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 Suppression of ARG kinase activity by STI571 induces cell cycle arrest through up-regulation of CDK inhibitor p18/INK4c

Oncogene ◽  
2003 ◽  
Vol 22 (26) ◽  
pp. 4074-4082 ◽  
Author(s):  
Noriko Nishimura ◽  
Yusuke Furukawa ◽  
Krittaya Sutheesophon ◽  
Mitsuru Nakamura ◽  
Kenji Kishi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Kinase Activity ◽  
Cdk Inhibitor ◽  
Cycle Arrest

Cdc7 inhibition as a novel approach for pancreas cancer therapy.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. e15059-e15059
Author(s):  
Mark G. Frattini ◽  
Lucia Regales ◽  
Ruth Santos ◽  
Diana Carrillo
Keyword(s):  
Cell Cycle ◽  
Pancreatic Cancer ◽  
Dna Damage ◽  
Cell Viability ◽  
Cell Cycle Arrest ◽  
Western Blotting ◽  
Kinase Activity ◽  
S Phase ◽  
Cycle Arrest ◽  
Cdc7 Kinase

e15059 Background: Pancreatic cancer is the fourth leading cause of cancer death in the USA. In 2012, 43,920 people will be diagnosed and 37,390 people will die of this disease. 95% of tumors reveal loss of the p16 protein, a regulator of the G1 to S phase transition. Cdc7 is a conserved kinase required for the initiation of DNA replication, is a target of the S-phase checkpoint, and has a role in controlling the DNA damage response. Downregulation of Cdc7 kinase activity resulted in slowing of S-phase and cell cycle arrest followed by accumulation of DNA damage. Cdc7 has been shown to be over-expressed in many different tumors including the majority of solid and liquid tumors. In our laboratory a novel natural product small molecule inhibitor (MSK-777) has been identified, developed and shown to be efficacious in cell based cytotoxicity assays and multiple animal models of cancer. Methods: We have examined the efficacy of Cdc7 kinase inhibition as a therapeutic approach for pancreatic cancer by examining the sensitivity of MSK-777 in Capan-1, BxPC3, and PANC-1 cell lines. These cells were treated with MSK-777, control (DMSO), or hydroxyurea and collected for viable cell counts, fluorescence-activated cell sorting (FACS), and western blotting. Results: Cell viability analyses revealed that MSK-777 had a dramatic effect after 24 hours, reducing cell viability to less then 20% in BxPC3 cells. FACS results demonstrated that MSK-777 exposure resulted in cell cycle arrest at G1/S in Capan-1 and PANC-1 cells by 48 hours while BxPC3 cells showed a significant sub-G1 population by 24 hours, indicating apoptotic cell death. Western blotting showed that in BxPC3 cells phosphorylation of the mini-chromosome maintenance 2 protein (Mcm2) disappeared by 24 hours, indicating inactivation of the helicase that unwinds the strands of DNA during replication. Western blots of Capan-1 and PANC-1 cells showed lower levels of phosphorylated Mcm2 by 48 hours. Conclusions: We are currently examining the efficacy of MSK-777 in mouse models of orthotopically injected pancreatic cancer cells. Based on these collective results, inhibition of Cdc7 kinase activity with MSK-777 represents a novel and promising therapy for this deadly disease.

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 A Fission Yeast Gene,him1+/dfp1+, Encoding a Regulatory Subunit for Hsk1 Kinase, Plays Essential Roles in S-Phase Initiation as Well as in S-Phase Checkpoint Control and Recovery from DNA Damage

1999 ◽  
Vol 19 (8) ◽  
pp. 5535-5547 ◽  
Author(s):  
Tadayuki Takeda ◽  
Keiko Ogino ◽  
Etsuko Matsui ◽  
Min Kwan Cho ◽  
Hiroyuki Kumagai ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Fission Yeast ◽  
Kinase Activity ◽  
S Phase ◽  
Regulatory Subunit ◽  
Checkpoint Control ◽  
Regulatory Subunits ◽  
Hydroxyurea Treatment ◽  
S Phase Checkpoint

ABSTRACT Saccharomyces cerevisiae CDC7 encodes a serine/threonine kinase required for G1/S transition, and its related kinases are present in fission yeast as well as in higher eukaryotes, including humans. Kinase activity of Cdc7 protein depends on the regulatory subunit, Dbf4, which also interacts with replication origins. We have identified him1+ from two-hybrid screening with Hsk1, a fission yeast homologue of Cdc7 kinase, and showed that it encodes a regulatory subunit of Hsk1. Him1, identical to Dfp1, previously identified as an associated molecule of Hsk1, binds to Hsk1 and stimulates its kinase activity, which phosphorylates both catalytic and regulatory subunits as well as recombinant MCM2 protein in vitro. him1+ is essential for DNA replication in fission yeast cells, and its transcription is cell cycle regulated, increasing at middle M to late G1. The protein level is low at START in G1, increases at the G1/S boundary, and is maintained at a high level throughout S phase. Him1 protein is hyperphosphorylated at G1/S through S during the cell cycle as well as in response to early S-phase arrest induced by nucleotide deprivation. Deletion of one of the motifs conserved in regulatory subunits for Cdc7-related kinases as well as alanine substitution of three serine and threonine residues present in the same motif resulted in a defect in checkpoint regulation normally induced by hydroxyurea treatment. The alanine mutant also showed growth retardation after UV irradiation and the addition of methylmethane sulfonate. In keeping with this result, a database search indicates that him1+ is identical to rad35+ . Our results reveal a novel function of the Cdc7/Dbf4-related kinase complex in S-phase checkpoint control as well as in growth recovery from DNA damage in addition to its predicted essential function in S-phase initiation.

