip2 is induced coincidentally with the transition to the endocycle in trophoblast giant cells.Kip2 mRNA is constitutively expressed during subsequent endocycles, but the protein level fluctuates. In trophoblast giant cells synchronized for the first few endocycles, the p57Kip2 protein accumulates only at the end of S-phase and then rapidly disappears a few hours before the onset of the next S-phase. The protein becomes stabilized by mutation of a C-terminal Cdk phosphorylation site. As a consequence, introduction of this stable form of p57Kip2 into giant cells blocks S-phase entry. These data imply that p57Kip2 is subject to phosphorylation-dependent turnover. Surprisingly, although this occurs in endoreduplicating giant cells, p57Kip2 is stable when ectopically expressed in proliferating trophoblast cells, indicating that these cells lack the mechanism for protein targeting and/or degradation. These data show that the appearance of p57Kip2punctuates the completion of DNA replication, whereas its turnover is subsequently required to initiate the next round of endoreduplication in trophoblast giant cells. Cyclical expression of a Cdk inhibitor, by terminating G1/S Cdk activity, may help promote the resetting of DNA replication machinery.

scholarly journals Human Cyclin a Is Required for Mitosis until Mid Prophase

1999 ◽  
Vol 147 (2) ◽  
pp. 295-306 ◽  
Author(s):  
Nobuaki Furuno ◽  
Nicole den Elzen ◽  
Jonathon Pines
Keyword(s):  
Cyclin A ◽  
Time Lapse ◽  
S Phase ◽  
Early Prophase ◽  
Cyclin Dependent Kinase ◽  
Late Prophase ◽  
Daughter Cells ◽  
G2 Phase ◽  
Rate Limiting

We have used microinjection and time-lapse video microscopy to study the role of cyclin A in mitosis. We have injected purified, active cyclin A/cyclin-dependent kinase 2 (CDK2) into synchronized cells at specific points in the cell cycle and assayed its effect on cell division. We find that cyclin A/CDK2 will drive G2 phase cells into mitosis within 30 min of microinjection, up to 4 h before control cells enter mitosis. Often this premature mitosis is abnormal; the chromosomes do not completely condense and daughter cells fuse. Remarkably, microinjecting cyclin A/CDK2 into S phase cells has no effect on progress through the following G2 phase or mitosis. In complementary experiments we have microinjected the amino terminus of p21Cip1/Waf1/Sdi1 (p21N) into cells to inhibit cyclin A/CDK2 activity. We find that p21N will prevent S phase or G2 phase cells from entering mitosis, and will cause early prophase cells to return to interphase. These results suggest that cyclin A/CDK2 is a rate-limiting component required for entry into mitosis, and for progress through mitosis until late prophase. They also suggest that cyclin A/CDK2 may be the target of the recently described prophase checkpoint.

scholarly journals BRCA1 Is Phosphorylated at Serine 1497 In Vivo at a Cyclin-Dependent Kinase 2 Phosphorylation Site

1999 ◽  
Vol 19 (7) ◽  
pp. 4843-4854 ◽  
Author(s):  
Heinz Ruffner ◽  
Wei Jiang ◽  
A. Grey Craig ◽  
Tony Hunter ◽  
Inder M. Verma
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Activity ◽  
Phosphorylation Site ◽  
Cyclin A ◽  
Dominant Negative ◽  
Protein Kinase Activity ◽  
Cyclin Dependent Kinase

ABSTRACT BRCA1 is a cell cycle-regulated nuclear protein that is phosphorylated mainly on serine and to a lesser extent on threonine residues. Changes in phosphorylation occur in response to cell cycle progression and DNA damage. Specifically, BRCA1 undergoes hyperphosphorylation during late G1 and S phases of the cell cycle. Here we report that BRCA1 is phosphorylated in vivo at serine 1497 (S1497), which is part of a cyclin-dependent kinase (CDK) consensus site. S1497 can be phosphorylated in vitro by CDK2-cyclin A or E. BRCA1 coimmunoprecipitates with an endogenous serine-threonine protein kinase activity that phosphorylates S1497 in vitro. This cellular kinase activity is sensitive to transfection of a dominant negative form of CDK2 as well as the application of the CDK inhibitors p21 and butyrolactone I but not p16. Furthermore, BRCA1 coimmunoprecipitates with CDK2 and cyclin A. These results suggest that the endogenous kinase activity is composed of CDK2-cyclin complexes, at least in part, concordant with the G1/S-specific increase in BRCA1 phosphorylation.

scholarly journals Noncatalytic Requirement for Cyclin A-cdk2 in p27 Turnover

2004 ◽  
Vol 24 (13) ◽  
pp. 6058-6066 ◽  
Author(s):  
Xin-Hua Zhu ◽  
Hoang Nguyen ◽  
H. Dorota Halicka ◽  
Frank Traganos ◽  
Andrew Koff
Keyword(s):  
Ubiquitin Ligase ◽  
Cyclin E ◽  
Flow Cytometric Analysis ◽  
E3 Ubiquitin Ligase ◽  
Cyclin A ◽  
Cyclin B1 ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Flow Cytometric

