scholarly journals The Wee1 protein kinase regulates T14 phosphorylation of fission yeast Cdc2.

1995 ◽  
Vol 6 (4) ◽  
pp. 371-385 ◽  
Author(s):  
G J Den Haese ◽  
N Walworth ◽  
A M Carr ◽  
K L Gould
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Fission Yeast ◽  
Protein Kinases ◽  
M Cell ◽  
Replication Checkpoint ◽  
Hydroxyurea Treatment ◽  
Normal Duration ◽  
Cdc25 Protein

The Cdc2 protein kinase is a key regulator of the G1-S and G2-M cell cycle transitions in the fission yeast Schizosaccharomyces pombe. The activation of Cdc2 at the G2-M transition is triggered by dephosphorylation at a conserved tyrosine residue Y15. The level of Y15 phosphorylation is controlled by the Wee1 and Mik1 protein kinases acting in opposition to the Cdc25 protein phosphatase. Here, we demonstrate that Wee1 overexpression leads to a high stoichiometry of phosphorylation at a previously undetected site in S. pombe Cdc2, T14. T14 phosphorylation was also detected in certain cell cycle mutants blocked in progression through S phase, indicating that T14 phosphorylation might normally occur at low stoichiometry during DNA replication or early G2. Strains in which the chromosomal copy of cdc2 was replaced with either a T14A or a T14S mutant allele were generated and the phenotypes of these strains are consistent with T14 phosphorylation playing an inhibitory role in the activation of Cdc2 as it does in higher eukaryotes. We have also obtained evidence that Wee1 but not Mik1 or Chk1 is required for phosphorylation at this site, that the Mik1 and Chk1 protein kinases are unable to drive T14 phosphorylation in vivo, that residue 14 phosphorylation requires previous phosphorylation at Y15, and that the T14A mutant, unlike Y15F, is recessive to wild-type Cdc2 activity. Finally, the normal duration of G2 delay after irradiation or hydroxyurea treatment in a T14A mutant strain indicates that T14 phosphorylation is not required for the DNA damage or replication checkpoint controls.

scholarly journals The Cdc2 protein kinase controls Cdc10/Sct1 complex formation.

1997 ◽  
Vol 8 (6) ◽  
pp. 1105-1115 ◽  
Author(s):  
T Connolly ◽  
M Caligiuri ◽  
D Beach
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Complex Formation ◽  
Fission Yeast ◽  
Biochemical Analysis ◽  
Binding Activity ◽  
G1 Arrest ◽  
Dna Binding Activity ◽  
Cdc2 Activity

In the fission yeast Schizosaccharomyces pombe, the execution of Start requires the activity of the Cdc2 protein kinase and the Cdc10/Sct1 transcription complex. The loss of any of these genes leads to G1 arrest and activation of the mating pathway under appropriate conditions. We have undertaken a genetic and biochemical analysis of these genes and their protein products to elucidate the molecular mechanism that governs the regulation of Start. We demonstrate that serine-196 of Cdc10 is phosphorylated in vivo and provide evidence that suggests that phosphorylation of this residue is required for Cdc10 function. Substitution of serine-196 of Cdc10 with alanine (Cdc10 S196A) leads to inactivation of Cdc10. We show that Cdc10 S196A is incapable of associating with Sct1 to form a heteromeric complex, whereas substitution of this serine with aspartic acid (S196D) restores DNA-binding activity by allowing Cdc10 to associate with Sct1. Furthermore, we demonstrate that Cdc2 activity is required for the formation of the heteromeric Sct1/Cdc10 transcription complex and that the Cdc10 S196D mutation alleviates this requirement. We thus provide biochemical evidence to demonstrate one mechanism by which the Cdc2 protein kinase may regulate Start in the fission yeast cell cycle.

scholarly journals The Direct Binding of Mrc1, a Checkpoint Mediator, to Mcm6, a Replication Helicase, Is Essential for the Replication Checkpoint against Methyl Methanesulfonate-Induced Stress

