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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

Analysis of Swe1 protein level in slt2 mutant cells after a replicative stress. A) Exponentially growing cells of the wild type (JCY1316) and slt2 (JCY1318) strains expressing a HA-epitope tagged Swe1 protein were incubated in the absence or presence of 200 mM hydroxyurea. Swe1 protein level and activation of the chekpoint kinase Rad53 was analyzed at the indicated time after the addition of HU by western blot. A non-specific band labelled with an asterisk that cross-react with the antibody is shown as loading control. Graph represents the relative amount of Swe1 protein related to the non-specific band in the HU-treated cells derived from three independent assays. B) Exponentially growing cultures of the slt2 (JCY1062) and the slt2 swe1 (JCY1633) strains were incubated in the presence of 200 mM hydroxyurea for 6 hours. Cell cycle distribution of cells and the presence of abnormal elongated bud were analyzed as described in Figure 7. Graphs represent cell distribution derived from at least three independent cultures. C) Aliquots from exponentially growing cultures of the swe1 (JCY1632) and the slt2 swe1 (JCY1633) strains were incubated for 90 min. at the indicated doses of HU, MMS and phleomycin or were exposed to different doses of UV radiation. Cells were plated on YPD and the percentage of surviving cells relative to untreated controls was determined.
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Figure 8: Analysis of Swe1 protein level in slt2 mutant cells after a replicative stress. A) Exponentially growing cells of the wild type (JCY1316) and slt2 (JCY1318) strains expressing a HA-epitope tagged Swe1 protein were incubated in the absence or presence of 200 mM hydroxyurea. Swe1 protein level and activation of the chekpoint kinase Rad53 was analyzed at the indicated time after the addition of HU by western blot. A non-specific band labelled with an asterisk that cross-react with the antibody is shown as loading control. Graph represents the relative amount of Swe1 protein related to the non-specific band in the HU-treated cells derived from three independent assays. B) Exponentially growing cultures of the slt2 (JCY1062) and the slt2 swe1 (JCY1633) strains were incubated in the presence of 200 mM hydroxyurea for 6 hours. Cell cycle distribution of cells and the presence of abnormal elongated bud were analyzed as described in Figure 7. Graphs represent cell distribution derived from at least three independent cultures. C) Aliquots from exponentially growing cultures of the swe1 (JCY1632) and the slt2 swe1 (JCY1633) strains were incubated for 90 min. at the indicated doses of HU, MMS and phleomycin or were exposed to different doses of UV radiation. Cells were plated on YPD and the percentage of surviving cells relative to untreated controls was determined.

Mentions: Recently, a morphogenetic function for the DNA integrity checkpoint has been described, which consists in switching off bud apical growth after damage [11]. This is achieved by the degradation of CDK inhibitor kinase Swe1. Cells with a defective checkpoint are unable to degrade Swe1 and as a consequence, they cannot induce the switch from polar to isotropic bud growth, resulting in the formation of elongated buds. By considering that Slt2 has been related to Swe1 regulation [26,27] and that slt2 mutant cells manifested a hyperpolarization defect in response to DNA damage, we wondered whether Slt2 is required for the morphogenetic response controlled by the Rad53 checkpoint kinase. To investigate this, the Swe1 protein level was analyzed after incubating cells with HU (Figure 8). As previously described, Swe1 is eliminated from wild-type cells after genotoxic stress to reduce to less than 20% of the initial protein level after 6 hours. It is remarkable to note that Swe1 protein decay was minimized in the absence of Slt2, and that more than 50% of the initial protein remained after 6 hours. This was not caused by cell cycle effects, as cell cycle distribution of slt2 mutant was roughly similar to that of wild type strain (see Figure 7A) neither to differences in checkpoint activation as Rad53 phosphorylation occurred with similar kinetics. This result indicates that Slt2 is involved in the morphogenetic response after DNA damage and is required for optimal Swe1 degradation in response to DNA damage. Interestingly, hyperpolarization of slt2 mutant cells is not observed when Swe1 kinase is inactivated (Figure 8B). This demonstrates that Slt2 control of bud morphogenesis in response to DNA damage is mediated by the Swe1 kinase. However, Slt2 inactivation caused loss of cell viability even in the absence of Swe1 (Figure 8C), indicating that the hipersensitivity to genotoxic stresses involves additional Swe1-independent mechanisms.


