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The yeast mitogen-activated protein kinase Slt2 is involved in the cellular response to genotoxic stress.

Soriano-Carot M, Bañó MC, Igual JC - Cell Div (2012)

Bottom Line: However, slt2 mutant cells showed an elongated bud and partially impaired Swe1 degradation after replicative stress, indicating that Slt2 could contribute, in parallel with Rad53, to bud morphogenesis control after genotoxic stresses.Slt2 function is important for bud morphogenesis and optimal Swe1 degradation under replicative stress.The MAPK Slt2 appears as a new player in the cellular response to genotoxic stresses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departament de Bioquímica i Biologia Molecular, Universitat de València, 46100 Burjassot (Valencia), Spain. jcigual@uv.es.

ABSTRACT

Background: The maintenance of genomic integrity is essential for cell viability. Complex signalling pathways (DNA integrity checkpoints) mediate the response to genotoxic stresses. Identifying new functions involved in the cellular response to DNA-damage is crucial. The Saccharomyces cerevisiae SLT2 gene encodes a member of the mitogen-activated protein kinase (MAPK) cascade whose main function is the maintenance of the cell wall integrity. However, different observations suggest that SLT2 may also have a role related to DNA metabolism.

Results: This work consisted in a comprehensive study to connect the Slt2 protein to genome integrity maintenance in response to genotoxic stresses. The slt2 mutant strain was hypersensitive to a variety of genotoxic treatments, including incubation with hydroxyurea (HU), methylmetanosulfonate (MMS), phleomycin or UV irradiation. Furthermore, Slt2 was activated by all these treatments, which suggests that Slt2 plays a central role in the cellular response to genotoxic stresses. Activation of Slt2 was not dependent on the DNA integrity checkpoint. For MMS and UV, Slt2 activation required progression through the cell cycle. In contrast, HU also activated Slt2 in nocodazol-arrested cells, which suggests that Slt2 may respond to dNTP pools alterations. However, neither the protein level of the distinct ribonucleotide reductase subunits nor the dNTP pools were affected in a slt2 mutant strain. An analysis of the checkpoint function revealed that Slt2 was not required for either cell cycle arrest or the activation of the Rad53 checkpoint kinase in response to DNA damage. However, slt2 mutant cells showed an elongated bud and partially impaired Swe1 degradation after replicative stress, indicating that Slt2 could contribute, in parallel with Rad53, to bud morphogenesis control after genotoxic stresses.

Conclusions: Slt2 is activated by several genotoxic treatments and is required to properly cope with DNA damage. Slt2 function is important for bud morphogenesis and optimal Swe1 degradation under replicative stress. The MAPK Slt2 appears as a new player in the cellular response to genotoxic stresses.

No MeSH data available.


Related in: MedlinePlus

Activation of Slt2 by genotoxic stress in DNA integrity checkpoint mutants. Exponentially growing cultures of the wild type (W303-1a), sml1 (JCY1144), rad53 sml1 (JCY1038), mec1 sml1 (JCY1039), mec1 tel1 sml1 (JCY1275) and tel1 (JCY1258) strains were split and incubated for 60 min in the absence or presence of 200 mM hydroxyurea or 0.04% MMS. Exponentially growing cultures of the tetO7:RAD53 (JCY1149) strain were incubated for 6 hours in the absence or presence of 5 μg/mL doxicycline in order to repress the tetO7 promoter followed by 60 min incubation in the absence or presence of 200 mM hydroxyurea. The level of phosphorylated Slt2, total Slt2 protein and the chekpoint kinase Rad53 was determined by western analysis. The ponceau staining of the membranes are shown as loading control.
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Figure 4: Activation of Slt2 by genotoxic stress in DNA integrity checkpoint mutants. Exponentially growing cultures of the wild type (W303-1a), sml1 (JCY1144), rad53 sml1 (JCY1038), mec1 sml1 (JCY1039), mec1 tel1 sml1 (JCY1275) and tel1 (JCY1258) strains were split and incubated for 60 min in the absence or presence of 200 mM hydroxyurea or 0.04% MMS. Exponentially growing cultures of the tetO7:RAD53 (JCY1149) strain were incubated for 6 hours in the absence or presence of 5 μg/mL doxicycline in order to repress the tetO7 promoter followed by 60 min incubation in the absence or presence of 200 mM hydroxyurea. The level of phosphorylated Slt2, total Slt2 protein and the chekpoint kinase Rad53 was determined by western analysis. The ponceau staining of the membranes are shown as loading control.

