Study of the higher eukaryotic gene function CDK2 using fission yeast

1994 ◽  
Vol 107 (3) ◽  
pp. 615-623 ◽  
Author(s):  
J. Paris ◽  
P. Leplatois ◽  
P. Nurse
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Fission Yeast ◽  
Temperature Sensitive ◽  
Protein Kinase Activity ◽  
Cycle Arrest ◽  
Eukaryotic Gene ◽  
G2 Block ◽  
New Gene ◽  
Transition Points

In the fission yeast Schizosaccharomyces pombe, cdc2 function is required both in G1 to enter the cell cycle and in G2 to initiate mitosis. In higher eukaryotes, these functions appeared to be shared between several cdc2-like genes including CDK2. Temperature-sensitive mutations in S. pombe cdc2 that arrest the cell cycle in both G1 and G2 phases are not complemented by CDK2. We have used S. pombe to investigate what functions CDK2 can perform. We found that overexpression of the human homologue (HsCDK2) caused cell cycle arrest in G2/M showing that HsCDK2 interfered with mitotic events. Xenopus CDK2 (XlCDK2) overexpression did not cause cell cycle arrest and could rescue the G1 block but not the G2 block of a cdc2-M26 ts strain. A mutant XlCDK2-R33, which is inactive as a kinase, failed to rescue the G1 block, suggesting that the protein kinase activity of CDK2 is required to enter the cell cycle in these circumstances. We designed screens to select mutants that would require XlCDK2 expression for viability, hoping to isolate new gene functions interacting with, or that could be replaced by, XlCDK2 in G1, or new cdc2 mutants altered solely in their G1 role. From these screens several cell cycle mutants were selected that were XlCDK2-dependent. These were all cdc2 mutants altered only in their G2/M function. Therefore XlCDK2 can influence both the G1/S and G2/M transition points of cdc2 in S. pombe.

scholarly journals Functions of Fission Yeast Orp2 in DNA Replication and Checkpoint Control

Genetics ◽  
2000 ◽  
Vol 154 (2) ◽  
pp. 599-607
Author(s):  
Joan Kiely ◽  
S B Haase ◽  
Paul Russell ◽  
Janet Leatherwood
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Cell Cycle Arrest ◽  
Fission Yeast ◽  
Replication Initiation ◽  
Initiation Factor ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Checkpoint Control ◽  
Cycle Arrest

Abstract orp2 is an essential gene of the fission yeast Schizosaccharomyces pombe with 22% identity to budding yeast ORC2. We isolated temperature-sensitive alleles of orp2 using a novel plasmid shuffle based on selection against thymidine kinase. Cells bearing the temperature-sensitive allele orp2-2 fail to complete DNA replication at a restrictive temperature and undergo cell cycle arrest. Cell cycle arrest depends on the checkpoint genes rad1 and rad3. Even when checkpoint functions are wild type, the orp2-2 mutation causes high rates of chromosome and plasmid loss. These phenotypes support the idea that Orp2 is a replication initiation factor. Selective spore germination allowed analysis of orp2 deletion mutants. These experiments showed that in the absence of orp2 function, cells proceed into mitosis despite a lack of DNA replication. This suggests either that the Orp2 protein is a part of the checkpoint machinery or more likely that DNA replication initiation is required to induce the replication checkpoint signal.

scholarly journals Fission Yeast Rad26 Is a Regulatory Subunit of the Rad3 Checkpoint Kinase

2002 ◽  
Vol 13 (2) ◽  
pp. 480-492 ◽  
Author(s):  
Tom D. Wolkow ◽  
Tamar Enoch
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Fission Yeast ◽  
Complex Analysis ◽  
Kinase Activity ◽  
Regulatory Subunit ◽  
Kinase Activation ◽  
Checkpoint Proteins ◽  
Cycle Arrest ◽  
Phosphoinositide 3 Kinase

Fission yeast Rad3 is a member of a family of phosphoinositide 3-kinase -related kinases required for the maintenance of genomic stability in all eukaryotic cells. In fission yeast, Rad3 regulates the cell cycle arrest and recovery activities associated with the G2/M checkpoint. We have developed an assay that directly measures Rad3 kinase activity in cells expressing physiological levels of the protein. Using the assay, we demonstrate directly that Rad3 kinase activity is stimulated by checkpoint signals. Of the five other G2/M checkpoint proteins (Hus1, Rad1, Rad9, Rad17, and Rad26), only Rad26 was required for Rad3 kinase activity. Because Rad26 has previously been shown to interact constitutively with Rad3, our results demonstrate that Rad26 is a regulatory subunit, and Rad3 is the catalytic subunit, of the Rad3/Rad26 kinase complex. Analysis of Rad26/Rad3 kinase activation in rad26.T12, a mutant that is proficient for cell cycle arrest, but defective in recovery, suggests that these two responses to checkpoint signals require quantitatively different levels of kinase activity from the Rad3/Rad26 complex.

scholarly journals Characterization of p96h2bk: immunoreaction with an anti-Erk(extracellular-signal-regulated kinase) peptide antibody and activity in Xenopus oocytes and eggs

