Pds1 phosphorylation in response to DNA damage is essential for its DNA damage checkpoint function

Abstract

In Saccharomyces cerevisiae, Pds1 is an anaphase inhibitor and plays an essential role in DNA damage and spindle checkpoint pathways. Pds1 is phosphorylated in response to DNA damage but not spindle disruption, indicating distinct mechanisms delaying anaphase entry. Phosphorylation of Pds1 is Mec1 and Chk1 dependent in vivo. Here, we show that Pds1 is phosphorylated at multiple sites in vivo in response to DNA damage by Chk1. Mutation of the Chk1 phosphorylation sites on Pds1 abolished most of its DNA damage-inducible phosphorylation and its checkpoint function, whereas its anaphase inhibitor functions and spindle checkpoint functions remain intact. Loss of Pds1 phosphorylation correlates with APC-dependent Pds1 destruction in response to DNA damage. We also show that APC(Cdc20) is active in preanaphase arrested cells after DNA damage. This suggests that Pds1 is stabilized by phosphorylation in response to DNA damage, but APC(Cdc20) activity is not altered. Our results indicate that phosphorylation of Pds1 by Chk1 is the key function of Chk1 required to prevent anaphase entry.

Figure 1

Identification of the Chk1 phosphorylation sites on Pds1. (A) Chk1-dependent phosphorylation of Pds1 in response to DNA damage. Y808 (WT) and Y1071 (Δchk1) cells expressing HA-tagged Pds1 were arrested before anaphase with nocodazole (10 μg/mL) for 2 h at 30°C, then the cultures were either left untreated (−) or exposed to γ-radiation (+, 6 krad). Protein samples were prepared 45 min after irradiation and processed for Western blot analysis (top). Y809 (cdc13-1) and Y811 (cdc13-1 Δchk1) cells expressing HA-tagged Pds1 were synchronized in G₁ by α-factor at 24°C and released at 32°C. Aliquots were withdrawn at indicated time to examine the Pds1-HA protein (bottom). (B) Phospho-Pds1 was purified from insect cell lysates and analyzed by mass spectrometry. Insect cells (Hi5) were coinfected with recombinant baculoviruses encoding GST-Chk1 and Pds1-Myc. GST-Chk1–Pds1 complexes were purified from cell lysates with glutathione beads and incubated with ATP. Proteins were separated on Tris-Glycine gradient gels (4–12%) and stained with coomassie blue. (C) Putative Chk1 phosphorylation sites on Pds1. Amino acid sequence of Pds1 with putative phospho-acceptor sites marked in black boxes. Chk1 phosphorylation consensus sequences are underlined. Six Ser residues mapped by mass spectrometry are marked with asterisks.

Figure 2

Pds1 is phosphorylated on multiple sites in response to DNA damage. (A) Plasmids encoding wild-type Pds1-HA (WT) or mutant Pds1-HA with Ala substitution at indicated sites were transformed into a Δpds1 strain. Transformants were cultured at 24°C and arrested with nocodazole (10 μg/mL) for 2 h, then exposed to γ-irradiation (+, 6 krad). Protein samples were prepared 45 min after irradiation and processed for Western blot analysis. (B) Combinatorial mutant alleles of Pds1-HA with Ala substitutions at the indicated putative Chk1 phosphorylation sites. (C) Plasmids encoding multiple alanine substitution Pds1-HA mutant proteins were transformed into a Δpds1 mutant strain. Cells then were processed as in A.

Figure 3

Pds1-m8 and Pds1-m9 mutant proteins are functional. (A) pds1-m8 and pds1-m9 are viable at 37°C. Y300 (WT), Y175 (Δpds1), Y1072 (pds1-m8), and Y1073 (pds1-m9) were struck onto YPD plates and incubated at either 24°C or 37°C. (B,C). pds1-m8 and pds1-m9 mutants are proficient for maintaining the cdc16-1 arrest. Y1076 (cdc16-1), Y1077 (cdc16-1 Δpds1), Y1078 (cdc16-1 pds1-m8), and Y1079 (cdc16-1 pds1-m9) cells were synchronized in G₁ with α-factor at 24°C and released into YPD containing 200 mM HU for 2.5 h to synchronize cells in S phase. During the last 30 min of the HU block, cells were shifted to 37°C to inactivate cdc16-1. Cells then were released from HU at 37°C. Aliquots were withdrawn at timed intervals to examine spindle morphology. Spindle morphology at 150 min for various strains are shown in B. Kinetics of anaphase entry was evaluated by the disappearance of short spindles (C). (D,E) pds1-m8 and pds1-m9 are proficient for the spindle checkpoint. Nocodazole (15 μg/mL) was added to exponentially growing cultures of Y974 (WT), Y1080 (Δpds1), Y1081 (pds1-m8), and Y1082 (pds1-m9) at 23°C. After 3 h, aliquots were withdrawn and fixed, and sister chromatids cohesion was examined. The percentage of nuclei with separated GFP signals was determined from the analysis of >100 cells (E).

