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Protein kinase C controls activation of the DNA integrity checkpoint.

Soriano-Carot M, Quilis I, Bañó MC, Igual JC - Nucleic Acids Res. (2014)

Bottom Line: This modification is a phosphorylation event mediated by Tel1.The expression of different mammalian PKC isoforms at the endogenous level in yeast pkc1 mutant cells revealed that PKCδ is able to activate the DNA integrity checkpoint.Our results indicate that the control of the DNA integrity checkpoint by PKC is a mechanism conserved from yeast to humans.

View Article: PubMed Central - PubMed

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

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Analysis of Mec1- and Tel1-dependent H2A and Xrs2 phosphorylation in pkc1 mutant strains. (A) Exponentially growing cultures of the wild-type (W303-1a), pkc1ts (JC6-3a), tel1 (JCY1258), tel1 pkc1ts (JCY1271), mec1 (JCY1039), mec1 pkc1ts (JCY1352) and mec1 tel1 (JCY1275) strains were incubated for 3 h at 37º followed by 1 h incubation in the absence or presence of 0.06% MMS. The level of phosphorylated H2A and total H2A protein was determined by western analysis using an antibody specific for H2A phosphorylated in Ser129 or an anti-H2A antibody, respectively. (B) Exponentially growing cultures of the wild-type (W303-1a), pkc1ts (JC6-3a), tel1 (JCY1258), tel1 pkc1ts (JCY1271) and mec1-1 (N1-3a) strains transformed with the pXRS2-HA plasmid were incubated for 3 h at 37º followed by 1 h incubation in the absence or presence of 25 μg/ml phleomycin. Phosphorylation of Xrs2 was determined by western analysis as slower migrating bands.
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Figure 4: Analysis of Mec1- and Tel1-dependent H2A and Xrs2 phosphorylation in pkc1 mutant strains. (A) Exponentially growing cultures of the wild-type (W303-1a), pkc1ts (JC6-3a), tel1 (JCY1258), tel1 pkc1ts (JCY1271), mec1 (JCY1039), mec1 pkc1ts (JCY1352) and mec1 tel1 (JCY1275) strains were incubated for 3 h at 37º followed by 1 h incubation in the absence or presence of 0.06% MMS. The level of phosphorylated H2A and total H2A protein was determined by western analysis using an antibody specific for H2A phosphorylated in Ser129 or an anti-H2A antibody, respectively. (B) Exponentially growing cultures of the wild-type (W303-1a), pkc1ts (JC6-3a), tel1 (JCY1258), tel1 pkc1ts (JCY1271) and mec1-1 (N1-3a) strains transformed with the pXRS2-HA plasmid were incubated for 3 h at 37º followed by 1 h incubation in the absence or presence of 25 μg/ml phleomycin. Phosphorylation of Xrs2 was determined by western analysis as slower migrating bands.

Mentions: For the histone H2A, it has been described that genotoxic stress induces the phosphorylation of Ser129 over a 50–100-kb region around the damage site and that this phosphorylation can be carried out by both Mec1 and Tel1 kinases (50–52). In order to examine whether the defects observed in checkpoint activation in the absence of Pkc1 were due to the defective activation of Mec1 and/or Tel1, we studied if H2A was properly phosphorylated in response to genotoxic stress in the pkc1 mutant cells. The results show that Ser129 phosphorylation in response to DNA damage can still be detected in single mec1 or tel1 mutant cells, and it is abolished only in mec1 tel1 double mutant strain (Figure 4A). Interestingly, H2A phosphorylation was drastically reduced in the absence of Pkc1 activity. The level of H2A phosphorylation observed in the pkc1 mutant strains was weaker than that detected in single mec1 and tel1 mutants, suggesting that both kinases are affected by Pkc1 inactivation. However, it has to be noted that the level of phosphorylated H2A detected in pkc1 mutant cells was somewhat higher than that observed in the double mec1 tel1 mutant strain. This may be due to the presence of residual Mec1 and/or Tel1 activity, which might be able to drive a basal level of H2A phosphorylation. In summary, cells need fully functional Pkc1 to correctly drive Mec1- and Tel1-dependent phosphorylation of histone H2A in response to DNA damage.


