scholarly journals Fine Tuning the Cell Cycle: Activation of the Cdk1 Inhibitory Phosphorylation Pathway during Mitotic Exit

2009 ◽  
Vol 20 (6) ◽  
pp. 1737-1748 ◽  
Author(s):  
Tamara A. Potapova ◽  
John R. Daum ◽  
Kendra S. Byrd ◽  
Gary J. Gorbsky
Keyword(s):  
Cell Cycle ◽  
Phosphorylation Site ◽  
Mitotic Exit ◽  
Fine Tuning ◽  
Cyclin B ◽  
Cyclin Dependent Kinase ◽  
Cell Cycle Activation ◽  
M Phase ◽  
Dependent Cell ◽  
Chemical Inhibition

Inactivation of cyclin-dependent kinase (Cdk) 1 promotes exit from mitosis and establishes G1. Proteolysis of cyclin B is the major known mechanism that turns off Cdk1 during mitotic exit. Here, we show that mitotic exit also activates pathways that catalyze inhibitory phosphorylation of Cdk1, a mechanism previously known to repress Cdk1 only during S and G2 phases of the cell cycle. We present evidence that down-regulation of Cdk1 activates Wee1 and Myt1 kinases and inhibits Cdc25 phosphatase during the M to G1 transition. If cyclin B/Cdk1 complex is present in G1, the inhibitory sites on Cdk1 become phosphorylated. Exit from mitosis induced by chemical Cdk inhibition can be reversed if cyclin B is preserved. However, this reversibility decreases with time after mitotic exit despite the continued presence of the cyclin. We show that this G1 block is due to phosphorylation of Cdk1 on inhibitory residues T14 and Y15. Chemical inhibition of Wee1 and Myt1 or expression of Cdk1 phosphorylation site mutants allows reversal to M phase even from late G1. This late Cdk1 reactivation often results in caspase-dependent cell death. Thus, in G1, the Cdk inhibitory phosphorylation pathway is functional and can lock Cdk1 in the inactive state.

scholarly journals Canonical and Alternative Pathways in Cyclin-Dependent Kinase 1/Cyclin B Inactivation upon M-Phase Exit in Xenopus laevis Cell-Free Extracts

2011 ◽  
Vol 2011 ◽  
pp. 1-8
Author(s):  
Jacek Z. Kubiak ◽  
Mohammed El Dika
Keyword(s):  
Cell Cycle ◽  
Xenopus Laevis ◽  
Cell Cycle Progression ◽  
Cyclin B ◽  
Cyclin Dependent Kinase ◽  
Cycle Progression ◽  
Alternative Pathways ◽  
Global View ◽  
M Phase

Cyclin-Dependent Kinase 1 (CDK1) is the major M-phase kinase known also as the M-phase Promoting Factor or MPF. Studies performed during the last decade have shown many details of how CDK1 is regulated and also how it regulates the cell cycle progression. Xenopus laevis cell-free extracts were widely used to elucidate the details and to obtain a global view of the role of CDK1 in M-phase control. CDK1 inactivation upon M-phase exit is a primordial process leading to the M-phase/interphase transition during the cell cycle. Here we discuss two closely related aspects of CDK1 regulation in Xenopus laevis cell-free extracts: firstly, how CDK1 becomes inactivated and secondly, how other actors, like kinases and phosphatases network and/or specific inhibitors, cooperate with CDK1 inactivation to assure timely exit from the M-phase.

scholarly journals Genetic and Biochemical Evaluation of the Importance of Cdc6 in Regulating Mitotic Exit

2003 ◽  
Vol 14 (11) ◽  
pp. 4592-4604 ◽  
Author(s):  
Vincent Archambault ◽  
Caihong X. Li ◽  
Alan J. Tackett ◽  
Ralph Wäsch ◽  
Brian T. Chait ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Mitotic Exit ◽  
Terminal Region ◽  
Cyclin Dependent Kinase ◽  
Replication Control ◽  
Mitotic Cyclin ◽  
Biochemical Evaluation ◽  
Inhibitory Domain ◽  
Late Mitosis ◽  
Control Protein

We evaluated the hypothesis that the N-terminal region of the replication control protein Cdc6 acts as an inhibitor of cyclin-dependent kinase (Cdk) activity, promoting mitotic exit. Cdc6 accumulation is restricted to the period from mid-cell cycle until the succeeding G1, due to proteolytic control that requires the Cdc6 N-terminal region. During late mitosis, Cdc6 is present at levels comparable with Sic1 and binds specifically to the mitotic cyclin Clb2. Moderate overexpression of Cdc6 promotes viability of CLB2Δdb strains, which otherwise arrest at mitotic exit, and rescue is dependent on the N-terminal putative Cdk-inhibitory domain. These observations support the potential for Cdc6 to inhibit Clb2-Cdk, thus promoting mitotic exit. Consistent with this idea, we observed a cytokinesis defect in cdh1Δ sic1Δ cdc6Δ2–49 triple mutants. However, we were able to construct viable strains, in three different backgrounds, containing neither SIC1 nor the Cdc6 Cdk-inhibitory domain, in contradiction to previous work. We conclude, therefore, that although both Cdc6 and Sic1 have the potential to facilitate mitotic exit by inhibiting Clb2-Cdk, mitotic exit nevertheless does not require any identified stoichiometric inhibitor of Cdk activity.

