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Pph3 dephosphorylation of Rad53 is required for cell recovery from MMS-induced DNA damage in Candida albicans.

Wang H, Gao J, Li W, Wong AH, Hu K, Chen K, Wang Y, Sang J - PLoS ONE (2012)

Bottom Line: The pathogenic fungus Candida albicans switches from yeast growth to filamentous growth in response to genotoxic stresses, in which phosphoregulation of the checkpoint kinase Rad53 plays a crucial role.Moreover, during this growth, Rad53 remained hyperphosphorylated, MBF-regulated genes were downregulated, and hypha-specific genes were upregulated.We have also identified S461 and S545 on Rad53 as potential dephosphorylation sites of Pph3/Psy2 that are specifically involved in cellular responses to MMS.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing, People's Republic of China.

ABSTRACT
The pathogenic fungus Candida albicans switches from yeast growth to filamentous growth in response to genotoxic stresses, in which phosphoregulation of the checkpoint kinase Rad53 plays a crucial role. Here we report that the Pph3/Psy2 phosphatase complex, known to be involved in Rad53 dephosphorylation, is required for cellular responses to the DNA-damaging agent methyl methanesulfonate (MMS) but not the DNA replication inhibitor hydroxyurea (HU) in C. albicans. Deletion of either PPH3 or PSY2 resulted in enhanced filamentous growth during MMS treatment and continuous filamentous growth even after MMS removal. Moreover, during this growth, Rad53 remained hyperphosphorylated, MBF-regulated genes were downregulated, and hypha-specific genes were upregulated. We have also identified S461 and S545 on Rad53 as potential dephosphorylation sites of Pph3/Psy2 that are specifically involved in cellular responses to MMS. Therefore, our studies have identified a novel molecular mechanism mediating DNA damage response to MMS in C. albicans.

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Rad53 undergoes hyperphosphorylation in response to HU and MMS.Fig 2A. Rad53 hyperphosphorylation in HU-treated cells. SJL9 (wild type with RAD53-Myc), SJL7 (pph3Δ RAD53-Myc), and SJL8 (psy2Δ RAD53-Myc) cells were incubated at 30°C in YPD containing 20 mM HU for 4 h. Cells were then washed and recovered with fresh YPD for the indicated times. Whole cell lysates were used for immunoblot analysis with anti-Myc antibody. Untreated cells were used as control. Cdc28 was probed with anti-PSTAIRE antibody as loading control. Fig 2B. Rad53 hyperphosphorylation in MMS-treated cells. SJL9 (wild type with RAD53-Myc), SJL7 (pph3Δ RAD53-Myc), and SJL8 (psy2Δ RAD53-Myc) cells were incubated at 30°C in YPD containing 0.02% MMS for 4 h. Cells were then washed and recovered with fresh YPD for the indicated times. Whole cell lysates were used for immunoblot analysis with anti-Myc antibody. Untreated cells were used as control. Cdc28 was probed with anti-PSTAIRE antibody as loading control.
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pone-0037246-g002: Rad53 undergoes hyperphosphorylation in response to HU and MMS.Fig 2A. Rad53 hyperphosphorylation in HU-treated cells. SJL9 (wild type with RAD53-Myc), SJL7 (pph3Δ RAD53-Myc), and SJL8 (psy2Δ RAD53-Myc) cells were incubated at 30°C in YPD containing 20 mM HU for 4 h. Cells were then washed and recovered with fresh YPD for the indicated times. Whole cell lysates were used for immunoblot analysis with anti-Myc antibody. Untreated cells were used as control. Cdc28 was probed with anti-PSTAIRE antibody as loading control. Fig 2B. Rad53 hyperphosphorylation in MMS-treated cells. SJL9 (wild type with RAD53-Myc), SJL7 (pph3Δ RAD53-Myc), and SJL8 (psy2Δ RAD53-Myc) cells were incubated at 30°C in YPD containing 0.02% MMS for 4 h. Cells were then washed and recovered with fresh YPD for the indicated times. Whole cell lysates were used for immunoblot analysis with anti-Myc antibody. Untreated cells were used as control. Cdc28 was probed with anti-PSTAIRE antibody as loading control.

Mentions: Rad53 was shown to be a substrate of the Pph3/Psy2 complex in both S. cerevisiae and C. albicans[34], [38]. Furthermore, Rad53 hyperphosphorylation was shown to cause cell cycle arrest and filamentous growth in C. albicans[38]. Next, we examined the phosphorylation state of Rad53 in response to MMS and HU treatment. Western blotting of C-terminally Myc-tagged Rad53 demonstrated that in wild-type cells Rad53 was hyperphosphorylated after both MMS (Fig. S3B) and HU treatment (Fig. S3A), and became dephosphorylated 6 h after HU or MMS removal (Fig. 2A&B). Both pph3Δ and psy2Δ mutants also showed a similar course of Rad53 dephosphorylation after HU removal (Fig. 2A). However, Rad53 hyperphosphorylation persisted in pph3Δ and psy2Δ mutants even at 6 h after MMS removal (Fig. 2B), indicating that Rad53 dephosphorylation during recovery from MMS treatment is dependent on the Pph3/Psy2 complex.


Pph3 dephosphorylation of Rad53 is required for cell recovery from MMS-induced DNA damage in Candida albicans.

