scholarly journals Cdk2 catalytic activity is essential for meiotic cell division in vivo

2016 ◽  
Vol 473 (18) ◽  
pp. 2783-2798 ◽  
Author(s):  
Sangeeta Chauhan ◽  
M. Kasim Diril ◽  
Joanna H.S. Lee ◽  
Xavier Bisteau ◽  
Vanessa Manoharan ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Kinase Activity ◽  
Gene Knockout ◽  
Mitotic Cell ◽  
Regulatory Proteins ◽  
Mouse Strains ◽  
Meiotic Cell ◽  
Independent Functions

Cyclin-dependent kinases (Cdks) control the eukaryotic cell cycle by phosphorylating serine and threonine residues in key regulatory proteins, but some Cdk family members may exert kinase-independent functions that cannot easily be assessed using gene knockout approaches. While Cdk2-deficient mice display near-normal mitotic cell proliferation due to the compensatory activities of Cdk1 and Cdk4, they are unable to undergo meiotic generation of gametes and are consequently sterile. To investigate whether Cdk2 regulates meiosis via protein phosphorylation or by alternative kinase-independent mechanisms, we generated two different knockin mouse strains in which Cdk2 point mutations ablated enzyme activity without altering protein expression levels. Mice homozygous for the mutations Cdk2D145N/D145N or Cdk2T160A/T160A expressed only ‘kinase-dead’ variants of Cdk2 under the control of the endogenous promoter, and despite exhibiting normal expression of cell cycle regulatory proteins and complexes, both mutations rendered mice sterile. Mouse cells that expressed only ‘kinase-dead’ variants of Cdk2 displayed normal mitotic cell cycle progression and proliferation both in vitro and in vivo, indicating that loss of Cdk2 kinase activity exerted little effect on this mode of cell division. In contrast, the reproductive organs of Cdk2 mutant mice exhibited abnormal morphology and impaired function associated with defective meiotic cell division and inability to produce gametes. Cdk2 mutant animals were therefore comparable to gene knockout mice, which completely lack the Cdk2 protein. Together, our data indicate that the essential meiotic functions of Cdk2 depend on its kinase activity, without which the generation of haploid cells is disrupted, resulting in sterility of otherwise healthy animals.

scholarly journals Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein.

1993 ◽  
Vol 13 (5) ◽  
pp. 2899-2908 ◽  
Author(s):  
A L Jackson ◽  
P M Pahl ◽  
K Harrison ◽  
J Rosamond ◽  
R A Sclafani
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Protein Interactions ◽  
Kinase Activity ◽  
Mitotic Cell ◽  
Common Point ◽  
Temperature Sensitive ◽  
Cdc7 Kinase

Yeast Cdc7 protein kinase and Dbf4 protein are both required for the initiation of DNA replication at the G1/S phase boundary of the mitotic cell cycle. Cdc7 kinase function is stage-specific in the cell cycle, but total Cdc7 protein levels remained unchanged. Therefore, regulation of Cdc7 function appears to be the result of posttranslational modification. In this study, we have attempted to elucidate the mechanism responsible for achieving this specific execution point of Cdc7. Cdc7 kinase activity was shown to be maximal at the G1/S boundary by using either cultures synchronized with alpha factor or Cdc- mutants or with inhibitors of DNA synthesis or mitosis. Therefore, Cdc7 kinase is regulated by a posttranslational mechanism that ensures maximal Cdc7 activity at the G1/S boundary, which is consistent with Cdc7 function in the cell cycle. This cell cycle-dependent regulation could be the result of association with the Dbf4 protein. In this study, the Dbf4 protein was shown to be required for Cdc7 kinase activity in that Cdc7 kinase activity is thermolabile in vitro when extracts prepared from a temperature-sensitive dbf4 mutant grown under permissive conditions are used. In vitro reconstitution assays, in addition to employment of the two-hybrid system for protein-protein interactions, have demonstrated that the Cdc7 and Dbf4 proteins interact both in vitro and in vivo. A suppressor mutation, bob1-1, which can bypass deletion mutations in both cdc7 and dbf4 was isolated. However, the bob1-1 mutation cannot bypass all events in G1 phase because it fails to suppress temperature-sensitive cdc4 or cdc28 mutations. This indicates that the Cdc7 and Dbf4 proteins act at a common point in the cell cycle. Therefore, because of the common point of function for the two proteins and the fact that the Dbf4 protein is essential for Cdc7 function, we propose that Dbf4 may represent a cyclin-like molecule specific for the activation of Cdc7 kinase.