Mentions: Slt2 is activated by phosphorylation in the activation loop. In previous works, we observed a dramatic increase in the phosphorylation state of the Slt2 MAP kinase after addition of hydroxyurea [43]. Here, we extended the analysis by testing whether other types of DNA damage also cause Slt2 activation. First, W303-1a cells were treated with HU or MMS, or were irradiated with different UV doses to induce DNA damage. Occurrence of damage was monitored by analyzing the phosphorylation state of the checkpoint kinase Rad53. The appearance of lower Rad53 electrophoretic mobility bands corresponding to the phosphorylated protein confirmed that the checkpoint was activated by these treatments. It is interesting to note that higher levels of phosphorylated Slt2 were detected in these cells, indicating that the MAP kinase Slt2 is activated in those cells incubated in the presence of HU or MMS, or those exposed to UV radiation (Figure 2). A similar result was obtained in a W303-derived RAD5 strain and in the SEY6211 genetic background (Figure 2). Slt2 activation was also observed after induction of double-strand breaks (DSB) with phleomycin. Next, we analyzed the response to a single DSB induced by the addition of galactose to raffinose-grown cells expressing the HO endonclease under GAL1 promoter control. As Figure 2 depicts the levels of phosphorylated Slt2 drastically increased after the induction of a single DSB. This in not due to the change in carbon source since Slt2 activation after addition of galactose is not observed in a wild type control strain. All these observations are consistent with the above-described slt2 mutant hypersensitivity to genotoxic stresses and indicate that Slt2 activation is a crucial step in the cellular response to all kinds of DNA damage. Interestingly, Slt2 activation by genetic stresses is mostly, if not totally, mediated by a post-translational mechanism since Slt2 protein level is not significantly affected (Figure 2, 4 and 5).


The yeast mitogen-activated protein kinase Slt2 is involved in the cellular response to genotoxic stress.

Soriano-Carot M, Bañó MC, Igual JC - Cell Div (2012)

Activation of Slt2 by genotoxic stress in DNA integrity checkpoint mutants. Exponentially growing cultures of the wild type (W303-1a), sml1 (JCY1144), rad53 sml1 (JCY1038), mec1 sml1 (JCY1039), mec1 tel1 sml1 (JCY1275) and tel1 (JCY1258) strains were split and incubated for 60 min in the absence or presence of 200 mM hydroxyurea or 0.04% MMS. Exponentially growing cultures of the tetO7:RAD53 (JCY1149) strain were incubated for 6 hours in the absence or presence of 5 μg/mL doxicycline in order to repress the tetO7 promoter followed by 60 min incubation in the absence or presence of 200 mM hydroxyurea. The level of phosphorylated Slt2, total Slt2 protein and the chekpoint kinase Rad53 was determined by western analysis. The ponceau staining of the membranes are shown as loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Activation of Slt2 by genotoxic stress in DNA integrity checkpoint mutants. Exponentially growing cultures of the wild type (W303-1a), sml1 (JCY1144), rad53 sml1 (JCY1038), mec1 sml1 (JCY1039), mec1 tel1 sml1 (JCY1275) and tel1 (JCY1258) strains were split and incubated for 60 min in the absence or presence of 200 mM hydroxyurea or 0.04% MMS. Exponentially growing cultures of the tetO7:RAD53 (JCY1149) strain were incubated for 6 hours in the absence or presence of 5 μg/mL doxicycline in order to repress the tetO7 promoter followed by 60 min incubation in the absence or presence of 200 mM hydroxyurea. The level of phosphorylated Slt2, total Slt2 protein and the chekpoint kinase Rad53 was determined by western analysis. The ponceau staining of the membranes are shown as loading control.
Mentions: Slt2 is activated by phosphorylation in the activation loop. In previous works, we observed a dramatic increase in the phosphorylation state of the Slt2 MAP kinase after addition of hydroxyurea [43]. Here, we extended the analysis by testing whether other types of DNA damage also cause Slt2 activation. First, W303-1a cells were treated with HU or MMS, or were irradiated with different UV doses to induce DNA damage. Occurrence of damage was monitored by analyzing the phosphorylation state of the checkpoint kinase Rad53. The appearance of lower Rad53 electrophoretic mobility bands corresponding to the phosphorylated protein confirmed that the checkpoint was activated by these treatments. It is interesting to note that higher levels of phosphorylated Slt2 were detected in these cells, indicating that the MAP kinase Slt2 is activated in those cells incubated in the presence of HU or MMS, or those exposed to UV radiation (Figure 2). A similar result was obtained in a W303-derived RAD5 strain and in the SEY6211 genetic background (Figure 2). Slt2 activation was also observed after induction of double-strand breaks (DSB) with phleomycin. Next, we analyzed the response to a single DSB induced by the addition of galactose to raffinose-grown cells expressing the HO endonclease under GAL1 promoter control. As Figure 2 depicts the levels of phosphorylated Slt2 drastically increased after the induction of a single DSB. This in not due to the change in carbon source since Slt2 activation after addition of galactose is not observed in a wild type control strain. All these observations are consistent with the above-described slt2 mutant hypersensitivity to genotoxic stresses and indicate that Slt2 activation is a crucial step in the cellular response to all kinds of DNA damage. Interestingly, Slt2 activation by genetic stresses is mostly, if not totally, mediated by a post-translational mechanism since Slt2 protein level is not significantly affected (Figure 2, 4 and 5).