2009 ◽  
Vol 29 (18) ◽  
pp. 5008-5019 ◽  
Author(s):  
Makiko Komata ◽  
Masashige Bando ◽  
Hiroyuki Araki ◽  
Katsuhiko Shirahige
Keyword(s):  
Amino Acid ◽  
Dna Replication ◽  
Terminal Region ◽  
Methyl Methanesulfonate ◽  
Checkpoint Activation ◽  
Replication Checkpoint ◽  
Hydroxyurea Treatment ◽  
Dna Replication Checkpoint ◽  
Direct Binding

ABSTRACT Mrc1 plays a role in mediating the DNA replication checkpoint. We surveyed replication elongation proteins that interact directly with Mrc1 and identified a replicative helicase, Mcm6, as a specific Mrc1-binding protein. The central portion of Mrc1, containing a conserved coiled-coil region, was found to be essential for interaction with the 168-amino-acid C-terminal region of Mcm6, and introduction of two amino acid substitutions in this C-terminal region abolished the interaction with Mrc1 in vivo. An mcm6 mutant bearing these substitutions showed a severe defect in DNA replication checkpoint activation in response to stress caused by methyl methanesulfonate. Interestingly, the mutant did not show any defect in DNA replication checkpoint activation in response to hydroxyurea treatment. The phenotype of the mcm6 mutant was suppressed when the mutant protein was physically fused with Mrc1. These results strongly suggest for the first time that an Mcm helicase acts as a checkpoint sensor for methyl methanesulfonate-induced DNA damage through direct binding to the replication checkpoint mediator Mrc1.

scholarly journals Human Enhancer of Invasion-Cluster, a Coiled-Coil Protein Required for Passage through Mitosis

2004 ◽  
Vol 24 (9) ◽  
pp. 3957-3971 ◽  
Author(s):  
Margret B. Einarson ◽  
Edna Cukierman ◽  
Duane A. Compton ◽  
Erica A. Golemis
Keyword(s):  
Cell Cycle ◽  
Coiled Coil ◽  
Cell Cycle Checkpoints ◽  
Mitotic Spindles ◽  
Human Genes ◽  
Hydroxyurea Treatment ◽  
Evolutionarily Conserved ◽  
Defects Analysis ◽  
Spindle Function

ABSTRACT In a cross-species overexpression approach, we used the pseudohyphal transition of Saccharomyces cerevisiae as a model screening system to identify human genes that regulate cell morphology and the cell cycle. Human enhancer of invasion-cluster (HEI-C), encoding a novel evolutionarily conserved coiled-coil protein, was isolated in a screen for human genes that induce agar invasion in S. cerevisiae. In human cells, HEI-C is primarily localized to the spindle during mitosis. Depletion of HEI-C in vivo with short interfering RNAs results in severe mitotic defects. Analysis by immunofluorescence, flow cytometry analysis, and videomicroscopy indicates that HEI-C-depleted cells form metaphase plates with normal timing after G2/M transition, although in many cases cells have disorganized mitotic spindles. Subsequently, severe defects occur at the metaphase-anaphase transition, characterized by a significant delay at this stage or, more commonly, cellular disintegration accompanied by the display of classic biochemical markers of apoptosis. These mitotic defects occur in spite of the fact that HEI-C-depleted cells retain functional cell cycle checkpoints, as these cells arrest normally following nocodazole or hydroxyurea treatment. These results place HEI-C as a novel regulator of spindle function and integrity during the metaphase-anaphase transition.

scholarly journals Dual effect of 2-methoxyestradiol on cell cycle events in human osteosarcoma 143B cells.

2002 ◽  
Vol 49 (1) ◽  
pp. 59-65 ◽  
Author(s):  
Justyna Gołebiewska ◽  
Piotr Rozwadowski ◽  
Jan Henryk Spodnik ◽  
Narcyz Knap ◽  
Takashi Wakabayashi ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Growth Inhibitor ◽  
Cytotoxic Action ◽  
M Cell ◽  
Human Osteosarcoma ◽  
Cycle Arrest ◽  
M Phase ◽  
The Stability