scholarly journals Specific Inhibition of Elm1 Kinase Activity Reveals Functions Required for Early G1 Events

2003 ◽  
Vol 23 (17) ◽  
pp. 6327-6337 ◽  
Author(s):  
Aparna Sreenivasan ◽  
Anthony C. Bishop ◽  
Kevan M. Shokat ◽  
Douglas R. Kellogg
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Cell Growth ◽  
Kinase Activity ◽  
Budding Yeast ◽  
Specific Inhibition ◽  
Bud Emergence ◽  
Wee1 Kinase ◽  
Yeast Homolog

ABSTRACT In budding yeast, the Elm1 kinase is required for coordination of cell growth and cell division at G2/M. Elm1 is also required for efficient cytokinesis and for regulation of Swe1, the budding yeast homolog of the Wee1 kinase. To further characterize Elm1 function, we engineered an ELM1 allele that can be rapidly and selectively inhibited in vivo. We found that inhibition of Elm1 kinase activity during G2 results in a phenotype similar to the phenotype caused by deletion of the ELM1 gene, as expected. However, inhibition of Elm1 kinase activity earlier in the cell cycle results in a prolonged G1 delay. The G1 requirement for Elm1 kinase activity occurs before bud emergence, polarization of the septins, and synthesis of G1 cyclins. Inhibition of Elm1 kinase activity during early G1 also causes defects in the organization of septins, and inhibition of Elm1 kinase activity in a strain lacking the redundant G1 cyclins CLN1 and CLN2 is lethal. These results demonstrate that the Elm1 kinase plays an important role in G1 events required for bud emergence and septin organization.

scholarly journals A RAD9-dependent cell cycle arrest in response to unresolved recombination intermediates in Saccharomyces cerevisiae

2019 ◽  
Author(s):  
Hardeep Kaur ◽  
GN Krishnaprasad ◽  
Michael Lichten
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Dna Damage ◽  
Cell Division ◽  
Cell Cycle Arrest ◽  
Mitotic Cell ◽  
Cycle Arrest ◽  
Associated Lesions

AbstractIn Saccharomyces cerevisiae, the conserved Sgs1-Top3-Rmi1 helicase-decatenase regulates homologous recombination by limiting accumulation of recombination intermediates that are precursors of crossovers. In vitro studies have suggested that the dissolution of double-Holliday junction joint molecules by Sgs1-driven convergent junction migration and Top3-Rmi1 mediated strand decatenation could be responsible for this. To ask if dissolution occurs in vivo, we conditionally depleted Sgs1 and/or Rmi1 during return to growth, a procedure where recombination intermediates formed during meiosis are resolved when cells resume the mitotic cell cycle. Sgs1 depletion during return to growth delayed joint molecule resolution, but ultimately most were resolved and cells divided normally. In contrast, Rmi1 depletion resulted in delayed and incomplete joint molecule resolution, and most cells did not divide. rad9Δ mutation restored cell division in Rmi1-depleted cells, indicating that the DNA damage checkpoint caused this cell cycle arrest. Restored cell division in rad9Δ, Rmi1-depleted cells frequently produced anucleate cells, consistent with the suggestion that persistent recombination intermediates prevented chromosome segregation. Our findings indicate that Sgs1-Top3-Rmi1 acts in vivo, as it does in vitro, to promote recombination intermediate resolution by dissolution. They also indicate that, in the absence of Top3-Rmi1 activity, unresolved recombination intermediates persist and activate the DNA damage response, which is usually thought to be activated by much earlier DNA damage-associated lesions.

Differential function and expression of Saccharomyces cerevisiae B-type cyclins in mitosis and meiosis

1993 ◽  
Vol 13 (4) ◽  
pp. 2113-2125
Author(s):  
N Grandin ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Kinase Activity ◽  
Mitotic Cell ◽  
S Phase ◽  
Mitotic Cell Cycle ◽  
Meiotic Cell ◽  
Cell Cycles ◽  
M Phase ◽  
Mitotic Cyclin