Protein kinase C controls activation of the DNA integrity checkpoint.

Soriano-Carot M, Quilis I, Bañó MC, Igual JC - Nucleic Acids Res. (2014)

Analysis of Mec1- and Tel1-dependent H2A and Xrs2 phosphorylation in pkc1 mutant strains. (A) Exponentially growing cultures of the wild-type (W303-1a), pkc1ts (JC6-3a), tel1 (JCY1258), tel1 pkc1ts (JCY1271), mec1 (JCY1039), mec1 pkc1ts (JCY1352) and mec1 tel1 (JCY1275) strains were incubated for 3 h at 37º followed by 1 h incubation in the absence or presence of 0.06% MMS. The level of phosphorylated H2A and total H2A protein was determined by western analysis using an antibody specific for H2A phosphorylated in Ser129 or an anti-H2A antibody, respectively. (B) Exponentially growing cultures of the wild-type (W303-1a), pkc1ts (JC6-3a), tel1 (JCY1258), tel1 pkc1ts (JCY1271) and mec1-1 (N1-3a) strains transformed with the pXRS2-HA plasmid were incubated for 3 h at 37º followed by 1 h incubation in the absence or presence of 25 μg/ml phleomycin. Phosphorylation of Xrs2 was determined by western analysis as slower migrating bands.
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Figure 4: Analysis of Mec1- and Tel1-dependent H2A and Xrs2 phosphorylation in pkc1 mutant strains. (A) Exponentially growing cultures of the wild-type (W303-1a), pkc1ts (JC6-3a), tel1 (JCY1258), tel1 pkc1ts (JCY1271), mec1 (JCY1039), mec1 pkc1ts (JCY1352) and mec1 tel1 (JCY1275) strains were incubated for 3 h at 37º followed by 1 h incubation in the absence or presence of 0.06% MMS. The level of phosphorylated H2A and total H2A protein was determined by western analysis using an antibody specific for H2A phosphorylated in Ser129 or an anti-H2A antibody, respectively. (B) Exponentially growing cultures of the wild-type (W303-1a), pkc1ts (JC6-3a), tel1 (JCY1258), tel1 pkc1ts (JCY1271) and mec1-1 (N1-3a) strains transformed with the pXRS2-HA plasmid were incubated for 3 h at 37º followed by 1 h incubation in the absence or presence of 25 μg/ml phleomycin. Phosphorylation of Xrs2 was determined by western analysis as slower migrating bands.
Mentions: For the histone H2A, it has been described that genotoxic stress induces the phosphorylation of Ser129 over a 50–100-kb region around the damage site and that this phosphorylation can be carried out by both Mec1 and Tel1 kinases (50–52). In order to examine whether the defects observed in checkpoint activation in the absence of Pkc1 were due to the defective activation of Mec1 and/or Tel1, we studied if H2A was properly phosphorylated in response to genotoxic stress in the pkc1 mutant cells. The results show that Ser129 phosphorylation in response to DNA damage can still be detected in single mec1 or tel1 mutant cells, and it is abolished only in mec1 tel1 double mutant strain (Figure 4A). Interestingly, H2A phosphorylation was drastically reduced in the absence of Pkc1 activity. The level of H2A phosphorylation observed in the pkc1 mutant strains was weaker than that detected in single mec1 and tel1 mutants, suggesting that both kinases are affected by Pkc1 inactivation. However, it has to be noted that the level of phosphorylated H2A detected in pkc1 mutant cells was somewhat higher than that observed in the double mec1 tel1 mutant strain. This may be due to the presence of residual Mec1 and/or Tel1 activity, which might be able to drive a basal level of H2A phosphorylation. In summary, cells need fully functional Pkc1 to correctly drive Mec1- and Tel1-dependent phosphorylation of histone H2A in response to DNA damage.

Bottom Line: This modification is a phosphorylation event mediated by Tel1.The expression of different mammalian PKC isoforms at the endogenous level in yeast pkc1 mutant cells revealed that PKCδ is able to activate the DNA integrity checkpoint.Our results indicate that the control of the DNA integrity checkpoint by PKC is a mechanism conserved from yeast to humans.

View Article: PubMed Central - PubMed

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

Show MeSH
Related in: MedlinePlus