1998 ◽  
Vol 335 (1) ◽  
pp. 43-50
Author(s):  
Dong-Hua CHEN ◽  
Chin-Tin CHEN ◽  
Yong ZHANG ◽  
Mei-Ann LIU ◽  
Roberto CAMPOS-GONZALEZ ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Xenopus Oocytes ◽  
Protein Kinase Activity ◽  
Cell Stage ◽  
Extracellular Signal Regulated Kinase ◽  
Oncogenic Ras ◽  
Cycle Arrest ◽  
Extracellular Signal ◽  
M Phase

We have shown previously that oncogenic Ras induces cell cycle arrest in activated Xenopus egg extracts [Pan, Chen and Lin (1994) J. Biol. Chem. 269, 5968–5975]. The cell cycle arrest correlates with the stimulation of a protein kinase activity that phosphorylates histone H2b in vitro (designated p96h2bk) [Chen and Pan (1994) J. Biol. Chem. 269, 28034–28043]. We report here that p96h2bk is likely to be p96ram, a protein of approx. 96 kDa that immunoreacts with a monoclonal antibody (Mk-1) raised against a synthetic peptide derived from a sequence highly conserved in Erk1/Erk2 (where Erk is extracellular-signal-regulated kinase). This is supported by two lines of evidence. First, activation/inactivation of p96h2bk correlates with upward/downward bandshifts of p96ram in polyacrylamide gels. Secondly, both p96h2bk and p96ram can be immunoprecipitated by antibody Mk-1. We also studied the activity of p96h2bk/p96ram in Xenopus oocytes and eggs. p96h2bk/p96ram was inactive in stage 6 oocytes, was active in unfertilized eggs, and became inactive again in eggs after fertilization. Since stage 6 oocytes are at G2-phase of the cell cycle, unfertilized eggs arrest at M-phase and eggs exit M-phase arrest after fertilization, the results thus indicate that p96h2bk/p96ram activity is cell cycle dependent. Moreover, microinjection of oncogenic Ras into fertilized eggs at the one-cell stage arrests the embryos at the two-cell stage, and this induced arrest is correlated with an inappropriate activation of p96h2bk/p96ram. The data are consistent with the concept that inappropriate activation of p96h2bk/p96ram plays a role in the cell cycle arrest induced by oncogenic Ras.

scholarly journals A single-point mutation in HCF causes temperature-sensitive cell-cycle arrest and disrupts VP16 function.

1997 ◽  
Vol 11 (6) ◽  
pp. 726-737 ◽  
Author(s):  
H Goto ◽  
S Motomura ◽  
A C Wilson ◽  
R N Freiman ◽  
Y Nakabeppu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Point Mutation ◽  
Single Point ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Sensitive Cell ◽  
Cycle Arrest

scholarly journals The Role of the RACK1 Ortholog Cpc2p in Modulating Pheromone-Induced Cell Cycle Arrest in Fission Yeast

PLoS ONE ◽  
2013 ◽  
Vol 8 (7) ◽  
pp. e65927 ◽  
Author(s):  
Magdalena Mos ◽  
Manuel A. Esparza-Franco ◽  
Emma L. Godfrey ◽  
Kathryn Richardson ◽  
John Davey ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Fission Yeast ◽  
Cycle Arrest

scholarly journals Id2 specifically alters regulation of the cell cycle by tumor suppressor proteins.

1996 ◽  
Vol 16 (6) ◽  
pp. 2570-2578 ◽  
Author(s):  
A Lasorella ◽  
A Iavarone ◽  
M A Israel
Keyword(s):  
Cell Cycle ◽  
Tumor Suppressor ◽  
Cyclin D1 ◽  
Cell Cycle Arrest ◽  
Cell Cycle Progression ◽  
D1 Protein ◽  
Protein Kinase Activity ◽  
Cycle Progression ◽  
Tumor Suppressor Proteins ◽  
Cycle Arrest

Cells which are highly proliferative typically lack expression of differentiated, lineage-specific characteristics. Id2, a member of the helix-loop-helix (HLH) protein family known to inhibit cell differentiation, binds to the retinoblastoma protein (pRb) and abolishes its growth-suppressing activity. We found that Id2 but not Id1 or Id3 was able to bind in vitro not only pRb but also the related proteins p107 and p130. Also, an association between Id2 and p107 or p130 was observed in vivo in transiently transfected Saos-2 cells. In agreement with these results, expression of Id1 or Id3 did not affect the block of cell cycle progression mediated by pRb. Conversely, expression of Id2 specifically reversed the cell cycle arrest induced by each of the three members of the pRb family. Furthermore, the growth-suppressive activities of cyclin-dependent kinase inhibitors p16 and p21 were efficiently antagonized by high levels of Id2 but not by Id1 Id3. Consistent with the role of p16 as a selective inhibitor of pRb and pRb-related protein kinase activity, p16-imposed cell cycle arrest was completely abolished by Id2. Only a partial reversal of p21-induced growth suppression was observed, which correlated with the presence of a functional pRb. We also documented decreased levels of cyclin D1 protein and mRNA and the loss of cyclin D1-cdk4 complexes in cells constitutively expressing Id2. These data provide evidence for important Id2-mediated alterations in cell cycle components normally involved in the regulatory events of cell cycle progression, and they highlight a specific role for Id2 as an antagonist of multiple tumor suppressor proteins.

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