We have studied the patterns of expression of four B-type cyclins (Clbs), Clb1, Clb2, Clb3, and Clb4, and their ability to activate p34cdc28 during the mitotic and meiotic cell cycles of Saccharomyces cerevisiae. During the mitotic cell cycle, Clb3 and Clb4 were expressed and induced a kinase activity in association with p34cdc28 from early S phase up to mitosis. On the other hand, Clb1 and Clb2 were expressed and activated p34cdc28 later in the mitotic cell cycle, starting in late S phase and continuing up to mitosis. The pattern of expression of Clb3 and Clb4 suggests a possible role in the regulation of DNA replication as well as mitosis. Clb1 and Clb2, whose pattern of expression is similar to that of other known Clbs, are likely to have a role predominantly in the regulation of M phase. During the meiotic cell cycle, Clb1, Clb3, and Clb4 were expressed and induced a p34cdc28-associated kinase activity just before the first meiotic division. The fact that Clb3 and Clb4 were not synthesized earlier, in S phase, suggests that these cyclins, which probably have a role in S phase during the mitotic cell cycle, are not implicated in premeiotic S phase. Clb2, the primary mitotic cyclin in S. cerevisiae, was not detectable during meiosis. Sporulation experiments on strains deleted for one, two, or three Clbs indicate, in agreement with the biochemical data, that Clb1 is the primary cyclin for the regulation of meiosis, while Clb2 is not involved at all.

scholarly journals Differential function and expression of Saccharomyces cerevisiae B-type cyclins in mitosis and meiosis.

1993 ◽  
Vol 13 (4) ◽  
pp. 2113-2125 ◽  
Author(s):  
N Grandin ◽  
S I Reed
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Cell Cycle ◽  
Kinase Activity ◽  
Mitotic Cell ◽  
S Phase ◽  
Mitotic Cell Cycle ◽  
Meiotic Cell ◽  
Cell Cycles ◽  
M Phase ◽  
Mitotic Cyclin

We have studied the patterns of expression of four B-type cyclins (Clbs), Clb1, Clb2, Clb3, and Clb4, and their ability to activate p34cdc28 during the mitotic and meiotic cell cycles of Saccharomyces cerevisiae. During the mitotic cell cycle, Clb3 and Clb4 were expressed and induced a kinase activity in association with p34cdc28 from early S phase up to mitosis. On the other hand, Clb1 and Clb2 were expressed and activated p34cdc28 later in the mitotic cell cycle, starting in late S phase and continuing up to mitosis. The pattern of expression of Clb3 and Clb4 suggests a possible role in the regulation of DNA replication as well as mitosis. Clb1 and Clb2, whose pattern of expression is similar to that of other known Clbs, are likely to have a role predominantly in the regulation of M phase. During the meiotic cell cycle, Clb1, Clb3, and Clb4 were expressed and induced a p34cdc28-associated kinase activity just before the first meiotic division. The fact that Clb3 and Clb4 were not synthesized earlier, in S phase, suggests that these cyclins, which probably have a role in S phase during the mitotic cell cycle, are not implicated in premeiotic S phase. Clb2, the primary mitotic cyclin in S. cerevisiae, was not detectable during meiosis. Sporulation experiments on strains deleted for one, two, or three Clbs indicate, in agreement with the biochemical data, that Clb1 is the primary cyclin for the regulation of meiosis, while Clb2 is not involved at all.

Cell cycle regulation of the yeast Cdc7 protein kinase by association with the Dbf4 protein

1993 ◽  
Vol 13 (5) ◽  
pp. 2899-2908 ◽  
Author(s):  
A L Jackson ◽  
P M Pahl ◽  
K Harrison ◽  
J Rosamond ◽  
R A Sclafani
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Protein Interactions ◽  
Kinase Activity ◽  
Mitotic Cell ◽  
Common Point ◽  
Temperature Sensitive ◽  
Cdc7 Kinase

