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- Alao, John Patrick, 1973, et al.
(författare)
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Caffeine stabilizes Cdc25 independently of Rad3 in Schizosaccharomyces pombe contributing to checkpoint override
- 2014
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Ingår i: Molecular Microbiology. - : Wiley. - 0950-382X .- 1365-2958. ; 92:4, s. 777-796
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Tidskriftsartikel (refereegranskat)abstract
- Cdc25 is required for Cdc2 dephosphorylation and is thus essential for cell cycle progression. Checkpoint activation requires dual inhibition of Cdc25 and Cdc2 in a Rad3-dependent manner. Caffeine is believed to override activation of the replication and DNA damage checkpoints by inhibiting Rad3-related proteins in both Schizosaccharomyces pombe and mammalian cells. In this study, we have investigated the impact of caffeine on Cdc25 stability, cell cycle progression and checkpoint override. Caffeine induced Cdc25 accumulation in S.pombe independently of Rad3. Caffeine delayed cell cycle progression under normal conditions but advanced mitosis in cells treated with replication inhibitors and DNA-damaging agents. In the absence of Cdc25, caffeine inhibited cell cycle progression even in the presence of hydroxyurea or phleomycin. Caffeine induces Cdc25 accumulation in S.pombe by suppressing its degradation independently of Rad3. The induction of Cdc25 accumulation was not associated with accelerated progression through mitosis, but rather with delayed progression through cytokinesis. Caffeine-induced Cdc25 accumulation appears to underlie its ability to override cell cycle checkpoints. The impact of Cdc25 accumulation on cell cycle progression is attenuated by Srk1 and Mad2. Together our findings suggest that caffeine overrides checkpoint enforcement by inducing the inappropriate nuclear localization of Cdc25.
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| 2. |
- Sjölander, Johanna J, 1980, et al.
(författare)
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A redox-sensitive thiol in Wis1 modulates the fission yeast MAPK response to H2O2 and is the target of a small molecule.
- 2020
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Ingår i: Molecular and Cellular Biology. - 0270-7306 .- 1098-5549. ; 40:7
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Tidskriftsartikel (refereegranskat)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 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 substitution 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 H2O2in vitro, and significantly increased Wis1 activation by low levels of H2O2in 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.
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| 3. |
- Sjölander, Johanna J, 1980
(författare)
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DNA checkpoint override and redox signaling in Schizosaccharomyces pombe
- 2019
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis covers intracellular stress signaling through genotoxic stress, overriding of checkpoint control, as well as cellular redox status in hypoxic and oxidative stress Papers I and II: Caffeine has been shown to override cell cycle checkpoints in humans as well as in the fission yeast Schizosaccharomyces pombe. Understanding of the mechanism may aid in the development of compounds with similar overriding mechanisms for sensitization in cancer therapy. We show that caffeine induces accumulation of the mitotic inducer protein Cdc25, which removes inhibitory phosphorylation from the CDK Cdc2. Deletion of genes encoding the fission yeast checkpoint proteins Rad3 or Cds1 resulted in a higher constitutive level of Cdc25, suggesting a constitutive role in regulation of the Cdc25 level. Importantly, however, caffeine-induced Cdc25 accumulation is Rad3 independent. Mechanistically our results indicate that caffeine stabilizes and induces nuclear accumulation of Cdc25 as well as preventing Wee1, the kinase phosphorylating the same residue that Cdc25 dephosphorylates, from increasing in response to DNA damage, thereby enforcing progression into mitosis. Our results are in agreement with the known caffeine inhibition of TORC1 contributing to checkpoint override. Paper III: FHIT, a human tumor suppressor, modulates DNA damage sensing, checkpoint control, proliferation and apoptosis. We investigated Aph1, the fission yeast homolog of FHIT, and found that deletion of the aph1+ gene led to enhanced proliferation in sublethal concentrations of genotoxins. This phenotype was accompanied by elevated chromosome fragmentation and/or missegregation. Moreover, we show that an aph1 deletion leads to knock-down of the checkpoint protein Rad1 in the 9-1-1 complex, and that Aph1 as well as all 9-1-1 proteins are downregulated in hypoxia. Paper IV: H2O2 induces oxidative stress, but is also a signaling molecule that exerts its function through reaction with selected thiols of protein cysteines. MAP kinase (MAPK) pathways are induced by H2O2 in both human and fission yeast. We observed that an active site cysteine, shown to be involved in negative regulation of a human MAP kinase kinase (MAPKK), is evolutionarily conserved in all MAPKKs of budding yeast, fission yeast and humans, indicating that regulation of kinase activity through this cysteine may be a conserved feature of MAPK signaling in these organisms. The active site cysteine C458 in fission yeast MAPKK has no plausible cysteine partner for intramolecular disulfide bond formation. However, Wis1 kinase activity was still inactivated by reversible thiol oxidation in a C458 dependent way. The synthetic allosteric MAPKK modulator molecule INR119, predicted to bind in a site next to C458, protected against negative oxidative regulation in vitro targeting C458, resulting in enhanced MAPK signaling in vivo.
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