We have demonstrated for the first time that the steroid metabolite, 2-methoxyestradiol (2-ME) is a powerful growth inhibitor of human osteosarcoma 143 B cell line by pleiotropic mechanisms involving cell cycle arrest at two different points and apoptosis. The ability of 2-ME to inhibit cell cycle at the respective points has been found concentration dependent. 1 microM 2-ME inhibited cell cycle at G1 phase while 10 microM 2-ME caused G2/M cell cycle arrest. As a natural estrogen metabolite 2-ME is expected to perturb the stability of microtubules (MT) in vivo analogously to Taxol--the MT binding anticancer agent. Contrary to 2-ME, Taxol induced accumulation of osteosarcoma cells in G2/M phase of cell cycle only. The presented data strongly suggest two different mechanisms of cytotoxic action of 2-ME at the level of a single cell.

scholarly journals Protein Kinase C Signaling Mediates a Program of Cell Cycle Withdrawal in the Intestinal Epithelium

2000 ◽  
Vol 151 (4) ◽  
pp. 763-778 ◽  
Author(s):  
Mark R. Frey ◽  
Jennifer A. Clark ◽  
Olga Leontieva ◽  
Joshua M. Uronis ◽  
Adrian R. Black ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Protein Kinase C ◽  
Protein Kinase ◽  
Intestinal Epithelium ◽  
Molecular Mechanisms ◽  
Model Systems ◽  
Intestinal Epithelial ◽  
Kinase C

Members of the protein kinase C (PKC) family of signal transduction molecules have been widely implicated in regulation of cell growth and differentiation, although the underlying molecular mechanisms involved remain poorly defined. Using combined in vitro and in vivo intestinal epithelial model systems, we demonstrate that PKC signaling can trigger a coordinated program of molecular events leading to cell cycle withdrawal into G0. PKC activation in the IEC-18 intestinal crypt cell line resulted in rapid downregulation of D-type cyclins and differential induction of p21waf1/cip1 and p27kip1, thus targeting all of the major G1/S cyclin-dependent kinase complexes. These events were associated with coordinated alterations in expression and phosphorylation of the pocket proteins p107, pRb, and p130 that drive cells to exit the cell cycle into G0 as indicated by concomitant downregulation of the DNA licensing factor cdc6. Manipulation of PKC isozyme levels in IEC-18 cells demonstrated that PKCα alone can trigger hallmark events of cell cycle withdrawal in intestinal epithelial cells. Notably, analysis of the developmental control of cell cycle regulatory molecules along the crypt–villus axis revealed that PKCα activation is appropriately positioned within intestinal crypts to trigger this program of cell cycle exit–specific events in situ. Together, these data point to PKCα as a key regulator of cell cycle withdrawal in the intestinal epithelium.

Microtubule-entrained kinase activities associated with the cortical cytoskeleton during cytokinesis

1997 ◽  
Vol 110 (12) ◽  
pp. 1373-1386 ◽  
Author(s):  
G.R. Walker ◽  
C.B. Shuster ◽  
D.R. Burgess
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Protein Phosphorylation ◽  
Immunoblot Analysis ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Mitotic Apparatus ◽  
Cortical Cytoskeleton

Research over the past few years has demonstrated the central role of protein phosphorylation in regulating mitosis and the cell cycle. However, little is known about how the mechanisms regulating the entry into mitosis contribute to the positional and temporal regulation of the actomyosin-based contractile ring formed during cytokinesis. Recent studies implicate p34cdc2 as a negative regulator of myosin II activity, suggesting a link between the mitotic cycle and cytokinesis. In an effort to study the relationship between protein phosphorylation and cytokinesis, we examined the in vivo and in vitro phosphorylation of actin-associated cortical cytoskeletal (CSK) proteins in an isolated model of the sea urchin egg cortex. Examination of cortices derived from eggs or zygotes labeled with 32P-orthophosphate reveals a number of cortex-associated phosphorylated proteins, including polypeptides of 20, 43 and 66 kDa. These three major phosphoproteins are also detected when isolated cortices are incubated with [32P]ATP in vitro, suggesting that the kinases that phosphorylate these substrates are also specifically associated with the cortex. The kinase activities in vivo and in vitro are stimulated by fertilization and display cell cycle-dependent activities. Gel autophosphorylation assays, kinase assays and immunoblot analysis reveal the presence of p34cdc2 as well as members of the mitogen-activated protein kinase family, whose activities in the CSK peak at cell division. Nocodazole, which inhibits microtubule formation and thus blocks cytokinesis, significantly delays the time of peak cortical protein phosphorylation as well as the peak in whole-cell histone H1 kinase activity. These results suggest that a key element regulating cortical contraction during cytokinesis is the timing of protein kinase activities associated with the cortical cytoskeleton that is in turn regulated by the mitotic apparatus.