Figure 4

pds1-m8 and pds1-m9 are defective in cell cycle arrest in response to DNA damage. (A) Y809 (cdc13-1), Y811 (cdc13-1 Δchk1), Y1074 (cdc13-1 pds1-m8), and Y1075 (cdc13-1 pds1-m9) cells were grown at 24°C in YPD and then plated on prewarmed YPD plates (30°C) and incubated at 30°C for 8 h. Cells were examined for microcolony formation. (dotted bars) Percentage of cells that showed a large-budded arrest; (hatched bars) percentage of cells that formed a microcolony. (B–D) Y809 (cdc13-1), Y811 (cdc13-1 Δchk1), Y817 (cdc13-1 Δpds1), Y1074 (cdc13-1 pds1-m8), and Y1075 (cdc13-1 pds1-m9) cells were synchronized in G₁ with α-factor at 24°C and released into YPD containing 200 mM HU for 2.5 h to synchronize cells in S phase. During the last 30 min of the HU block, cells were shifted to 32°C to inactivate cdc13-1. Cells then were released from HU at 32°C, and α-factor was added to prevent cell cycle reentry. Aliquots were withdrawn at timed intervals to examine DNA content by fluorescence-activated cell sorting analysis (B) and spindle morphology. Spindle morphology of various strains at 120 min is shown in C. Kinetics of anaphase entry is evaluated by the disappearance of short spindles (D).

Figure 5

Pds1-m8 and Pds1-m9 proteins are degraded by the APC in the presence of DNA damage. (A) Pds1-m8 and Pds1-m9 proteins are degraded in the presence of DNA damage. Y809 (cdc13-1), Y811 (cdc13-1 Δchk1), Y1074 (cdc13-1 pds1-m8), and Y1075 (cdc13-1 pds1-m9) cells expressing HA-tagged wild-type Pds1, Pds1-m8, or Pds1-m9 were synchronized in G₁ with α-factor at 24°C. During the last 30 min of the α-factor block, cells were shifted to 32°C to inactivate cdc13-1. Cells were released into YPD at 32°C and α-factor was added to these cultures 55 min after the release to prevent cell cycle reentry. Protein samples were prepared at the indicated times, and Pds1 protein was analyzed by Western blotting. Levels of Rna1 are used as loading control. (B,C) Degradation of Pds1-m8 and Pds1-m9 mutant proteins is dependent on the APC, and apc mutants rescued the anaphase entry defect of pds1-m8 and pds1-m9 mutants in the presence of DNA damage. Y1074 (cdc13-1 pds1-m8), Y1083 (cdc13-1 cdc16-1 pds1-m8), Y1075 (cdc13-1 pds1-m9), and Y1084 (cdc13-1 cdc16-1 pds1-m9) cells were treated as in A except that cultures were shifted to 37°C instead of 32°C. Protein samples were prepared at the indicated times, and Pds1 protein was analyzed by Western blotting. Levels of Rna1 are used as a loading control. Aliquots also were withdrawn to examine DNA content by fluorescence-activated cell sorting and spindle morphology. Kinetics of anaphase entry is evaluated by the disappearance of short spindles (C).

Figure 6

APC^Cdc20 is active in the presence of DNA damage. (A,B) Pds1-m9 mutant protein is degraded during the preanaphase arrest maintained by wild-type Pds1 in the presence of DNA damage. Y1085 (cdc13-1 PDS1-Myc PDS1-HA) and Y1086 (cdc13-1 PDS1-Myc pds1-m9-HA) cells were synchronized in G₁ with α-factor at 24°C. During the last 30 min of the α-factor block, cells were shifted to 32°C to inactivate cdc13-1. Then cells were released into YPD at 32°C. Aliquots were withdrawn at the indicated times to examine spindle morphology. Spindle morphology at 240 min after release indicated that cells maintained a preanaphase arrest (A). Protein samples also were prepared at the indicated times, and Pds1 protein was analyzed by Western blotting with both anti-Myc and anti-HA antibodies (B). (C) Pds1-m9 mutant protein is stabilized like wild-type Pds1 in the presence of spindle damage. Y1090 (PDS1-Myc PDS1-HA) and Y1091 (PDS1-Myc pds1-m9-HA) cells were synchronized in G₁ with α-factor, then released into YPD with nocodazole (15 μg/mL) at 24°C. Protein samples were prepared at the indicated times, and Pds1 protein was analyzed by Western blotting with both anti-Myc and anti-HA antibodies. Aliquots also were withdrawn to examine the budding index. The percentage of large budded cells is shown below the Western blots. (D,E) Clb5 is degraded in the presence of DNA damage. Y1087 (wild type), Y1088 (cdc13-1), and Y1089 (cdc13-1 Δchk1) cells expressing HA-tagged Clb5 were synchronized in G₁ with α-factor at 24°C. During the last 30 min of the α-factor block, cells were shifted to 34°C to inactivate cdc13-1. Cells were released into YPD at 34°C, and α-factor was added to these cultures 55 min after release to prevent cell cycle reentry. At the indicated times, protein samples were prepared to analyze the level of Clb5 protein, and aliquots were withdrawn to check the budding index. Levels of Rna1 are used as a loading control.