scholarly journals The N-Terminal 24 Amino Acids of the p55 Gamma Regulatory Subunit of Phosphoinositide 3-Kinase Binds Rb and Induces Cell Cycle Arrest

2003 ◽  
Vol 23 (5) ◽  
pp. 1717-1725 ◽  
Author(s):  
Xianmin Xia ◽  
Aiwu Cheng ◽  
Damilola Akinmade ◽  
Anne W. Hamburger
Keyword(s):  
Cell Cycle ◽  
Amino Acid ◽  
Cell Cycle Arrest ◽  
Cell Cycle Progression ◽  
Molecular Mechanisms ◽  
Biological Effects ◽  
Cycle Progression ◽  
Regulatory Subunits ◽  
Cycle Arrest ◽  
Phosphoinositide 3 Kinase

ABSTRACT Although phosphoinositide 3-kinase (PI 3-kinase) is essential for cell cycle progression, the molecular mechanisms that regulate its diverse biological effects are poorly understood. We demonstrate here that Rb, a key regulator of cell cycle progression, associates with p55 kDa (p55α and p55γ) regulatory subunits of PI 3-kinase in vivo and in vitro. Both confocal microscopy and biochemical analysis demonstrated the presence of p55γ in the nucleus. The 24-amino-acid N-terminal end of p55γ, which is unique among PI 3-kinase regulatory subunits, was sufficient to bind Rb. Addition of serum or growth factors to quiescent cells triggered the dissociation of Rb from p55. Ectopic expression of the 24-amino-acid N-terminal end of p55γ inhibited cell cycle progression, as evidenced by induction of cell growth arrest at the G0/G1 phase, inhibition of DNA synthesis, inhibition of cyclin D and cyclin E promoter activity, and changes in the expression of cell cycle-related proteins. The inhibitory effects of the N-terminal end of p55γ on cell cycle progression depended on the presence of functional Rb. These data demonstrate for the first time an association of p55γ with Rb and show that modification of this association can lead to cell cycle arrest.

scholarly journals Requirement for p27KIP1 in Retinoblastoma Protein-Mediated Senescence

2001 ◽  
Vol 21 (11) ◽  
pp. 3616-3631 ◽  
Author(s):  
Kamilah Alexander ◽  
Philip W. Hinds
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Cyclin E ◽  
Ectopic Expression ◽  
Retinoblastoma Protein ◽  
Morphological Transformation ◽  
Cycle Arrest

ABSTRACT In vivo and in vitro evidence indicate that cells do not divide indefinitely but instead stop growing and undergo a process termed cellular proliferative senescence. Very little is known about how senescence occurs, but there are several indications that the retinoblastoma protein (pRb) is involved, the most striking being that reintroduction of RB into RB −/−tumor cell lines induces senescence. In investigating the mechanism by which pRb induces senescence, we have found that pRb causes a posttranscriptional accumulation of the cyclin-dependent kinase inhibitor p27KIP1 that is accompanied by an increase in p27KIP1 specifically bound to cyclin E and a concomitant decrease in cyclin E-associated kinase activity. In contrast, pRb-related proteins p107 and p130, which also decrease cyclin E-kinase activity, do not cause an accumulation of p27KIP1 and induce senescence poorly. In addition, the use of pRb proteins mutated in the pocket domain demonstrates that pRb upregulation of p27KIP1 and senescence induction do not require the interaction of pRb with E2F. Furthermore, ectopic expression of p21CIP1 or p27KIP1 induces senescence but not the morphology change associated with pRb-mediated senescence, uncoupling senescence from the morphological transformation. Finally, the ability of pRb to maintain cell cycle arrest and induce senescence is reversibly abrogated by ablation of p27KIP1 expression. These findings suggest that prolonged cell cycle arrest through the persistent and specific inhibition of cdk2 activity by p27KIP1 is critical for pRb-induced senescence.

scholarly journals AP-1 blockade in breast cancer cells causes cell cycle arrest by suppressing G1 cyclin expression and reducing cyclin-dependent kinase activity

Oncogene ◽  
2004 ◽  
Vol 23 (50) ◽  
pp. 8238-8246 ◽  
Author(s):  
Yongmin Liu ◽  
Chunhua Lu ◽  
Qiang Shen ◽  
Debbie Munoz-Medellin ◽  
Heetae Kim ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Cancer Cells ◽  
Breast Cancer Cells ◽  
Kinase Activity ◽  
Cyclin Dependent Kinase ◽  
Cycle Arrest ◽  
G1 Cyclin
Sign in / Sign up

Export Citation Format

Share Document