scholarly journals A Redox-Sensitive Thiol in Wis1 Modulates the Fission Yeast Mitogen-Activated Protein Kinase Response to H2O2 and Is the Target of a Small Molecule

2020 ◽  
Vol 40 (7) ◽  
Author(s):  
Johanna J. Sjölander ◽  
Agata Tarczykowska ◽  
Cecilia Picazo ◽  
Itziar Cossio ◽  
Itedale Namro Redwan ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Fission Yeast ◽  
Inhibitory Effect ◽  
Mitogen Activated Protein Kinase ◽  
Activation Loop ◽  
Content Type ◽  
Kinase Activation ◽  
Mitogen Activated Protein

ABSTRACT Oxidation of a highly conserved cysteine (Cys) residue located in the kinase activation loop of mitogen-activated protein kinase kinases (MAPKK) inactivates mammalian MKK6. This residue is conserved in the fission yeast Schizosaccharomyces pombe MAPKK Wis1, which belongs to the H2O2-responsive MAPK Sty1 pathway. Here, we show that H2O2 reversibly inactivates Wis1 through this residue (C458) in vitro. We found that C458 is oxidized in vivo and that serine replacement of this residue significantly enhances Wis1 activation upon addition of H2O2. The allosteric MAPKK inhibitor INR119, which binds in a pocket next to the activation loop and C458, prevented the inhibition of Wis1 by H2O2 in vitro and significantly increased Wis1 activation by low levels of H2O2 in vivo. We propose that oxidation of C458 inhibits Wis1 and that INR119 cancels out this inhibitory effect by binding close to this residue. Kinase inhibition through the oxidation of a conserved Cys residue in MKK6 (C196) is thus conserved in the S. pombe MAPKK Wis1.

scholarly journals Membrane localization of the kinase which phosphorylates p34cdc2 on threonine 14.

1994 ◽  
Vol 5 (3) ◽  
pp. 273-282 ◽  
Author(s):  
S Kornbluth ◽  
B Sebastian ◽  
T Hunter ◽  
J Newport
Keyword(s):  
Cell Cycle ◽  
Protein Kinase ◽  
Schizosaccharomyces Pombe ◽  
Protein Kinases ◽  
Membrane Fraction ◽  
Kinase Activity ◽  
Serine Threonine Kinase ◽  
Threonine Kinase ◽  
Dual Specificity ◽  
Detergent Extraction

The key regulator of entry into mitosis is the serine/threonine kinase p34cdc2. This kinase is regulated both by association with cyclins and by phosphorylation at several sites. Phosphorylation at Tyr 15 and Thr 14 are believed to inhibit the kinase activity of cdc2. In Schizosaccharomyces pombe, the wee1 (and possibly mik1) protein kinase catalyzes phosphorylation of Tyr 15. It is not clear whether these or other, as yet unidentified, protein kinases phosphorylate Thr 14. In this report we show, using extracts of Xenopus eggs, that the Thr 14-directed kinase is tightly membrane associated. Specifically, we have shown that a purified membrane fraction, in the absence of cytoplasm, can promote phosphorylation of cdc2 on both Thr 14 and Tyr 15. In contrast, the cytoplasm can phosphorylate cdc2 only on Tyr 15, suggesting the existence of at least two distinctly localized subpopulations of cdc2 Tyr 15-directed kinases. The membrane-associated Tyr 15 and Thr 14 kinase activities behaved similarly during salt or detergent extraction and were similarly regulated during the cell cycle and by the checkpoint machinery that delays mitosis while DNA is being replicated. This suggests the possibility that a dual-specificity membrane-associated protein kinase may catalyze phosphorylation of both Tyr 15 and Thr 14.

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.

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