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.

scholarly journals A Protein Encoded by the Latency-Related Gene of Bovine Herpesvirus 1 Is Expressed in Trigeminal Ganglionic Neurons of Latently Infected Cattle and Interacts with Cyclin-Dependent Kinase 2 during Productive Infection

1998 ◽  
Vol 72 (10) ◽  
pp. 8133-8142 ◽  
Author(s):  
Yunquan Jiang ◽  
Ashfaque Hossain ◽  
Maria Teresa Winkler ◽  
Todd Holt ◽  
Alan Doster ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Acute Infection ◽  
Cyclin A ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Bovine Herpesvirus 1 ◽  
Cycle Progression ◽  
Latently Infected ◽  
Herpesvirus 1

ABSTRACT Despite productive viral gene expression in the peripheral nervous system during acute infection, the bovine herpesvirus 1 (BHV-1) infection cycle is blocked in sensory ganglionic neurons and consequently latency is established. The only abundant viral transcript expressed during latency is the latency-related (LR) RNA. LR gene products inhibit S-phase entry, and binding of the LR protein (LRP) to cyclin A was hypothesized to block cell cycle progression. This study demonstrates LRP is a nuclear protein which is expressed in neurons of latently infected cattle. Affinity chromatography indicated that LRP interacts with cyclin-dependent kinase 2 (cdk2)-cyclin complexes or cdc2-cyclin complexes in transfected human cells or infected bovine cells. After partial purification using three different columns (DEAE-Sepharose, Econo S, and heparin-agarose), LRP was primarily associated with cdk2-cyclin E complexes, an enzyme which is necessary for G1-to-S-phase cell cycle progression. During acute infection of trigeminal ganglia or following dexamethasone-induced reactivation, BHV-1 induces expression of cyclin A in neurons (L. M. Schang, A. Hossain, and C. Jones, J. Virol. 70:3807–3814, 1996). Expression of S-phase regulatory proteins (cyclin A, for example) leads to neuronal apoptosis. Consequently, we hypothesize that interactions between LRP and cell cycle regulatory proteins promote survival of postmitotic neurons during acute infection and/or reactivation.

Chromatin-bound Cdc6 persists in S and G2 phases in human cells, while soluble Cdc6 is destroyed in a cyclin A-cdk2 dependent process

2000 ◽  
Vol 113 (11) ◽  
pp. 1929-1938 ◽  
Author(s):  
D. Coverley ◽  
C. Pelizon ◽  
S. Trewick ◽  
R.A. Laskey
Keyword(s):  
Dna Replication ◽  
Protein Kinase ◽  
Mammalian Cell ◽  
Cyclin A ◽  
S Phase ◽  
Human Cells ◽  
Cell Extracts ◽  
In Vitro Replication ◽  
Initiation Of Dna Replication

Cdc6 is essential for the initiation of DNA replication in all organisms in which it has been studied. In addition, recombinant Cdc6 can stimulate initiation in G(1) nuclei in vitro. We have analysed the behaviour of recombinant Cdc6 in mammalian cell extracts under in vitro replication conditions. We find that Cdc6 is imported into the nucleus in G(1)phase, where it binds to chromatin and remains relatively stable. In S phase, exogenous Cdc6 is destroyed in a process that requires import into the nucleus and phosphorylation by a chromatin-bound protein kinase. Recombinant cyclin A-cdk2 can completely substitute for the nucleus in promoting destruction of soluble Xenopus and human Cdc6. Despite this regulated destruction, endogenous Cdc6 persists in the nucleus after initiation, although the amount falls. Cdc6 levels remain constant in G(2) then fall again before mitosis. We propose that cyclin A-cdk2 phosphorylation results in destruction of any Cdc6 not assembled into replication complexes, but that assembled proteins remain, in the phosphorylated state, in the nucleus. This process could contribute to the prevention of reinitiation in human cells by making free Cdc6 unavailable for re-assembly into replication complexes after G(1) phase.

scholarly journals v-Jun Overrides the Mitogen Dependence of S-Phase Entry by Deregulating Retinoblastoma Protein Phosphorylation and E2F-Pocket Protein Interactions as a Consequence of Enhanced Cyclin E-cdk2 Catalytic Activity

2000 ◽  
Vol 20 (7) ◽  
pp. 2529-2542 ◽  
Author(s):  
W. Clark ◽  
E. J. Black ◽  
A. MacLaren ◽  
U. Kruse ◽  
N. LaThangue ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Protein Interactions ◽  
Cyclin E ◽  
Specific Inhibitor ◽  
Cyclin A ◽  
S Phase ◽  
Pocket Protein ◽  
Cdk2 Activity ◽  
Rb Phosphorylation ◽  
Phase Entry