Yeast Cdc7 protein kinase and Dbf4 protein are both required for the initiation of DNA replication at the G1/S phase boundary of the mitotic cell cycle. Cdc7 kinase function is stage-specific in the cell cycle, but total Cdc7 protein levels remained unchanged. Therefore, regulation of Cdc7 function appears to be the result of posttranslational modification. In this study, we have attempted to elucidate the mechanism responsible for achieving this specific execution point of Cdc7. Cdc7 kinase activity was shown to be maximal at the G1/S boundary by using either cultures synchronized with alpha factor or Cdc- mutants or with inhibitors of DNA synthesis or mitosis. Therefore, Cdc7 kinase is regulated by a posttranslational mechanism that ensures maximal Cdc7 activity at the G1/S boundary, which is consistent with Cdc7 function in the cell cycle. This cell cycle-dependent regulation could be the result of association with the Dbf4 protein. In this study, the Dbf4 protein was shown to be required for Cdc7 kinase activity in that Cdc7 kinase activity is thermolabile in vitro when extracts prepared from a temperature-sensitive dbf4 mutant grown under permissive conditions are used. In vitro reconstitution assays, in addition to employment of the two-hybrid system for protein-protein interactions, have demonstrated that the Cdc7 and Dbf4 proteins interact both in vitro and in vivo. A suppressor mutation, bob1-1, which can bypass deletion mutations in both cdc7 and dbf4 was isolated. However, the bob1-1 mutation cannot bypass all events in G1 phase because it fails to suppress temperature-sensitive cdc4 or cdc28 mutations. This indicates that the Cdc7 and Dbf4 proteins act at a common point in the cell cycle. Therefore, because of the common point of function for the two proteins and the fact that the Dbf4 protein is essential for Cdc7 function, we propose that Dbf4 may represent a cyclin-like molecule specific for the activation of Cdc7 kinase.

scholarly journals Analysis of Cell Cycle and Replication of Mouse Macrophages afterIn VivoandIn VitroCryptococcus neoformans Infection Using Laser Scanning Cytometry

2012 ◽  
Vol 80 (4) ◽  
pp. 1467-1478 ◽  
Author(s):  
Carolina Coelho ◽  
Lydia Tesfa ◽  
Jinghang Zhang ◽  
Johanna Rivera ◽  
Teresa Gonçalves ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Cytotoxic Effect ◽  
Laser Scanning ◽  
Cell Cycle Progression ◽  
Cycle Progression ◽  
Content Type ◽  
Cycle Arrest ◽  
Laser Scanning Cytometry

ABSTRACTWe investigated the outcome of the interaction ofCryptococcus neoformanswith murine macrophages using laser scanning cytometry (LSC). Previous results in our lab had shown that phagocytosis ofC. neoformanspromoted cell cycle progression. LSC allowed us to simultaneously measure the phagocytic index, macrophage DNA content, and 5-ethynyl-2′-deoxyuridine (EdU) incorporation such that it was possible to study host cell division as a function of phagocytosis. LSC proved to be a robust, reliable, and high-throughput method for quantifying phagocytosis. Phagocytosis ofC. neoformanspromoted cell cycle progression, but infected macrophages were significantly less likely to complete mitosis. Hence, we report a new cytotoxic effect associated with intracellularC. neoformansresidence that manifested itself in impaired cell cycle completion as a consequence of a block in the G2/M stage of the mitotic cell cycle. Cell cycle arrest was not due to increased cell membrane permeability or DNA damage. We investigated alveolar macrophage replicationin vivoand demonstrated that these cells are capable of low levels of cell division in the presence or absence ofC. neoformansinfection. In summary, we simultaneously studied phagocytosis, the cell cycle state of the host cell and pathogen-mediated cytotoxicity, and our results demonstrate a new cytotoxic effect ofC. neoformansinfection on murine macrophages: fungus-induced cell cycle arrest. Finally, we provide evidence for alveolar macrophage proliferationin vivo.

scholarly journals Tyrosine phosphorylation and activation of homologous protein kinases during oocyte maturation and mitogenic activation of fibroblasts.

1991 ◽  
Vol 11 (5) ◽  
pp. 2517-2528 ◽  
Author(s):  
J Posada ◽  
J Sanghera ◽  
S Pelech ◽  
R Aebersold ◽  
J A Cooper
Keyword(s):  
Cell Cycle ◽  
Map Kinase ◽  
Tyrosine Phosphorylation ◽  
Oocyte Maturation ◽  
Mammalian Cells ◽  
Map Kinases ◽  
Kinase Activity ◽  
Mitotic Cell ◽  
Meiotic Maturation ◽  
Sea Star