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)

Analysis of Swe1 protein level in slt2 mutant cells after a replicative stress. A) Exponentially growing cells of the wild type (JCY1316) and slt2 (JCY1318) strains expressing a HA-epitope tagged Swe1 protein were incubated in the absence or presence of 200 mM hydroxyurea. Swe1 protein level and activation of the chekpoint kinase Rad53 was analyzed at the indicated time after the addition of HU by western blot. A non-specific band labelled with an asterisk that cross-react with the antibody is shown as loading control. Graph represents the relative amount of Swe1 protein related to the non-specific band in the HU-treated cells derived from three independent assays. B) Exponentially growing cultures of the slt2 (JCY1062) and the slt2 swe1 (JCY1633) strains were incubated in the presence of 200 mM hydroxyurea for 6 hours. Cell cycle distribution of cells and the presence of abnormal elongated bud were analyzed as described in Figure 7. Graphs represent cell distribution derived from at least three independent cultures. C) Aliquots from exponentially growing cultures of the swe1 (JCY1632) and the slt2 swe1 (JCY1633) strains were incubated for 90 min. at the indicated doses of HU, MMS and phleomycin or were exposed to different doses of UV radiation. Cells were plated on YPD and the percentage of surviving cells relative to untreated controls was determined.
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Related In: Results  -  Collection

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Figure 8: Analysis of Swe1 protein level in slt2 mutant cells after a replicative stress. A) Exponentially growing cells of the wild type (JCY1316) and slt2 (JCY1318) strains expressing a HA-epitope tagged Swe1 protein were incubated in the absence or presence of 200 mM hydroxyurea. Swe1 protein level and activation of the chekpoint kinase Rad53 was analyzed at the indicated time after the addition of HU by western blot. A non-specific band labelled with an asterisk that cross-react with the antibody is shown as loading control. Graph represents the relative amount of Swe1 protein related to the non-specific band in the HU-treated cells derived from three independent assays. B) Exponentially growing cultures of the slt2 (JCY1062) and the slt2 swe1 (JCY1633) strains were incubated in the presence of 200 mM hydroxyurea for 6 hours. Cell cycle distribution of cells and the presence of abnormal elongated bud were analyzed as described in Figure 7. Graphs represent cell distribution derived from at least three independent cultures. C) Aliquots from exponentially growing cultures of the swe1 (JCY1632) and the slt2 swe1 (JCY1633) strains were incubated for 90 min. at the indicated doses of HU, MMS and phleomycin or were exposed to different doses of UV radiation. Cells were plated on YPD and the percentage of surviving cells relative to untreated controls was determined.
Mentions: Recently, a morphogenetic function for the DNA integrity checkpoint has been described, which consists in switching off bud apical growth after damage [11]. This is achieved by the degradation of CDK inhibitor kinase Swe1. Cells with a defective checkpoint are unable to degrade Swe1 and as a consequence, they cannot induce the switch from polar to isotropic bud growth, resulting in the formation of elongated buds. By considering that Slt2 has been related to Swe1 regulation [26,27] and that slt2 mutant cells manifested a hyperpolarization defect in response to DNA damage, we wondered whether Slt2 is required for the morphogenetic response controlled by the Rad53 checkpoint kinase. To investigate this, the Swe1 protein level was analyzed after incubating cells with HU (Figure 8). As previously described, Swe1 is eliminated from wild-type cells after genotoxic stress to reduce to less than 20% of the initial protein level after 6 hours. It is remarkable to note that Swe1 protein decay was minimized in the absence of Slt2, and that more than 50% of the initial protein remained after 6 hours. This was not caused by cell cycle effects, as cell cycle distribution of slt2 mutant was roughly similar to that of wild type strain (see Figure 7A) neither to differences in checkpoint activation as Rad53 phosphorylation occurred with similar kinetics. This result indicates that Slt2 is involved in the morphogenetic response after DNA damage and is required for optimal Swe1 degradation in response to DNA damage. Interestingly, hyperpolarization of slt2 mutant cells is not observed when Swe1 kinase is inactivated (Figure 8B). This demonstrates that Slt2 control of bud morphogenesis in response to DNA damage is mediated by the Swe1 kinase. However, Slt2 inactivation caused loss of cell viability even in the absence of Swe1 (Figure 8C), indicating that the hipersensitivity to genotoxic stresses involves additional Swe1-independent mechanisms.

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