ABSTRACT v-Jun accelerates G1 progression and shares the capacity of the Myc, E2F, and E1A oncoproteins to sustain S-phase entry in the absence of mitogens; however, how it does so is unknown. To gain insight into the mechanism, we investigated how v-Jun affects mitogen-dependent processes which control the G1/S transition. We show that v-Jun enables cells to express cyclin A and cyclin A-cdk2 kinase activity in the absence of growth factors and that deregulation of cdk2 is required for S-phase entry. Cyclin A expression is repressed in quiescent cells by E2F acting in conjunction with its pocket protein partners Rb, p107, and p130; however, v-Jun overrides this control, causing phosphorylated Rb and proliferation-specific E2F-p107 complexes to persist after mitogen withdrawal. Dephosphorylation of Rb and destruction of cyclin A nevertheless occur normally at mitosis, indicating that v-Jun enables cells to rephosphorylate Rb and reaccumulate cyclin A without exogenous mitogenic stimulation each time the mitotic “clock” is reset. D-cyclin–cdk activity is required for Rb phosphorylation in v-Jun-transformed cells, since ectopic expression of the cdk4- and cdk6-specific inhibitor p16 INK4A inhibits both DNA synthesis and cell proliferation. Despite this, v-Jun does not stimulate D-cyclin–cdk activity but does induce a marked deregulation of cyclin E-cdk2. In particular, hormonal activation of a conditional v-Jun–estrogen receptor fusion protein in quiescent, growth factor-deprived cells stimulates cyclin E-cdk2 activity and triggers Rb phosphorylation and DNA synthesis. Thus, v-Jun overrides the mitogen dependence of S-phase entry by deregulating Rb phosphorylation, E2F-pocket protein interactions, and ultimately cyclin A-cdk2 activity. This is the first report, however, that cyclin E-cdk2, rather than D-cyclin–cdk, is likely to be the critical Rb kinase target of v-Jun.

scholarly journals Direct regulation of Treslin by cyclin-dependent kinase is essential for the onset of DNA replication

2011 ◽  
Vol 193 (6) ◽  
pp. 995-1007 ◽  
Author(s):  
Akiko Kumagai ◽  
Anna Shevchenko ◽  
Andrej Shevchenko ◽  
William G. Dunphy
Keyword(s):  
Dna Replication ◽  
Deoxyribonucleic Acid ◽  
S Phase ◽  
Human Cells ◽  
Target Sequence ◽  
Cyclin Dependent Kinase ◽  
Functional Importance ◽  
Interacting Protein ◽  
Egg Extracts

Treslin, a TopBP1-interacting protein, is necessary for deoxyribonucleic acid (DNA) replication in vertebrates. Association between Treslin and TopBP1 requires cyclin-dependent kinase (Cdk) activity in Xenopus laevis egg extracts. We investigated the mechanism and functional importance of Cdk for this interaction using both X. laevis egg extracts and human cells. We found that Treslin also associated with TopBP1 in a Cdk-regulated manner in human cells and that Treslin was phosphorylated within a conserved Cdk consensus target sequence (on S976 in X. laevis and S1000 in humans). Recombinant human Cdk2–cyclin E also phosphorylated this residue of Treslin in vitro very effectively. Moreover, a mutant of Treslin that cannot undergo phosphorylation on this site showed significantly diminished binding to TopBP1. Finally, human cells harboring this mutant were severely deficient in DNA replication. Collectively, these results indicate that Cdk-mediated phosphorylation of Treslin during S phase is necessary for both its effective association with TopBP1 and its ability to promote DNA replication in human cells.

scholarly journals How cyclin A destruction escapes the spindle assembly checkpoint

2010 ◽  
Vol 190 (4) ◽  
pp. 501-509 ◽  
Author(s):  
Barbara Di Fiore ◽  
Jonathon Pines
Keyword(s):  
Ubiquitin Ligase ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Cyclin A ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
N Terminus ◽  
Specific Proteins ◽  
Necessary And Sufficient

The anaphase-promoting complex/cyclosome (APC/C) is the ubiquitin ligase essential to mitosis, which ensures that specific proteins are degraded at specific times to control the order of mitotic events. The APC/C coactivator, Cdc20, is targeted by the spindle assembly checkpoint (SAC) to restrict APC/C activity until metaphase, yet early substrates, such as cyclin A, are degraded in the presence of the active checkpoint. Cdc20 and the cyclin-dependent kinase cofactor, Cks, are required for cyclin A destruction, but how they enable checkpoint-resistant destruction has not been elucidated. In this study, we answer this problem: we show that the N terminus of cyclin A binds directly to Cdc20 and with sufficient affinity that it can outcompete the SAC proteins. Subsequently, the Cks protein is necessary and sufficient to promote cyclin A degradation in the presence of an active checkpoint by binding cyclin A–Cdc20 to the APC/C.

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