Meiotic maturation of Xenopus and sea star oocytes involves the activation of a number of protein-serine/threonine kinase activities, including a myelin basic protein (MBP) kinase. A 44-kDa MBP kinase (p44mpk) purified from mature sea star oocytes is shown here to be phosphorylated at tyrosine. Antiserum to purified sea star p44mpk was used to identify antigenically related proteins in Xenopus oocytes. Two tyrosine-phosphorylated 42-kDa proteins (p42) were detected with this antiserum in Xenopus eggs. Xenopus p42 chromatographs with MBP kinase activity on a Mono Q ion-exchange column. Tyrosine phosphorylation of Xenopus p42 approximately parallels MBP kinase activity during meiotic maturation. These results suggest that related MBP kinases are activated during meiotic maturation of Xenopus and sea star oocytes. Previous studies have suggested that Xenopus p42 is related to the mitogen-activated protein (MAP) kinases of culture mammalian cells. We have cloned a MAP kinase relative from a Xenopus ovary cDNA library and demonstrate that this clone encodes the Xenopus p42 that is tyrosine phosphorylated during oocyte maturation. Comparison of the sequences of Xenopus p42 and a rat MAP kinase (ERK1) and peptide sequences from sea star p44mpk indicates that these proteins are close relatives. The family members appear to be tyrosine phosphorylated, and activated, in different contexts, with the murine MAP kinase active during the transition from quiescence to the G1 stage of the mitotic cell cycle and the sea star and Xenopus kinases being active during M phase of the meiotic cell cycle.

Cyclin D: CDK4/6 Kinase Activity, Regulated by GATA-1, Is Required for Megakaryocyte Polyploidization.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 780-780
Author(s):  
Andrew G. Muntean ◽  
Liyan Pang ◽  
Mortimer Poncz ◽  
Steve Dowdy ◽  
Gerd Blobel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factors ◽  
Cyclin D1 ◽  
Kinase Activity ◽  
Fetal Liver ◽  
Cyclin D3 ◽  
Luciferase Reporter ◽  
Cell Cycle Regulators ◽  
Wild Type

Abstract Megakaryocytes, which fragment to give rise to platelets, undergo a unique form of cell cycle, termed endomitosis, to become polyploid and terminally differentiate. During this process, cells transverse the cell cycle but the late stages of mitosis are bypassed to lead to accumulation of DNA up to 128N. While the mechanisms of polyploidization in megakaryocytes are poorly understood, a few cell cycle regulators, such as cyclin D3, have been implicated in this process. Hematopoietic transcription factors, including GATA-1 and RUNX1 are also essential for polyploidization, as both GATA1-deficient and RUNX1-null megakaryocytes undergo fewer rounds of endomitosis. Interestingly, GATA-1 deficient megakaryocytes are also smaller than their wild-type counterparts. However, the link between transcription factors and the growth and polyploidization of megakaryocytes has not been established. In our studies to identify key downstream targets of GATA-1 in the megakaryocyte lineage, we discovered that the cell cycle regulators cyclin D1 and p16 were aberrantly expressed in the absence of GATA-1: cyclin D1 expression was reduced nearly 10-fold, while that of p16ink4a was increased 10-fold. Luciferase reporter assays revealed that GATA-1, but not the leukemic isoform GATA-1s, promotes cyclinD1 expression. Consistent with these observations, megakaryocytes that express GATA-1s in place of full-length GATA-1 are smaller than their wild-type counterparts. Chromatin immunoprecipitation studies revealed that GATA-1 is bound to the cyclin D1 promoter in vivo, in primary fetal liver derived megakaryocytes. In contrast, GATA-1 is not associated with the cyclin D1 promoter in erythroid cells, which do not become polyploid. Thus, cyclin D1 is a bona fide GATA-1 target gene in megakaryocytes. To investigate whether restoration of cyclin D1 expression could rescue the polyploidization defect in GATA-1 deficient cells, we infected fetal liver progenitors isolated from GATA-1 knock-down mice with retroviruses harboring the cyclin D1 cDNA (and GFP via an IRES element) or GFP alone. Surprisingly, expression of cyclin D1 did not increase the extent of polyploidization of the GATA-1 deficient megakaryocytes. However, co-overexpression of cyclin D1 and Cdk4 resulted in a dramatic increase in polyploidization. Consistent with the model that cyclinD:Cdk4/6 also regulates cellular metabolism, we observed that the size of the doubly infected cells was also significantly increased. Finally, in support of our model that cyclin D:Cdk4/6 kinase activity is essential for endomitosis, we discovered that introduction of wild-type p16 TAT fusion protein, but not a mutant that fails to interact with Cdk4/6, significantly blocked polyploidization of primary fetal liver derived megakaryocytes. Taken together, our data reveal that the process of endomitosis and cell growth relies heavily on cyclinD:Cdk4/6 kinase activity and that the maturation defects in GATA-1 deficient megakaryocytes are due, in part, to reduced Cyclin D1 and increase p16 expression.

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