Wang H, Gao J, Li W, Wong AH, Hu K, Chen K, Wang Y, Sang J - PLoS ONE (2012)

Rad53 undergoes hyperphosphorylation in response to HU and MMS.Fig 2A. Rad53 hyperphosphorylation in HU-treated cells. SJL9 (wild type with RAD53-Myc), SJL7 (pph3Δ RAD53-Myc), and SJL8 (psy2Δ RAD53-Myc) cells were incubated at 30°C in YPD containing 20 mM HU for 4 h. Cells were then washed and recovered with fresh YPD for the indicated times. Whole cell lysates were used for immunoblot analysis with anti-Myc antibody. Untreated cells were used as control. Cdc28 was probed with anti-PSTAIRE antibody as loading control. Fig 2B. Rad53 hyperphosphorylation in MMS-treated cells. SJL9 (wild type with RAD53-Myc), SJL7 (pph3Δ RAD53-Myc), and SJL8 (psy2Δ RAD53-Myc) cells were incubated at 30°C in YPD containing 0.02% MMS for 4 h. Cells were then washed and recovered with fresh YPD for the indicated times. Whole cell lysates were used for immunoblot analysis with anti-Myc antibody. Untreated cells were used as control. Cdc28 was probed with anti-PSTAIRE antibody as loading control.
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pone-0037246-g002: Rad53 undergoes hyperphosphorylation in response to HU and MMS.Fig 2A. Rad53 hyperphosphorylation in HU-treated cells. SJL9 (wild type with RAD53-Myc), SJL7 (pph3Δ RAD53-Myc), and SJL8 (psy2Δ RAD53-Myc) cells were incubated at 30°C in YPD containing 20 mM HU for 4 h. Cells were then washed and recovered with fresh YPD for the indicated times. Whole cell lysates were used for immunoblot analysis with anti-Myc antibody. Untreated cells were used as control. Cdc28 was probed with anti-PSTAIRE antibody as loading control. Fig 2B. Rad53 hyperphosphorylation in MMS-treated cells. SJL9 (wild type with RAD53-Myc), SJL7 (pph3Δ RAD53-Myc), and SJL8 (psy2Δ RAD53-Myc) cells were incubated at 30°C in YPD containing 0.02% MMS for 4 h. Cells were then washed and recovered with fresh YPD for the indicated times. Whole cell lysates were used for immunoblot analysis with anti-Myc antibody. Untreated cells were used as control. Cdc28 was probed with anti-PSTAIRE antibody as loading control.
Mentions: Rad53 was shown to be a substrate of the Pph3/Psy2 complex in both S. cerevisiae and C. albicans[34], [38]. Furthermore, Rad53 hyperphosphorylation was shown to cause cell cycle arrest and filamentous growth in C. albicans[38]. Next, we examined the phosphorylation state of Rad53 in response to MMS and HU treatment. Western blotting of C-terminally Myc-tagged Rad53 demonstrated that in wild-type cells Rad53 was hyperphosphorylated after both MMS (Fig. S3B) and HU treatment (Fig. S3A), and became dephosphorylated 6 h after HU or MMS removal (Fig. 2A&B). Both pph3Δ and psy2Δ mutants also showed a similar course of Rad53 dephosphorylation after HU removal (Fig. 2A). However, Rad53 hyperphosphorylation persisted in pph3Δ and psy2Δ mutants even at 6 h after MMS removal (Fig. 2B), indicating that Rad53 dephosphorylation during recovery from MMS treatment is dependent on the Pph3/Psy2 complex.

Bottom Line: The pathogenic fungus Candida albicans switches from yeast growth to filamentous growth in response to genotoxic stresses, in which phosphoregulation of the checkpoint kinase Rad53 plays a crucial role.Moreover, during this growth, Rad53 remained hyperphosphorylated, MBF-regulated genes were downregulated, and hypha-specific genes were upregulated.We have also identified S461 and S545 on Rad53 as potential dephosphorylation sites of Pph3/Psy2 that are specifically involved in cellular responses to MMS.

View Article: PubMed Central - PubMed

Affiliation: Key Laboratory of Cell Proliferation and Regulation Biology, Ministry of Education, College of Life Sciences, Beijing Normal University, Beijing, People's Republic of China.

ABSTRACT
The pathogenic fungus Candida albicans switches from yeast growth to filamentous growth in response to genotoxic stresses, in which phosphoregulation of the checkpoint kinase Rad53 plays a crucial role. Here we report that the Pph3/Psy2 phosphatase complex, known to be involved in Rad53 dephosphorylation, is required for cellular responses to the DNA-damaging agent methyl methanesulfonate (MMS) but not the DNA replication inhibitor hydroxyurea (HU) in C. albicans. Deletion of either PPH3 or PSY2 resulted in enhanced filamentous growth during MMS treatment and continuous filamentous growth even after MMS removal. Moreover, during this growth, Rad53 remained hyperphosphorylated, MBF-regulated genes were downregulated, and hypha-specific genes were upregulated. We have also identified S461 and S545 on Rad53 as potential dephosphorylation sites of Pph3/Psy2 that are specifically involved in cellular responses to MMS. Therefore, our studies have identified a novel molecular mechanism mediating DNA damage response to MMS in C. albicans.

Show MeSH
Related in: MedlinePlus