scholarly journals Akt/Protein Kinase B-Dependent Phosphorylation and Inactivation of WEE1Hu Promote Cell Cycle Progression at G2/M Transition

2005 ◽  
Vol 25 (13) ◽  
pp. 5725-5737 ◽  
Author(s):  
Kazuhiro Katayama ◽  
Naoya Fujita ◽  
Takashi Tsuruo
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Kinase Inhibitor ◽  
Phosphorylation Site ◽  
Cytoplasmic Localization ◽  
M Cell ◽  
Serine Threonine Kinase ◽  
Cycle Progression ◽  
M Phase ◽  
Promote Cell Cycle Progression

ABSTRACT The serine/threonine kinase Akt is known to promote cell growth by regulating the cell cycle in G1 phase through activation of cyclin/Cdk kinases and inactivation of Cdk inhibitors. However, how the G2/M phase is regulated by Akt remains unclear. Here, we show that Akt counteracts the function of WEE1Hu. Inactivation of Akt by chemotherapeutic drugs or the phosphatidylinositide-3-OH kinase inhibitor LY294002 induced G2/M arrest together with the inhibitory phosphorylation of Cdc2. Because the increased Cdc2 phosphorylation was completely suppressed by wee1hu gene silencing, WEE1Hu was associated with G2/M arrest induced by Akt inactivation. Further analyses revealed that Akt directly bound to and phosphorylated WEE1Hu during the S to G2 phase. Serine-642 was identified as an Akt-dependent phosphorylation site. WEE1Hu kinase activity was not affected by serine-642 phosphorylation. We revealed that serine-642 phosphorylation promoted cytoplasmic localization of WEE1Hu. The nuclear-to-cytoplasmic translocation was mediated by phosphorylation-dependent WEE1Hu binding to 14-3-3θ but not 14-3-3β or -σ. These results indicate that Akt promotes G2/M cell cycle progression by inducing phosphorylation-dependent 14-3-3θ binding and cytoplasmic localization of WEE1Hu.

scholarly journals Disruption of Centrosome Structure, Chromosome Segregation, and Cytokinesis by Misexpression of Human Cdc14A Phosphatase

2002 ◽  
Vol 13 (7) ◽  
pp. 2289-2300 ◽  
Author(s):  
Brett K. Kaiser ◽  
Zachary A. Zimmerman ◽  
Harry Charbonneau ◽  
Peter K. Jackson
Keyword(s):  
Cell Cycle ◽  
Phosphatase Activity ◽  
Chromosome Segregation ◽  
Mitotic Spindle ◽  
Genomic Stability ◽  
Mitotic Exit ◽  
Protein Abundance ◽  
Cyclin Dependent Kinase ◽  
Chromosome Partitioning

In budding yeast, the Cdc14p phosphatase activates mitotic exit by dephosphorylation of specific cyclin-dependent kinase (Cdk) substrates and seems to be regulated by sequestration in the nucleolus until its release in mitosis. Herein, we have analyzed the two human homologs of Cdc14p, hCdc14A and hCdc14B. We demonstrate that the human Cdc14A phosphatase is selective for Cdk substrates in vitro and that although the protein abundance and intrinsic phosphatase activity of hCdc14A and B vary modestly during the cell cycle, their localization is cell cycle regulated. hCdc14A dynamically localizes to interphase but not mitotic centrosomes, and hCdc14B localizes to the interphase nucleolus. These distinct patterns of localization suggest that each isoform of human Cdc14 likely regulates separate cell cycle events. In addition, hCdc14A overexpression induces the loss of the pericentriolar markers pericentrin and γ-tubulin from centrosomes. Overproduction of hCdc14A also causes mitotic spindle and chromosome segregation defects, defective karyokinesis, and a failure to complete cytokinesis. Thus, the hCdc14A phosphatase appears to play a role in the regulation of the centrosome cycle, mitosis, and cytokinesis, thereby influencing chromosome partitioning and genomic stability in human cells.

The Polo-like kinase Plx1 is a component of the MPF amplification loop at the G2/M-phase transition of the cell cycle in Xenopus eggs

1998 ◽  
Vol 111 (12) ◽  
pp. 1751-1757 ◽  
Author(s):  
A. Abrieu ◽  
T. Brassac ◽  
S. Galas ◽  
D. Fisher ◽  
J.C. Labbe ◽  
...  
Keyword(s):  
Phase Transition ◽  
Cell Cycle ◽  
Cyclin A ◽  
Cyclin B ◽  
Cdc2 Kinase ◽  
C Terminus ◽  
M Phase ◽  
Egg Extracts ◽  
Amplification Loop

We have investigated whether Plx1, a kinase recently shown to phosphorylate cdc25c in vitro, is required for activation of cdc25c at the G2/M-phase transition of the cell cycle in Xenopus. Using immunodepletion or the mere addition of an antibody against the C terminus of Plx1, which suppressed its activation (not its activity) at G2/M, we show that Plx1 activity is required for activation of cyclin B-cdc2 kinase in both interphase egg extracts receiving recombinant cyclin B, and cycling extracts that spontaneously oscillate between interphase and mitosis. Furthermore, a positive feedback loop allows cyclin B-cdc2 kinase to activate Plx1 at the G2/M-phase transition. In contrast, activation of cyclin A-cdc2 kinase does not require Plx1 activity, and cyclin A-cdc2 kinase fails to activate Plx1 and its consequence, cdc25c activation in cycling extracts.