Bottom Line: However, slt2 mutant cells showed an elongated bud and partially impaired Swe1 degradation after replicative stress, indicating that Slt2 could contribute, in parallel with Rad53, to bud morphogenesis control after genotoxic stresses.Slt2 function is important for bud morphogenesis and optimal Swe1 degradation under replicative stress.The MAPK Slt2 appears as a new player in the cellular response to genotoxic stresses.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departament de Bioquímica i Biologia Molecular, Universitat de València, 46100 Burjassot (Valencia), Spain. jcigual@uv.es.

ABSTRACT

Background: The maintenance of genomic integrity is essential for cell viability. Complex signalling pathways (DNA integrity checkpoints) mediate the response to genotoxic stresses. Identifying new functions involved in the cellular response to DNA-damage is crucial. The Saccharomyces cerevisiae SLT2 gene encodes a member of the mitogen-activated protein kinase (MAPK) cascade whose main function is the maintenance of the cell wall integrity. However, different observations suggest that SLT2 may also have a role related to DNA metabolism.

Results: This work consisted in a comprehensive study to connect the Slt2 protein to genome integrity maintenance in response to genotoxic stresses. The slt2 mutant strain was hypersensitive to a variety of genotoxic treatments, including incubation with hydroxyurea (HU), methylmetanosulfonate (MMS), phleomycin or UV irradiation. Furthermore, Slt2 was activated by all these treatments, which suggests that Slt2 plays a central role in the cellular response to genotoxic stresses. Activation of Slt2 was not dependent on the DNA integrity checkpoint. For MMS and UV, Slt2 activation required progression through the cell cycle. In contrast, HU also activated Slt2 in nocodazol-arrested cells, which suggests that Slt2 may respond to dNTP pools alterations. However, neither the protein level of the distinct ribonucleotide reductase subunits nor the dNTP pools were affected in a slt2 mutant strain. An analysis of the checkpoint function revealed that Slt2 was not required for either cell cycle arrest or the activation of the Rad53 checkpoint kinase in response to DNA damage. However, slt2 mutant cells showed an elongated bud and partially impaired Swe1 degradation after replicative stress, indicating that Slt2 could contribute, in parallel with Rad53, to bud morphogenesis control after genotoxic stresses.

Conclusions: Slt2 is activated by several genotoxic treatments and is required to properly cope with DNA damage. Slt2 function is important for bud morphogenesis and optimal Swe1 degradation under replicative stress. The MAPK Slt2 appears as a new player in the cellular response to genotoxic stresses.

No MeSH data available.


Related in: MedlinePlus