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 Phosphorylation of ORC2 Protein Dissociates Origin Recognition Complex from Chromatin and Replication Origins

2012 ◽  
Vol 287 (15) ◽  
pp. 11891-11898 ◽  
Author(s):  
Kyung Yong Lee ◽  
Sung Woong Bang ◽  
Sang Wook Yoon ◽  
Seung-Hoon Lee ◽  
Jong-Bok Yoon ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Origin Recognition Complex ◽  
S Phase ◽  
Cyclin Dependent Kinase ◽  
Replication Origins ◽  
Eukaryotic Chromosome ◽  
M Phase ◽  
Cycle Dependent ◽  
Prereplicative Complex ◽  
Origin Recognition

During the late M to the G1 phase of the cell cycle, the origin recognition complex (ORC) binds to the replication origin, leading to the assembly of the prereplicative complex for subsequent initiation of eukaryotic chromosome replication. We found that the cell cycle-dependent phosphorylation of human ORC2, one of the six subunits of ORC, dissociates ORC2, -3, -4, and -5 (ORC2–5) subunits from chromatin and replication origins. Phosphorylation at Thr-116 and Thr-226 of ORC2 occurs by cyclin-dependent kinase during the S phase and is maintained until the M phase. Phosphorylation of ORC2 at Thr-116 and Thr-226 dissociated the ORC2–5 from chromatin. Consistent with this, the phosphomimetic ORC2 protein exhibited defective binding to replication origins as well as to chromatin, whereas the phosphodefective protein persisted in binding throughout the cell cycle. These results suggest that the phosphorylation of ORC2 dissociates ORC from chromatin and replication origins and inhibits binding of ORC to newly replicated DNA.

scholarly journals Endoreduplication in Maize Endosperm: An Approach for Increasing Crop Productivity

2000 ◽  
Author(s):  
Gideon Grafi ◽  
Brian Larkins
Keyword(s):  
Cell Cycle ◽  
Dna Synthesis ◽  
Storage Protein ◽  
Molecular Mechanisms ◽  
S Phase ◽  
Cyclin B ◽  
Regulatory Subunit ◽  
Maize Endosperm ◽  
M Phase

The focus of this research project is to investigate the role of endoreduplication in maize endosperm development and the extent to which this process contributes to high levels of starch and storage protein synthesis. Although endoreduplication has been widely observed in many cells and tissues, especially those with high levels of metabolic activity, the molecular mechanisms through which the cell cycle is altered to produce consecutive cycles of S-phase without an intervening M-phase are unknown. Our previous research has shown that changes in the expression of several cell cycle regulatory genes coincide with the onset of endoreduplication. During this process, there is a sharp reduction in the activity of the mitotic cyclin-dependent kinase (CDK) and activation of the S-phase CDK. It appears the M-phase CDK is stable, but its activity is blocked by a proteinaceous inhibitor. Coincidentally, the S-phase checkpoint protein, retinoblastoma (ZmRb), becomes phosphorylated, presumably releasing an E2F-type transcriptional regulator which promotes the expression of genes responsible for DNA synthesis. To investigate the role of these cell cycle proteins in endoreduplication, we have created transgenic maize plants that express various genes in an endosperm-specific manner using a storage protein (g-zein) promoter. During the first year of the grant, we constructed point mutations of the maize M-phase kinase, p34cdc2. One alteration replaced aspartic acid at position 146 with asparagine (p3630-CdcD146N), while another changed threonine 161 to alanine (p3630-CdcT161A). These mutations abolish the activity of the CDK. We hypothesized that expression of the mutant forms of p34cdc2 in endoreduplicating endosperm, compared to a control p34cdc2, would lead to extra cycles of DNA synthesis. We also fused the gene encoding the regulatory subunit of the M- phase kinase, cyclin B, under the g-zein promoter. Normally, cyclin B is expected to be destroyed prior to the onset of endoreduplication. By producing high levels of this protein in developing endosperm, we hypothesized that the M-phase would be extended, potentially reducing the number of cycles of endoreduplication. Finally, we genetically engineered the wheat dwarf virus RepA protein for endosperm-specific expression. RepA binds to the maize retinoblastoma protein and presumably releases E2F-like transcription factors that activate DNA synthesis. We anticipated that inactivation of ZmRb by RepA would lead to additional cycles of DNA synthesis.

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